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https://socialsci.libretexts.org/Courses/Sacramento_City_College/LIBR_324%3A_Critical_Thinking_and_Information_Literacy/3%3A_SLO_3/3.4%3A_Rhetorical_Modes/Description	Description Learning Objectives - Determine the purpose and structure of the description essay. - Understand how to write a description essay. THE PURPOSE OF DESCRIPTION IN WRITING Writers use description in writing to make sure that their audience is fully immersed in the words on the page. This requires a concerted effort by the writer to describe his or her world through the use of sensory details. As mentioned earlier in this chapter, sensory details are descriptions that appeal to our sense of sight, sound, smell, taste, and touch. Your descriptions should try to focus on the five senses because we all rely on these senses to experience the world. The use of sensory details, then, provides you the greatest possibility of relating to your audience and thus engaging them in your writing, making descriptive writing important not only during your education but also during everyday situations. tip Avoid empty descriptors if possible. Empty descriptors are adjectives that can mean different things to different people. Good, beautiful, terrific, and nice are examples. The use of such words in descriptions can lead to misreads and confusion. A good day, for instance, can mean far different things depending on one’s age, personality, or tastes. WRITING AT WORK Whether you are presenting a new product or service to a client, training new employees, or brainstorming ideas with colleagues, the use of clear, evocative detail is crucial. Make an effort to use details that express your thoughts in a way that will register with others. Sharp, concise details are always impressive. Exercise $\PageIndex{1}$ On a separate sheet of paper, describe the following five items in a short paragraph. Use at least three of the five senses for each description. - Night - Beach - City - Dinner - Stranger THE STRUCTURE OF A DESCRIPTION ESSAY Description essays typically describe a person, a place, or an object using sensory details. The structure of a descriptive essay is more flexible than in some of the other rhetorical modes. The introduction of a description essay should set up the tone and point of the essay. The thesis should convey the writer’s overall impression of the person, place, or object described in the body paragraphs. The organization of the essay may best follow spatial order, an arrangement of ideas according to physical characteristics or appearance. Depending on what the writer describes, the organization could move from top to bottom, left to right, near to far, warm to cold, frightening to inviting, and so on. For example, if the subject were a client’s kitchen in the midst of renovation, you might start at one side of the room and move slowly across to the other end, describing appliances, cabinetry, and so on. Or you might choose to start with older remnants of the kitchen and progress to the new installations. Maybe start with the floor and move up toward the ceiling. Exercise $\PageIndex{2}$ On a separate sheet of paper, choose an organizing strategy and then execute it in a short paragraph for three of the following six items: - Train station - Your office - Your car - A coffee shop - Lobby of a movie theater - Mystery Option - Choose an object to describe but do not indicate it. Describe it, but preserve the mystery. WRITING A DESCRIPTION ESSAY Choosing a subject is the first step in writing a description essay. Once you have chosen the person, place, or object you want to describe, your challenge is to write an effective thesis statement to guide your essay. The remainder of your essay describes your subject in a way that best expresses your thesis. Remember, you should have a strong sense of how you will organize your essay. Choose a strategy and stick to it. Every part of your essay should use vivid sensory details. The more you can appeal to your readers’ senses, the more they will be engaged in your essay. Exercise $\PageIndex{3}$ On a separate sheet of paper, choose one of the topics that you started in Exercise 2, and expand it into a five-paragraph essay. Expanding on ideas in greater detail can be difficult. Sometimes it is helpful to look closely at each of the sentences in a summary paragraph. Those sentences can often serve as topic sentences to larger paragraphs. Mystery Option: Here is an opportunity to collaborate. Please share with a classmate and compare your thoughts on the mystery descriptions. Did your classmate correctly guess your mystery topic? If not, how could you provide more detail to describe it and lead them to the correct conclusion? key takeaways - Description essays should describe something vividly to the reader using strong sensory details. - Sensory details appeal to the five human senses: sight, sound, smell, taste, and touch. - A description essay should start with the writer’s main impression of a person, a place, or an object. - Use spatial order to organize your descriptive writing. References - This section was originally from Writing for Success, found at the University of Minnesota open textbook project. Full license information: This is a derivative of Writing for Success by a publisher who has requested that they and the original author not receive attribution, originally released and is used under CC BY-NC-SA. This work, unless otherwise expressly stated, is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.	libretexts	2025-03-17T22:27:23.808721	2019-09-23T22:10:35	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://socialsci.libretexts.org/Courses/Sacramento_City_College/LIBR_324%3A_Critical_Thinking_and_Information_Literacy/3%3A_SLO_3/3.4%3A_Rhetorical_Modes/Description", "book_url": "https://commons.libretexts.org/book/socialsci-23897", "title": "Description", "author": null }
https://socialsci.libretexts.org/Courses/Sacramento_City_College/LIBR_324%3A_Critical_Thinking_and_Information_Literacy/3%3A_SLO_3/3.4%3A_Rhetorical_Modes/Illustration	Illustration Learning Objectives - Determine the purpose and structure of the illustration essay. - Understand how to write an illustration essay. THE PURPOSE OF ILLUSTRATION IN WRITING To illustrate means to show or demonstrate something clearly. An effective illustration essay clearly demonstrates and supports a point through the use of evidence. The controlling idea of an essay is called a thesis. A writer can use different types of evidence to support his or her thesis. Using scientific studies, experts in a particular field, statistics, historical events, current events, analogies, and personal anecdotes are all ways in which a writer can illustrate a thesis. Ultimately, you want the evidence to help the reader “see” your point, as one would see a good illustration in a magazine or on a website. The stronger your evidence is, the more clearly the reader will consider your point. Using evidence effectively can be challenging, though. The evidence you choose will usually depend on your subject and who your reader is (your audience). When writing an illustration essay, keep in mind the following: - Use evidence that is appropriate to your topic as well as appropriate for your audience. - Assess how much evidence you need to adequately explain your point depending on the complexity of the subject and the knowledge of your audience regarding that subject. For example, if you were writing about a new communication software and your audience was a group of English-major undergrads, you might want to use an analogy or a personal story to illustrate how the software worked. You might also choose to add a few more pieces of evidence to make sure the audience understands your point. However, if you were writing about the same subject and you audience members were information technology (IT) specialists, you would likely use more technical evidence because they would be familiar with the subject. Keeping in mind your subject in relation to your audience will increase your chances of effectively illustrating your point. tip You never want to insult your readers’ intelligence by overexplaining concepts the audience members may already be familiar with, but it may be necessary to clearly articulate your point. When in doubt, add an extra example to illustrate your idea. Exercise $\PageIndex{1}$ On a separate piece of paper, form a thesis based on each of the following three topics. Then list the types of evidence that would best explain your point for each of the two audiences. - Topic: Combat and mental health Audience: family members of veterans, doctors - Topic: Video games and teen violence Audience: parents, children - Topic: Architecture and earthquakes Audience: engineers, local townspeople THE STRUCTURE OF AN ILLUSTRATION ESSAY The controlling idea, or thesis, belongs at the beginning of the essay. Evidence is then presented in the essay’s body paragraphs to support the thesis. You can start supporting your main point with your strongest evidence first, or you can start with evidence of lesser importance and have the essay build to increasingly stronger evidence. This type of organization—order of importance—you learned about in Chapter 8 “The Writing Process: How Do I Begin?” and Chapter 9 “Writing Essays: From Start to Finish”. The time transition words listed in Table 10.1 “Transition Words and Phrases for Expressing Time” are also helpful in ordering the presentation of evidence. Words like first, second, third, currently, next, and finally all help orient the reader and sequence evidence clearly. Because an illustration essay uses so many examples, it is also helpful to have a list of words and phrases to present each piece of evidence. Table 10.2 “Phrases of Illustration” provides a list of phrases for illustration. \| case in point \| for instance \| specifically \| to illustrate \| \| for example \| in particular \| in this case \| one example/ another example \| tip Vary the phrases of illustration you use. Do not rely on just one. Variety in choice of words and phrasing is critical when trying to keep readers engaged in your writing and your ideas. WRITING AT WORK In the workplace, it is often helpful to keep the phrases of illustration in mind as a way to incorporate them whenever you can. Whether you are writing out directives that colleagues will have to follow or requesting a new product or service from another company, making a conscious effort to incorporate a phrase of illustration will force you to provide examples of what you mean. Exercise $\PageIndex{2}$ On a separate sheet of paper, form a thesis based on one of the following topics. Then support that thesis with three pieces of evidence. Make sure to use a different phrase of illustration to introduce each piece of evidence you choose. - Cooking - Baseball - Work hours - Exercise - Traffic Collaboration Please share with a classmate and compare your answers. Discuss which topic you like the best or would like to learn more about. Indicate which thesis statement you perceive as the most effective. WRITING AN ILLUSTRATION ESSAY First, decide on a topic that you feel interested in writing about. Then create an interesting introduction to engage the reader. The main point, or thesis, should be stated at the end of the introduction. Gather evidence that is appropriate to both your subject and your audience. You can order the evidence in terms of importance, either from least important to most important or from most important to least important. Be sure to fully explain all of your examples using strong, clear supporting details. See Chapter 15 “Readings: Examples of Essays” to read a sample illustration essay. Exercise $\PageIndex{3}$ On a separate sheet of paper, write a five-paragraph illustration essay. You can choose one of the topics from “Exercise 1” or “Exercise 2”, or you can choose your own. key takeaways - An illustration essay clearly explains a main point using evidence. - When choosing evidence, always gauge whether the evidence is appropriate for the subject as well as the audience. - Organize the evidence in terms of importance, either from least important to most important or from most important to least important. - Use time transitions to order evidence. - Use phrases of illustration to call out examples. References - This section was originally from Writing for Success, found at the University of Minnesota open textbook project. Full license information: This is a derivative of Writing for Success by a publisher who has requested that they and the original author not receive attribution, originally released and is used under CC BY-NC-SA. This work, unless otherwise expressly stated, is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.	libretexts	2025-03-17T22:27:23.877049	2019-09-23T22:10:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://socialsci.libretexts.org/Courses/Sacramento_City_College/LIBR_324%3A_Critical_Thinking_and_Information_Literacy/3%3A_SLO_3/3.4%3A_Rhetorical_Modes/Illustration", "book_url": "https://commons.libretexts.org/book/socialsci-23897", "title": "Illustration", "author": null }
https://socialsci.libretexts.org/Courses/Sacramento_City_College/LIBR_324%3A_Critical_Thinking_and_Information_Literacy/3%3A_SLO_3/3.4%3A_Rhetorical_Modes/Narrative	Narrative Learning Objectives - Determine the purpose and structure of narrative writing. - Understand how to write a narrative essay. THE PURPOSE OF NARRATIVE WRITING Narration means the art of storytelling, and the purpose of narrative writing is to tell stories. Any time you tell a story to a friend or family member about an event or incident in your day, you engage in a form of narration. In addition, a narrative can be factual or fictional. A factual story is one that is based on, and tries to be faithful to, actual events as they unfolded in real life. A fictional story is a made-up, or imagined, story; the writer of a fictional story can create characters and events as he or she sees fit. The big distinction between factual and fictional narratives is based on a writer’s purpose. The writers of factual stories try to recount events as they actually happened, but writers of fictional stories can depart from real people and events because the writers’ intents are not to retell a real-life event. Biographies and memoirs are examples of factual stories, whereas novels and short stories are examples of fictional stories. Note Because the line between fact and fiction can often blur, it is helpful to understand what your purpose is from the beginning. Is it important that you recount history, either your own or someone else’s? Or does your interest lie in reshaping the world in your own image—either how you would like to see it or how you imagine it could be? Your answers will go a long way in shaping the stories you tell. Ultimately, whether the story is fact or fiction, narrative writing tries to relay a series of events in an emotionally engaging way. You want your audience to be moved by your story, which could mean through laughter, sympathy, fear, anger, and so on. The more clearly you tell your story, the more emotionally engaged your audience is likely to be. Exercise $\PageIndex{1}$ On a separate sheet of paper, start brainstorming ideas for a narrative. First, decide whether you want to write a factual or fictional story. Then, freewrite for five minutes. Be sure to use all five minutes, and keep writing the entire time. Do not stop and think about what to write. The following are some topics to consider as you get going: - Childhood - School - Adventure - Work - Love - Family - Friends - Vacation - Nature - Space THE STRUCTURE OF A NARRATIVE ESSAY Major narrative events are most often conveyed in chronological order, the order in which events unfold from first to last. Stories typically have a beginning, a middle, and an end, and these events are typically organized by time. Certain transitional words and phrases aid in keeping the reader oriented in the sequencing of a story. Some of these phrases are listed in Table 10.1 “Transition Words and Phrases for Expressing Time”. \| after/afterward \| as soon as \| at last \| before \| \| currently \| during \| eventually \| meanwhile \| \| next \| now \| since \| soon \| \| finally \| later \| still \| then \| \| until \| when/whenever \| while \| first, second, third \| The following are the other basic components of a narrative: - Plot. The events as they unfold in sequence. - Characters. The people who inhabit the story and move it forward. Typically, there are minor characters and main characters. The minor characters generally play supporting roles to the main character, or theprotagonist. - Conflict. The primary problem or obstacle that unfolds in the plot that the protagonist must solve or overcome by the end of the narrative. The way in which the protagonist resolves the conflict of the plot results in the theme of the narrative. - Theme. The ultimate message the narrative is trying to express; it can be either explicit or implicit. WRITING AT WORK When interviewing candidates for jobs, employers often ask about conflicts or problems a potential employee has had to overcome. They are asking for a compelling personal narrative. To prepare for this question in a job interview, write out a scenario using the narrative mode structure. This will allow you to troubleshoot rough spots, as well as better understand your own personal history. Both processes will make your story better and your self-presentation better, too. Exercise $\PageIndex{2}$ Take your freewriting exercise from the last section and start crafting it chronologically into a rough plot summary. To read more about a summary, see Chapter 6 “Writing Paragraphs: Separating Ideas and Shaping Content”. Be sure to use the time transition words and phrases listed in Table 10.1 “Transition Words and Phrases for Expressing Time” to sequence the events. Collaboration Please share with a classmate and compare your rough plot summary. WRITING A NARRATIVE ESSAY When writing a narrative essay, start by asking yourself if you want to write a factual or fictional story. Then freewrite about topics that are of general interest to you. Once you have a general idea of what you will be writing about, you should sketch out the major events of the story that will compose your plot. Typically, these events will be revealed chronologically and climax at a central conflict that must be resolved by the end of the story. The use of strong details is crucial as you describe the events and characters in your narrative. You want the reader to emotionally engage with the world that you create in writing. tip To create strong details, keep the human senses in mind. You want your reader to be immersed in the world that you create, so focus on details related to sight, sound, smell, taste, and touch as you describe people, places, and events in your narrative. As always, it is important to start with a strong introduction to hook your reader into wanting to read more. Try opening the essay with an event that is interesting to introduce the story and get it going. Finally, your conclusion should help resolve the central conflict of the story and impress upon your reader the ultimate theme of the piece. Exercise $\PageIndex{3}$ On a separate sheet of paper, add two or three paragraphs to the plot summary you started in the last section. Describe in detail the main character and the setting of the first scene. Try to use all five senses in your descriptions. key takeaways - Narration is the art of storytelling. - Narratives can be either factual or fictional. In either case, narratives should emotionally engage the reader. - Most narratives are composed of major events sequenced in chronological order. - Time transition words and phrases are used to orient the reader in the sequence of a narrative. - The four basic components to all narratives are plot, character, conflict, and theme. - The use of sensory details is crucial to emotionally engaging the reader. - A strong introduction is important to hook the reader. A strong conclusion should add resolution to the conflict and evoke the narrative’s theme. References - This section was originally from Writing for Success, found at the University of Minnesota open textbook project. Full license information: This is a derivative of Writing for Success by a publisher who has requested that they and the original author not receive attribution, originally released and is used under CC BY-NC-SA. This work, unless otherwise expressly stated, is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.	libretexts	2025-03-17T22:27:23.947232	2019-09-23T22:10:34	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://socialsci.libretexts.org/Courses/Sacramento_City_College/LIBR_324%3A_Critical_Thinking_and_Information_Literacy/3%3A_SLO_3/3.4%3A_Rhetorical_Modes/Narrative", "book_url": "https://commons.libretexts.org/book/socialsci-23897", "title": "Narrative", "author": null }
https://socialsci.libretexts.org/Courses/Sacramento_City_College/LIBR_324%3A_Critical_Thinking_and_Information_Literacy/3%3A_SLO_3/3.4%3A_Rhetorical_Modes/Persuasion/Argument	Argument Learning Objectives - Determine the purpose and structure of persuasion in writing. - Identify bias in writing. - Assess various rhetorical devices. - Distinguish between fact and opinion. - Understand the importance of visuals to strengthen arguments. - Write a persuasive essay. THE PURPOSE OF PERSUASIVE WRITING The purpose of persuasion in writing is to convince, motivate, or move readers toward a certain point of view, or opinion. The act of trying to persuade automatically implies more than one opinion on the subject can be argued. The idea of an argument often conjures up images of two people yelling and screaming in anger. In writing, however, an argument is very different. An argument is a reasoned opinion supported and explained by evidence. To argue in writing is to advance knowledge and ideas in a positive way. Written arguments often fail when they employ ranting rather than reasoning. tip Most of us feel inclined to try to win the arguments we engage in. On some level, we all want to be right, and we want others to see the error of their ways. More times than not, however, arguments in which both sides try to win end up producing losers all around. The more productive approach is to persuade your audience to consider your opinion as a valid one, not simply the right one. THE STRUCTURE OF A PERSUASIVE ESSAY The following five features make up the structure of a persuasive essay: - Introduction and thesis - Opposing and qualifying ideas - Strong evidence in support of claim - Style and tone of language - A compelling conclusion CREATING AN INTRODUCTION AND THESIS The persuasive essay begins with an engaging introduction that presents the general topic. The thesis typically appears somewhere in the introduction and states the writer’s point of view. tip Avoid forming a thesis based on a negative claim. For example, “The hourly minimum wage is not high enough for the average worker to live on.” This is probably a true statement, but persuasive arguments should make a positive case. That is, the thesis statement should focus on how the hourly minimum wage is low or insufficient. ACKNOWLEDGING OPPOSING IDEAS AND LIMITS TO YOUR ARGUMENT Because an argument implies differing points of view on the subject, you must be sure to acknowledge those opposing ideas. Avoiding ideas that conflict with your own gives the reader the impression that you may be uncertain, fearful, or unaware of opposing ideas. Thus it is essential that you not only address counterarguments but also do so respectfully. Try to address opposing arguments earlier rather than later in your essay. Rhetorically speaking, ordering your positive arguments last allows you to better address ideas that conflict with your own, so you can spend the rest of the essay countering those arguments. This way, you leave your reader thinking about your argument rather than someone else’s. You have the last word. Acknowledging points of view different from your own also has the effect of fostering more credibility between you and the audience. They know from the outset that you are aware of opposing ideas and that you are not afraid to give them space. It is also helpful to establish the limits of your argument and what you are trying to accomplish. In effect, you are conceding early on that your argument is not the ultimate authority on a given topic. Such humility can go a long way toward earning credibility and trust with an audience. Audience members will know from the beginning that you are a reasonable writer, and audience members will trust your argument as a result. For example, in the following concessionary statement, the writer advocates for stricter gun control laws, but she admits it will not solve all of our problems with crime: Although tougher gun control laws are a powerful first step in decreasing violence in our streets, such legislation alone cannot end these problems since guns are not the only problem we face. Such a concession will be welcome by those who might disagree with this writer’s argument in the first place. To effectively persuade their readers, writers need to be modest in their goals and humble in their approach to get readers to listen to the ideas. See Table 10.5 “Phrases of Concession” for some useful phrases of concession. \| although \| granted that \| of course \| \| still \| though \| yet \| Exercise $\PageIndex{1}$ Try to form a thesis for each of the following topics. Remember the more specific your thesis, the better. - Foreign policy - Television and advertising - Stereotypes and prejudice - Gender roles and the workplace - Driving and cell phones Collaboration Please share with a classmate and compare your answers. Choose the thesis statement that most interests you and discuss why. BIAS IN WRITING Everyone has various biases on any number of topics. For example, you might have a bias toward wearing black instead of brightly colored clothes or wearing jeans rather than formal wear. You might have a bias toward working at night rather than in the morning, or working by deadlines rather than getting tasks done in advance. These examples identify minor biases, of course, but they still indicate preferences and opinions. Handling bias in writing and in daily life can be a useful skill. It will allow you to articulate your own points of view while also defending yourself against unreasonable points of view. The ideal in persuasive writing is to let your reader know your bias, but do not let that bias blind you to the primary components of good argumentation: sound, thoughtful evidence and a respectful and reasonable address of opposing sides. The strength of a personal bias is that it can motivate you to construct a strong argument. If you are invested in the topic, you are more likely to care about the piece of writing. Similarly, the more you care, the more time and effort you are apt to put forth and the better the final product will be. The weakness of bias is when the bias begins to take over the essay—when, for example, you neglect opposing ideas, exaggerate your points, or repeatedly insert yourself ahead of the subject by using I too often. Being aware of all three of these pitfalls will help you avoid them. THE USE OF I IN WRITING The use of I in writing is often a topic of debate, and the acceptance of its usage varies from instructor to instructor. It is difficult to predict the preferences for all your present and future instructors, but consider the effects it can potentially have on your writing. Be mindful of the use of I in your writing because it can make your argument sound overly biased. There are two primary reasons: - Excessive repetition of any word will eventually catch the reader’s attention—and usually not in a good way. The use of I is no different. - The insertion of I into a sentence alters not only the way a sentence might sound but also the composition of the sentence itself. I is often the subject of a sentence. If the subject of the essay is supposed to be, say, smoking, then by inserting yourself into the sentence, you are effectively displacing the subject of the essay into a secondary position. In the following example, the subject of the sentence is underlined: Smoking is bad. I think smoking is bad. In the first sentence, the rightful subject, smoking, is in the subject position in the sentence. In the second sentence, the insertion of I and think replaces smoking as the subject, which draws attention to I and away from the topic that is supposed to be discussed. Remember to keep the message (the subject) and the messenger (the writer) separate. CHECKLIST Developing Sound Arguments Does my essay contain the following elements? - An engaging introduction - A reasonable, specific thesis that is able to be supported by evidence - A varied range of evidence from credible sources - Respectful acknowledgement and explanation of opposing ideas - A style and tone of language that is appropriate for the subject and audience - Acknowledgement of the argument’s limits - A conclusion that will adequately summarize the essay and reinforce the thesis FACT AND OPINION Facts are statements that can be definitely proven using objective data. The statement that is a fact is absolutely valid. In other words, the statement can be pronounced as true or false. For example, 2 + 2 = 4. This expression identifies a true statement, or a fact, because it can be proved with objective data. Opinions are personal views, or judgments. An opinion is what an individual believes about a particular subject. However, an opinion in argumentation must have legitimate backing; adequate evidence and credibility should support the opinion. Consider the credibility of expert opinions. Experts in a given field have the knowledge and credentials to make their opinion meaningful to a larger audience. For example, you seek the opinion of your dentist when it comes to the health of your gums, and you seek the opinion of your mechanic when it comes to the maintenance of your car. Both have knowledge and credentials in those respective fields, which is why their opinions matter to you. But the authority of your dentist may be greatly diminished should he or she offer an opinion about your car, and vice versa. In writing, you want to strike a balance between credible facts and authoritative opinions. Relying on one or the other will likely lose more of your audience than it gains. tip The word prove is frequently used in the discussion of persuasive writing. Writers may claim that one piece of evidence or another proves the argument, but proving an argument is often not possible. No evidence proves a debatable topic one way or the other; that is why the topic is debatable. Facts can be proved, but opinions can only be supported, explained, and persuaded. Exercise $\PageIndex{2}$ On a separate sheet of paper, take three of the theses you formed in Exercise 1, and list the types of evidence you might use in support of that thesis. Exercise $\PageIndex{3}$ Using the evidence you provided in support of the three theses in Exercise 2, come up with at least one counterargument to each. Then write a concession statement, expressing the limits to each of your three arguments. USING VISUAL ELEMENTS TO STRENGTHEN ARGUMENTS Adding visual elements to a persuasive argument can often strengthen its persuasive effect. There are two main types of visual elements: quantitative visuals and qualitative visuals. Quantitative visuals present data graphically. They allow the audience to see statistics spatially. The purpose of using quantitative visuals is to make logical appeals to the audience. For example, sometimes it is easier to understand the disparity in certain statistics if you can see how the disparity looks graphically. Bar graphs, pie charts, Venn diagrams, histograms, and line graphs are all ways of presenting quantitative data in spatial dimensions. Qualitative visuals present images that appeal to the audience’s emotions. Photographs and pictorial images are examples of qualitative visuals. Such images often try to convey a story, and seeing an actual example can carry more power than hearing or reading about the example. For example, one image of a child suffering from malnutrition will likely have more of an emotional impact than pages dedicated to describing that same condition in writing. WRITING AT WORK When making a business presentation, you typically have limited time to get across your idea. Providing visual elements for your audience can be an effective timesaving tool. Quantitative visuals in business presentations serve the same purpose as they do in persuasive writing. They should make logical appeals by showing numerical data in a spatial design. Quantitative visuals should be pictures that might appeal to your audience’s emotions. You will find that many of the rhetorical devices used in writing are the same ones used in the workplace. WRITING A PERSUASIVE ESSAY Choose a topic that you feel passionate about. If your instructor requires you to write about a specific topic, approach the subject from an angle that interests you. Begin your essay with an engaging introduction. Your thesis should typically appear somewhere in your introduction. Start by acknowledging and explaining points of view that may conflict with your own to build credibility and trust with your audience. Also state the limits of your argument. This too helps you sound more reasonable and honest to those who may naturally be inclined to disagree with your view. By respectfully acknowledging opposing arguments and conceding limitations to your own view, you set a measured and responsible tone for the essay. Make your appeals in support of your thesis by using sound, credible evidence. Use a balance of facts and opinions from a wide range of sources, such as scientific studies, expert testimony, statistics, and personal anecdotes. Each piece of evidence should be fully explained and clearly stated. Make sure that your style and tone are appropriate for your subject and audience. Tailor your language and word choice to these two factors, while still being true to your own voice. Finally, write a conclusion that effectively summarizes the main argument and reinforces your thesis. Exercise $\PageIndex{4}$ Choose one of the topics you have been working on throughout this section. Use the thesis, evidence, opposing argument, and concessionary statement as the basis for writing a full persuasive essay. Be sure to include an engaging introduction, clear explanations of all the evidence you present, and a strong conclusion. key takeaways - The purpose of persuasion in writing is to convince or move readers toward a certain point of view, or opinion. - An argument is a reasoned opinion supported and explained by evidence. To argue, in writing, is to advance knowledge and ideas in a positive way. - A thesis that expresses the opinion of the writer in more specific terms is better than one that is vague. - It is essential that you not only address counterarguments but also do so respectfully. - It is also helpful to establish the limits of your argument and what you are trying to accomplish through a concession statement. - To persuade a skeptical audience, you will need to use a wide range of evidence. Scientific studies, opinions from experts, historical precedent, statistics, personal anecdotes, and current events are all types of evidence that you might use in explaining your point. - Make sure that your word choice and writing style is appropriate for both your subject and your audience. - You should let your reader know your bias, but do not let that bias blind you to the primary components of good argumentation: sound, thoughtful evidence and respectfully and reasonably addressing opposing ideas. - You should be mindful of the use of I in your writing because it can make your argument sound more biased than it needs to. - Facts are statements that can be proven using objective data. - Opinions are personal views, or judgments, that cannot be proven. - In writing, you want to strike a balance between credible facts and authoritative opinions. - Quantitative visuals present data graphically. The purpose of using quantitative visuals is to make logical appeals to the audience. - Qualitative visuals present images that appeal to the audience’s emotions. References - This section was originally from Writing for Success, found at the University of Minnesota open textbook project. Full license information: This is a derivative of Writing for Success by a publisher who has requested that they and the original author not receive attribution, originally released and is used under CC BY-NC-SA. This work, unless otherwise expressly stated, is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.	libretexts	2025-03-17T22:27:24.053832	2019-09-23T22:10:40	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://socialsci.libretexts.org/Courses/Sacramento_City_College/LIBR_324%3A_Critical_Thinking_and_Information_Literacy/3%3A_SLO_3/3.4%3A_Rhetorical_Modes/Persuasion/Argument", "book_url": "https://commons.libretexts.org/book/socialsci-23897", "title": "Argument", "author": null }
https://socialsci.libretexts.org/Courses/Sacramento_City_College/LIBR_324%3A_Critical_Thinking_and_Information_Literacy/3%3A_SLO_3/3.4%3A_Rhetorical_Modes/Process_Analysis	Process Analysis Learning Objectives - Determine the purpose and structure of the process analysis essay. - Understand how to write a process analysis essay. THE PURPOSE OF PROCESS ANALYSIS IN WRITING The purpose of a process analysis essay is to explain how to do something or how something works. In either case, the formula for a process analysis essay remains the same. The process is articulated into clear, definitive steps. Almost everything we do involves following a step-by-step process. From riding a bike as children to learning various jobs as adults, we initially needed instructions to effectively execute the task. Likewise, we have likely had to instruct others, so we know how important good directions are—and how frustrating it is when they are poorly put together. WRITING AT WORK The next time you have to explain a process to someone at work, be mindful of how clearly you articulate each step. Strong communication skills are critical for workplace satisfaction and advancement. Effective process analysis plays a critical role in developing that skill set. Exercise $\PageIndex{1}$ On a separate sheet of paper, make a bulleted list of all the steps that you feel would be required to clearly illustrate three of the following four processes: - Tying a shoelace - Parallel parking - Planning a successful first date - Being an effective communicator THE STRUCTURE OF A PROCESS ANALYSIS ESSAY The process analysis essay opens with a discussion of the process and a thesis statement that states the goal of the process. The organization of a process analysis essay typically follows chronological order. The steps of the process are conveyed in the order in which they usually occur. Body paragraphs will be constructed based on these steps. If a particular step is complicated and needs a lot of explaining, then it will likely take up a paragraph on its own. But if a series of simple steps is easier to understand, then the steps can be grouped into a single paragraph. The time transition phrases covered in the Narration and Illustration sections are also helpful in organizing process analysis essays (see Table 10.1 “Transition Words and Phrases for Expressing Time” and Table 10.2 “Phrases of Illustration”). Words such as first, second, third, next, and finally are helpful cues to orient reader and organize the content of essay. tip Always have someone else read your process analysis to make sure it makes sense. Once we get too close to a subject, it is difficult to determine how clearly an idea is coming across. Having a friend or coworker read it over will serve as a good way to troubleshoot any confusing spots. Exercise $\PageIndex{2}$ Choose two of the lists you created in Exercise 1 and start writing out the processes in paragraph form. Try to construct paragraphs based on the complexity of each step. For complicated steps, dedicate an entire paragraph. If less complicated steps fall in succession, group them into a single paragraph. WRITING A PROCESS ANALYSIS ESSAY Choose a topic that is interesting, is relatively complex, and can be explained in a series of steps. As with other rhetorical writing modes, choose a process that you know well so that you can more easily describe the finer details about each step in the process. Your thesis statement should come at the end of your introduction, and it should state the final outcome of the process you are describing. Body paragraphs are composed of the steps in the process. Each step should be expressed using strong details and clear examples. Use time transition phrases to help organize steps in the process and to orient readers. The conclusion should thoroughly describe the result of the process described in the body paragraphs. Exercise $\PageIndex{3}$ Choose one of the expanded lists from Exercise 2. Construct a full process analysis essay from the work you have already done. That means adding an engaging introduction, a clear thesis, time transition phrases, body paragraphs, and a solid conclusion. key takeaways - A process analysis essay explains how to do something, how something works, or both. - The process analysis essay opens with a discussion of the process and a thesis statement that states the outcome of the process. - The organization of a process analysis essay typically follows a chronological sequence. - Time transition phrases are particularly helpful in process analysis essays to organize steps and orient reader. References - This section was originally from Writing for Success, found at the University of Minnesota open textbook project. Full license information: This is a derivative of Writing for Success by a publisher who has requested that they and the original author not receive attribution, originally released and is used under CC BY-NC-SA. This work, unless otherwise expressly stated, is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.	libretexts	2025-03-17T22:27:24.116903	2019-09-23T22:10:38	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://socialsci.libretexts.org/Courses/Sacramento_City_College/LIBR_324%3A_Critical_Thinking_and_Information_Literacy/3%3A_SLO_3/3.4%3A_Rhetorical_Modes/Process_Analysis", "book_url": "https://commons.libretexts.org/book/socialsci-23897", "title": "Process Analysis", "author": null }
https://socialsci.libretexts.org/Courses/Sacramento_City_College/LIBR_324%3A_Critical_Thinking_and_Information_Literacy/3%3A_SLO_3/3.5%3A_Research_and_Critical_Reading_(Part_1)	3.5: Research and Critical Reading (Part 1) Learning Objectives - Read critically to discover the meaning, purpose, and content of a piece - Respond critically to written works using reading strategy INTRODUCTION Good researchers and writers examine their sources critically and actively. They do not just compile and summarize these research sources in their writing, but use them to create their own ideas, theories, and, ultimately, their own, new understanding of the topic they are researching. Such an approach means not taking the information and opinions that the sources contain at face value and for granted, but to investigate, test, and even doubt every claim, every example, every story, and every conclusion. It means not to sit back and let your sources control you, but to engage in active conversation with them and their authors. In order to be a good researcher and writer, one needs to be a critical and active reader. This chapter is about the importance of critical and active reading. It is also about the connection between critical reading and active, strong writing. Much of the discussion you will find in this chapter in fundamental to research and writing, no matter what writing genre, medium, or academic discipline you read and write in. Every other approach to research writing, every other research method and assignment offered elsewhere in this book is, in some way, based upon the principles discussed in this chapter. Reading is at the heart of the research process. No matter what kinds of research sources and, methods you use, you are always reading and interpreting text. Most of us are used to hearing the word “reading” in relation to secondary sources, such as books, journals, magazines, websites, and so on. But even if you are using other research methods and sources, such as interviewing someone or surveying a group of people, you are reading. You are reading their subjects’ ideas and views on the topic you are investigating. Even if you are studying photographs, cultural artifacts, and other non-verbal research sources, you are reading them, too by trying to connect them to their cultural and social contexts and to understand their meaning. Principles of critical reading which we are about to discuss in this chapter apply to those research situations as well. I like to think about reading and writing as not two separate activities but as two tightly connected parts of the same whole. That whole is the process of learning and making of new meaning. It may seem that reading and writing are complete opposite of one another. According to the popular view, when we read, we “consume” texts, and when we write, we “produce” texts. But this view of reading and writing is true only if you see reading as a passive process of taking in information from the text and not as an active and energetic process of making new meaning and new knowledge. Similarly, good writing does not come from nowhere but is usually based upon, or at least influenced by ideas, theories, and stories that come from reading. So, if, as a college student, you have ever wondered why your writing teachers have asked you to read books and articles and write responses to them, it is because writers who do not read and do not actively engage with their reading, have little to say to others. We will begin this chapter with the definition of the term “critical reading.” We will consider its main characteristics and briefly touch upon ways to become an active and critical reader. Next, we will discuss the importance of critical reading for research and how reading critically can help you become a better researcher and make the research process more enjoyable. Also in this chapter, a student-writer offers us an insight into his critical reading and writing processes. This chapter also shows how critical reading can and should be used for critical and strong writing. And, as all other chapters, this one offers you activities and projects designed to help you implement the advice presented here into practice. WHAT KIND OF READER ARE YOU? You read a lot, probably more that you think. You read school textbooks, lecture notes, your classmates’ papers, and class websites. When school ends, you probably read some fiction, magazines. But you also read other texts. These may include CD liner notes, product reviews, grocery lists, maps, driving directions, road signs, and the list can go on and on. And you don’t read all these texts in the same way. You read them with different purposes and using different reading strategies and techniques. The first step towards becoming a critical and active reader is examining your reading process and your reading preferences. Therefore, you are invited to complete the following exploration activity. Exercise $\PageIndex{1}$: Analyzing Your Reading Habits List all the reading you have done in the last week. Include both “school” and “out-of school” reading. Try to list as many texts as you can think of, no matter how short and unimportant they might seem. Now, answer the following questions. • What was your purpose in reading each of those texts? Did you read for information, to pass a test, for enjoyment, to decide on a product you wanted to buy, and so on? Or, did you read to figure out some complex problem that keeps you awake at night? • You have probably come up with a list of different purposes. How did each of those purposes influence your reading strategies? Did you take notes or try to memorize what you read? How long did it take you to read different texts? Did you begin at the beginning and read till you reached the end, or did you browse some texts? Consider the time of day you were reading. Consider even whether some texts tired you out or whether you thought they were “boring.” Why? • What did you do with the results of your reading? Did you use them for some practical purpose, such as buying a new product or finding directions, or did you use them for a less practical purpose, such as understanding some topic better r learning something about yourself and others? When you finish, share your results with the rest of the class and with your instructor. Having answered the questions above, you have probably noticed that your reading strategies differed depending on the reading task you were facing and on what you planned to do with the results of the reading. If, for example, you read lecture notes in order to pass a test, chances are you “read for information,” or “for the main” point, trying to remember as much material as possible and anticipating possible test questions. If, on the other hand, you read a good novel, you probably just focused on following the story. Finally, if you were reading something that you hoped would help you answer some personal question or solve some personal problem, it is likely that you kept comparing and contrasting the information that you read your own life and your own experiences. You may have spent more time on some reading tasks than others. For example, when we are interested in one particular piece of information or fact from a text, we usually put that text aside once we have located the information we were looking for. In other cases, you may have been reading for hours on end taking careful notes and asking questions. If you share the results of your investigation into your reading habits with your classmates, you may also notice that some of their reading habits and strategies were different from yours. Like writing strategies, approaches to reading may vary from person to person depending on our previous experiences with different topics and types of reading materials, expectations we have of different texts, and, of course, the purpose with which we are reading. Life presents us with a variety of reading situations which demand different reading strategies and techniques. Sometimes, it is important to be as efficient as possible and read purely for information or “the main point.” At other times, it is important to just “let go” and turn the pages following a good story, although this means not thinking about the story you are reading. At the heart of writing and research, however, lies the kind of reading known as critical reading. Critical examination of sources is what makes their use in research possible and what allows writers to create rhetorically effective and engaging texts. KEY FEATURES OF CRITICAL READING Critical readers are able to interact with the texts they read through carefully listening, writing, conversation, and questioning. They do not sit back and wait for the meaning of a text to come to them, but work hard in order to create such meaning. Critical readers are not made overnight. Becoming a critical reader will take a lot of practice and patience. Depending on your current reading philosophy and experiences with reading, becoming a critical reader may require a significant change in your whole understanding of the reading process. The trade-off is worth it, however. By becoming a more critical and active reader, you will also become a better researcher and a better writer. Last but not least, you will enjoy reading and writing a whole lot more because you will become actively engaged in both. One of my favorite passages describing the substance of critical and active reading comes from the introduction to their book Ways of Reading , whose authors David Bartholomae and Anthony Petrosky write: Reading involves a fair measure of push and shove. You make your mark on the book and it makes its mark on you. Reading is not simply a matter of hanging back and waiting for a piece, or its author, to tell you what the writing has to say. In fact, one of the difficult things about reading is that the pages before you will begin to speak only when the authors are silent and you begin to speak in their place, sometimes for them—doing their work, continuing their projects—and sometimes for yourself, following your own agenda (1). Notice that Bartholomae and Petrosky describe reading process in pro-active terms. Meaning of every text is “made,” not received. Readers need to “push and shove” in order to create their own, unique content of every text they read. It is up the you as a reader to make the pages in front of you “speak” by talking with and against the text, by questioning and expanding it. Critical reading, then, is a two-way process. As reader, you are not a consumer of words, waiting patiently for ideas from the printed page or a web-site to fill your head and make you smarter. Instead, as a critical reader, you need to interact with what you read, asking questions of the author, testing every assertion, fact, or idea, and extending the text by adding your own understanding of the subject and your own personal experiences to your reading. The following are key features of the critical approach to reading: - No text, however well written and authoritative, contains its own, pre-determined meaning. - Readers must work hard to create meaning from every text. - Critical readers interact with the texts they read by questioning them, responding to them, and expanding them, usually in writing. - To create meaning, critical readers use a variety of approaches, strategies, and techniques which include applying their personal experiences and existing knowledge to the reading process. - Critical readers seek actively out other texts, related to the topic of their investigation. The following section is an examination of these claims about critical reading in more detail. TEXTS PRESENT IDEAS, NOT ABSOLUTE TRUTHS In order to understand the mechanisms and intellectual challenges of critical reading, we need to examine some of our deepest and long-lasting assumptions about reading. Perhaps the two most significant challenges facing anyone who wants to become a more active and analytical reader is understanding that printed texts doe not contain inarguable truths and learning to questions and talk back to those texts. Students in my writing classes often tell me that the biggest challenge they face in trying to become critical readers is getting away from the idea that they have to believe everything they read on a printed page. Years of schooling have taught many of us to believe that published texts present inarguable, almost absolute truths. The printed page has authority because, before publishing his or her work, every writer goes through a lengthy process of approval, review, revision, fact-checking, and so on. Consequently, this theory goes, what gets published must be true. And if it is true, it must be taken at face value, not questioned, challenged, or extended in any way. Perhaps, the ultimate authority among the readings materials encountered by college belongs to the textbook. As students, we all have had to read and almost memorize textbook chapters in order to pass an exam. We read textbooks “for information,” summarizing their chapters, trying to find “the main points” and then reproducing these main points during exams. I have nothing against textbook as such, in fact, I am writing one right now. And it is certainly possible to read textbooks critically and actively. But, as I think about the challenges which many college students face trying to become active and critical readers, I come to the conclusion that the habit to read every text as if they were preparing for an exam on it, as if it was a source of unquestionable truth and knowledge prevents many from becoming active readers. Treating texts as if they were sources of ultimate and unquestionable knowledge and truth represents the view of reading as consumption. According to this view, writers produce ideas and knowledge, and we, readers, consume them. Of course, sometimes we have to assume this stance and read for information or the “main point” of a text. But it is critical reading that allows us to create new ideas from what we read and to become independent and creative learners. Critical reading is a collaboration between the reader and the writer. It offers readers the ability to be active participants in the construction of meaning of every text they read and to use that meaning for their own learning and self-fulfillment. Not even the best researched and written text is absolutely complete and finished. Granted, most fields of knowledge have texts which are called “definitive.” Such texts usually represent our best current knowledge on their subjects. However, even the definitive works get revised over time and they are always open to questioning and different interpretations. READING IS A RHETORICAL TOOL To understand how the claim that every reader makes his or her meaning from texts works, it is necessary to examine what is know as the rhetorical theory of reading. The work that best describes and justifies the rhetorical reading theory is Douglas Brent’s 1992 book Reading as Rhetorical Invention: Knowledge, Persuasion, and the Teaching of Research-Based Writing . I like to apply Brent’s ideas to my discussions of critical reading because I think that they do a good job demystifying critical reading’s main claims. Brent’s theory of reading is a rhetorical device puts significant substance behind the somewhat abstract ideas of active and critical reading, explaining how the mechanisms of active interaction between readers and texts actually work. Briefly explained, Brent treats reading not only as a vehicle for transmitting information and knowledge, but also as a means of persuasion. In fact, according to Brent, knowledge equals persuasion because, in his words, “Knowledge is not simply what one has been told. Knowledge is what one believes, what one accepts as being at least provisionally true.” (xi). This short passage contains two assertions which are key to the understanding of mechanisms of critical reading. Firstly, notice that simply reading “for the main point” will not necessarily make you “believe” what you read. Surely, such reading can fill our heads with information, but will that information become our knowledge in a true sense, will we be persuaded by it, or will we simply memorize it to pass the test and forget it as soon as we pass it? Of course not! All of us can probably recall many instances in which we read a lot to pass a test only to forget, with relief, what we read as soon as we left the classroom where that test was held. The purpose of reading and research, then, is not to get as much as information out of a text as possible but to change and update one’s system of beliefs on a given subject (Brent 55-57). Brent further states: The way we believe or disbelieve certain texts clearly varies from one individual to the next. If you present a text that is remotely controversial to a group of people, some will be convinced by it and some not, and those who are convinced will be convinced in different degrees. The task of a rhetoric of reading is to explain systematically how these differences arise— how people are persuaded differently by texts (18). Critical and active readers not only accept the possibility that the same texts will have different meanings for different people, but welcome this possibility as an inherent and indispensable feature of strong, engaged, and enjoyable reading process. To answer his own questions about what factors contribute to different readers’ different interpretations of the same texts, Brent offers us the following principles that I have summarized from his book: • Readers are guided by personal beliefs, assumptions, and pre-existing knowledge when interpreting texts. You can read more on the role of the reader’s pre-existing knowledge in the construction of meaning later on in this chapter. • Readers react differently to the logical proofs presented by the writers of texts. • Readers react differently to emotional and ethical proofs presented by writers. For example, an emotional story told by a writer may resonate with one person more than with another because the first person lived through a similar experience and the second one did not, and so on. The idea behind the rhetorical theory of reading is that when we read, we not only take in ideas, information, and facts, but instead we “update our view of the world.” You cannot force someone to update their worldview, and therefore, the purpose of writing is persuasion and the purpose of reading is being persuaded. Persuasion is possible only when the reader is actively engaged with the text and understands that much more than simple retrieval of information is at stake when reading. One of the primary factors that influence our decision to accept or not to accept an argument is what Douglas Brent calls our “repertoire of experience, much of [which] is gained through prior interaction with texts” (56). What this means is that when we read a new text, we do not begin with a clean slate, an empty mind. However unfamiliar the topic of this new reading may seem to us, we approach it with a large baggage of previous knowledge, experiences, points of view, and so on. When an argument “comes in” into our minds from a text, this text, by itself, cannot change our view on the subject. Our prior opinions and knowledge about the topic of the text we are reading will necessarily “filter out” what is incompatible with those views (Brent 56-57). This, of course, does not mean that, as readers, we should persist in keeping our old ideas about everything and actively resist learning new things. Rather, it suggests that the reading process is an interaction between the ideas in the text in front of us and our own ideas and pre-conceptions about the subject of our reading. We do not always consciously measure what we read according to our existing systems of knowledge and beliefs, but we measure it nevertheless. Reading, according to Brent, is judgment, and, like in life where we do not always consciously examine and analyze the reasons for which we make various decisions, evaluating a text often happens automatically or subconsciously (59). Applied to research writing, Brent’s theory or reading means the following: - The purpose of research is not simply to retrieve data, but to participate in a conversation about it. Simple summaries of sources is not research, and writers should be aiming for active interpretation of sources instead - There is no such thing as an unbiased source. Writers make claims for personal reasons that critical readers need to learn to understand and evaluate. - Feelings can be a source of shareable good reason for belief. Readers and writers need to use, judiciously, ethical and pathetic proofs in interpreting texts and in creating their own. - Research is recursive. Critical readers and researchers never stop asking questions about their topic and never consider their research finished. ACTIVE READERS LOOK FOR CONNECTIONS BETWEEN TEXTS Earlier on, I mentioned that one of the traits of active readers is their willingness to seek out other texts and people who may be able to help them in their research and learning. I find that for many beginning researchers and writers, the inability to seek out such connections often turns into a roadblock on their research route. Here is what I am talking about. Recently, I asked my writing students to investigate some problem on campus and to propose a solution to it. I asked them to use both primary (interviews, surveys, etc.) and secondary (library, Internet, etc.) research. Conducting secondary research allows a writer to connect a local problem he or she is investigating and a local solution he or she is proposing with a national and even global context, and to see whether the local situation is typical or a-typical. One group of students decided to investigate the issue of racial and ethnic diversity on our campus. The lack of diversity is a “hot” issue on our campus, and recently an institutional task force was created to investigate possible ways of making our university more diverse. The students had no trouble designing research questions and finding people to interview and survey. Their subjects included students and faculty as well as the university vice-president who was changed with overseeing the work of the diversity task force. Overall, these authors have little trouble conducting and interpreting primary research that led them to conclude that, indeed, our campus is not diverse enough and that most students would like to see the situation change. The next step these writers took was to look at the websites of some other schools similar in size and nature to ours, to see how our university compared on the issue of campus diversity with others. They were able to find some statistics on the numbers of minorities at other colleges and universities that allowed them to create a certain backdrop for their primary research that they had conducted earlier. But good writing goes beyond the local situation. Good writing tries to connect the local and the national and the global. It tries to look beyond the surface of the problem, beyond simply comparing numbers and other statistics. It seeks to understand the roots of a problem and propose a solution based on a local and well as a global situation and research. The primary and secondary research conducted by these students was not allowing them to make that step from analyzing local data to understanding their problem in context. They needed some other type of research sources. At that point, however, those writers hit an obstacle. How and where, they reasoned, would we find other secondary sources, such as books, journals, and websites, about the lack of diversity on our campus? The answer to that question was that, at this stage in their research and writing, they did not need to look for more sources about our local problem with the lack of diversity. They needed to look at diversity and ways to increase it as a national and global issue. They needed to generalize the problem and, instead of looking at a local example, to consider its implications for the issue they were studying overall. Such research would not only have allowed these writers to examine the problem as a whole but also to see how it was being solved in other places. This, in turn, might have helped them to propose a local solution. Critical readers and researchers understand that it is not enough to look at the research question locally or narrowly. After conducting research and understanding their problem locally, or as it applies specifically to them, active researchers contextualize their investigation by seeking out texts and other sources which would allow them to see the big picture. Sometimes, it is hard to understand how external texts which do not seem to talk directly about you can help you research and write about questions, problems, and issues in your own life. In her 2004 essay, “Developing ‘Interesting Thoughts’: Reading for Research,” writing teacher and my former colleague Janette Martin tells a story of a student who was writing a paper about what it is like to be a collegiate athlete. The emerging theme in that paper was that of discipline and sacrifice required of student athletes. Simultaneously, that student was reading a chapter from the book by the French philosopher Michel Foucault called Discipline and Punish. Foucault’s work is a study of the western penitentiary system, which, of course cannot be directly compared to experiences of a student athlete. At the same time, one of the leading themes in Foucault’s work is discipline. Martin states that the student was able to see some connection between Foucault and her own life and use the reading for her research and writing (6). In addition to showing how related texts can be used to explore various aspects of the writer’s own life, this example highlights the need to read texts critically and interpret them creatively. Such reading and research goes beyond simply comparing of facts and numbers and towards relating ideas and concepts with one another. FROM READING TO WRITING Reading and writing are the two essential tools of learning. Critical reading is not a process of passive consumption, but one of interaction and engagement between the reader and the text. Therefore, when reading critically and actively, it is important not only to take in the words on the page, but also to interpret and to reflect upon what you read through writing and discussing it with others. CRITICAL READERS UNDERSTAND THE DIFFERENCE BETWEEN REACTING AND RESPONDING TO A TEXT As stated earlier in this chapter, actively responding to difficult texts, posing questions, and analyzing ideas presented in them is the key to successful reading. The goal of an active reader is to engage in a conversation with the text he or she is reading. In order to fulfill this goal, it is important to understand the difference between reacting to the text and responding to it. Reacting to a text is often done on an emotional, rather than on an intellectual level. It is quick and shallow. For example, if we encounter a text that advances arguments with which we strongly disagree, it is natural to dismiss those ideas out of hand as not wrong and not worthy of our attention. Doing so would be reacting to the text based only on emotions and on our pre-set opinions about its arguments. It is easy to see that reacting in this way does not take the reader any closer to understanding the text. A wall of disagreement that existed between the reader and the text before the reading continues to exist after the reading. Responding to a text, on the other hand, requires a careful study of the ideas presented and arguments advanced in it. Critical readers who possess this skill are not willing to simply reject or accept the arguments presented in the text after the first reading right away. To continue with our example from the preceding paragraph, a reader who responds to a controversial text rather than reacting to it might apply several of the following strategies before forming and expressing an opinion about that text. - Read the text several times, taking notes, asking questions, and underlining key places. - Study why the author of the text advances ideas, arguments, and convictions, so different from the reader’s own. For example, is the text’s author advancing an agenda of some social, political, religious, or economic group of which he or she is a member? - Study the purpose and the intended audience of the text. - Study the history of the argument presented in the text as much as possible. For example, modern texts on highly controversial issues such as the death penalty, abortion, or euthanasia often use past events, court cases, and other evidence to advance their claims. Knowing the history of the problem will help you to construct meaning of a difficult text. - Study the social, political, and intellectual context in which the text was written. Good writers use social conditions to advance controversial ideas. Compare the context in which the text was written to the one in which it is read. For example, have social conditions changed, thus invalidating the argument or making it stronger? - Consider the author’s (and your own) previous knowledge of the issue at the center of the text and your experiences with it. How might such knowledge or experience have influenced your reception of the argument? Taking all these steps will help you to move away from simply reacting to a text and towards constructing informed and critical response to it. CRITICAL READERS RESIST OVERSIMPLIFIED BINARY RESPONSES Critical readers learn to avoid simple “agree-disagree” responses to complex texts. Such way of thinking and arguing is often called “binary” because is allows only two answers to every statement and every questions. But the world of ideas is complex and, a much more nuanced approach is needed when dealing with complex arguments. When you are asked to “critique” a text, which readers are often asked to do, it does not mean that you have to “criticize” it and reject its argument out of hand. What you are being asked to do instead is to carefully evaluate and analyze the text’s ideas, to understand how and why they are constructed and presented, and only then develop a response to that text. Not every text asks for an outright agreement or disagreement. Sometimes, we as readers are not in a position to either simply support an argument or reject it. What we can do in such cases, though, is to learn more about the text’s arguments by carefully considering all of their aspects and to construct a nuanced, sophisticated response to them. After you have done all that, it will still be possible to disagree with the arguments presented in the reading, but your opinion about the text will be much more informed and nuanced than if you have taken the binary approach from the start.	libretexts	2025-03-17T22:27:24.194640	2019-09-23T22:10:41	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://socialsci.libretexts.org/Courses/Sacramento_City_College/LIBR_324%3A_Critical_Thinking_and_Information_Literacy/3%3A_SLO_3/3.5%3A_Research_and_Critical_Reading_(Part_1)", "book_url": "https://commons.libretexts.org/book/socialsci-23897", "title": "3.5: Research and Critical Reading (Part 1)", "author": null }
https://socialsci.libretexts.org/Courses/Sacramento_City_College/LIBR_324%3A_Critical_Thinking_and_Information_Literacy/3%3A_SLO_3/3.6%3A_Research_and_Critical_Reading_(Part_2)	3.6: Research and Critical Reading (Part 2) TWO SAMPLE STUDENT RESPONSES To illustrate the principles laid out in this section, consider the following two reading responses. Both texts respond to a very well known piece, “A Letter from Birmingham Jail,” by Martin Luther King, Jr. In the letter, King responds to criticism from other clergymen who had called his methods of civil rights struggle “unwise and untimely.” Both student writers were given the same response prompt: After reading King’s piece several times and with a pen or pencil in hand, consider what shapes King’s letter. Specifically, what rhetorical strategies is he using to achieve a persuasive effect on his readers? In making your decisions, consider such factors as background information that he gives, ways in which he addresses his immediate audience, and others. Remember that your goal is to explore King’s text, thus enabling you to understand his rhetorical strategies better. Example Student A Martin Luther King Jr’s “Letter from Birmingham Jail” is a very powerful text. At the time when minorities in America were silenced and persecuted, King had the courage to lead his people in the struggle for equality. After being jailed in Birmingham, Alabama, King wrote a letter to his “fellow clergymen” describing his struggle for civil rights. In the letter, King recounts a brief history of that struggle and rejects the accusation that it is “unwise and untimely.” Overall, I think that King’s letter is a very rhetorically effective text, one that greatly helped Americans to understand the civil rights movement. Example Student B King begins his “Letter from Birmingham Jail” by addressing it to his “fellow clergymen.” Thus, he immediately sets the tone of inclusion rather than exclusion. By using the word “fellow” in the address, I think he is trying to do two things. First of all, he presents himself as a colleague and a spiritual brother of his audience. That, in effect, says “you can trust me,” “I am one of your kind.” Secondly, by addressing his readers in that way, King suggests that everyone, even those Americans who are not directly involved in the struggle for civil rights, should be concerned with it. Hence the word “fellow.” King’s opening almost invokes the phrase “My fellow Americans” or “My fellow citizens” used so often by American Presidents when they address the nation. King then proceeds to give a brief background of his actions as a civil rights leader. As I read this part of the letter, I was wondering whether his readers would really have not known what he had accomplished as a civil rights leader. Then I realized that perhaps he gives all that background information as a rhetorical move. His immediate goal is to keep reminding his readers about his activities. His ultimate goal is to show to his audience that his actions were non-violent but peaceful. In reading this passage by King, I remembered once again that it is important not to assume that your audience knows anything about the subject of the writing. I will try to use this strategy more in my own papers. In the middle of the letter, King states: “The purpose of our direct-action program is to create a situation so crisis-packed that it will inevitably open the door to negotiation.” This sentence looks like a thesis statement and I wonder why he did not place it towards the beginning of the text, to get his point across right away. After thinking about this for a few minutes and re- reading several pages from our class textbook, I think he leaves his “thesis” till later in his piece because he is facing a not- so-friendly (if not hostile) audience. Delaying the thesis and laying out some background information and evidence first helps a writer to prepare his or her audience for the coming argument. That is another strategy I should probably use more often in my own writing, depending on the audience I am facing. REFLECTING ON THE RESPONSES To be sure, much more can be said about King’s letter than either of these writers have said. However, these two responses allow us to see two dramatically different approaches to reading. After studying both responses, consider the questions below. - Which response fulfills the goals set in the prompt better and why? - Which responses shows a deeper understanding of the texts by the reader and why? - Which writer does a better job at avoiding binary thinking and creating a sophisticated reading of King’s text and why? - Which writer is more likely to use the results of the reading in his or her own writing in the future and why? - Which writer leaves room for response to his text by others and why? CRITICAL READERS DO NOT READ ALONE AND IN SILENCE One of the key principles of critical reading is that active readers do not read silently and by themselves. By this I mean that they take notes and write about what they read. They also discuss the texts they are working with, with others and compare their own interpretations of those texts with the interpretations constructed by their colleagues. As a college student, you are probably used to taking notes of what you read. When I was in college, my favorite way of preparing for a test was reading a chapter or two from my textbook, then closing the book, then trying to summarize what I have read on a piece of paper. I tried to get the main points of the chapters down and the explanations and proofs that the textbooks’ authors used. Sometimes, I wrote a summary of every chapter in the textbook and then studied for the test from those summaries rather than from the textbook itself. I am sure you have favorite methods of note taking and studying from your notes, too. But now it strikes me that what I did with those notes was not critical reading. I simply summarized my textbooks in a more concise, manageable form and then tried to memorize those summaries before the test. I did not take my reading of the textbooks any further than what was already on their pages. Reading for information and trying to extract the main points, I did not talk back to the texts, did not question them, and did not try to extend the knowledge which they offered in any way. I also did not try to connect my reading with my personal experiences or pre-existing knowledge in any way. I also read in silence, without exchanging ideas with other readers of the same texts. Of course, my reading strategies and techniques were dictated by my goal, which was to pass the test. Critical reading has other goals, one of which is entering an on-going intellectual exchange. Therefore it demands different reading strategies, approaches, and techniques. One of these new approaches is not reading in silence and alone. Instead, critical readers read with a pen or pencil in hand. They also discuss what they read with others. STRATEGIES FOR CONNECTING READING AND WRITING If you want to become a critical reader, you need to get into a habit of writing as you read. You also need to understand that complex texts cannot be read just once. Instead, they require multiple readings, the first of which may be a more general one during which you get acquainted with the ideas presented in the text, its structure and style. During the second and any subsequent readings, however, you will need to write, and write a lot. The following are some critical reading and writing techniques which active readers employ as they work to create meanings from texts they read. UNDERLINE INTERESTING AND IMPORTANT PLACES IN THE TEXT Underline words, sentences, and passages that stand out, for whatever reason. Underline the key arguments that you believe the author of the text is making as well as any evidence, examples, and stories that seem interesting or important. Don’t be afraid to “get it wrong.” There is no right or wrong here. The places in the text that you underline may be the same or different from those noticed by your classmates, and this difference of interpretation is the essence of critical reading. TAKE NOTES Take notes on the margins. If you do not want to write on your book or journal, attach post-it notes with your comments to the text. Do not be afraid to write too much. This is the stage of the reading process during which you are actively making meaning. Writing about what you read is the best way to make sense of it, especially, if the text is difficult. Do not be afraid to write too much. This is the stage of the reading process during which you are actively making meaning. Writing about what you read will help you not only to remember the argument which the author of the text is trying to advance (less important for critical reading), but to create your own interpretations of the text you are reading (more important). Here are some things you can do in your comments - Ask questions. - Agree or disagree with the author. - Question the evidence presented in the text - Offer counter-evidence - Offer additional evidence, examples, stories, and so on that support the author’s argument - Mention other texts which advance the same or similar arguments - Mention personal experiences that enhance your reading of the text WRITE EXPLORATORY RESPONSES Write extended responses to readings. Writing students are often asked to write one or two page exploratory responses to readings, but they are not always clear on the purpose of these responses and on how to approach writing them. By writing reading responses, you are continuing the important work of critical reading which you began when you underlined interesting passages and took notes on the margins. You are extending the meaning of the text by creating your own commentary to it and perhaps even branching off into creating your own argument inspired by your reading. Your teacher may give you a writing prompt, or ask you to come up with your own topic for a response. In either case, realize that reading responses are supposed to be exploratory, designed to help you delve deeper into the text you are reading than note-taking or underlining will allow. When writing extended responses to the readings, it is important to keep one thing in mind, and that is their purpose. The purpose of these exploratory responses, which are often rather informal, is not to produce a complete argument, with an introduction, thesis, body, and conclusion. It is not to impress your classmates and your teacher with “big” words and complex sentences. On the contrary, it is to help you understand the text you are working with at a deeper level. The verb “explore” means to investigate something by looking at it more closely. Investigators get leads, some of which are fruitful and useful and some of which are dead-ends. As you investigate and create the meaning of the text you are working with, do not be afraid to take different directions with your reading response. In fact, it is important resist the urge to make conclusions or think that you have found out everything about your reading. When it comes to exploratory reading responses, lack of closure and presence of more leads at the end of the piece is usually a good thing. Of course, you should always check with your teacher for standards and format of reading responses. Try the following guidelines to write a successful response to a reading: Remember your goal—exploration. The purpose of writing a response is to construct the meaning of a difficult text. It is not to get the job done as quickly as possible and in as few words as possible. As you write, “talk back to the text.” Make comments, ask questions, and elaborate on complex thoughts. This part of the writing becomes much easier if, prior to writing your response, you had read the assignment with a pen in hand and marked important places in the reading. If your teacher provides a response prompt, make sure you understand it. Then try to answer the questions in the prompt to the best of your ability. While you are doing that, do not be afraid of bringing in related texts, examples, or experiences. Active reading is about making connections, and your readers will appreciate your work because it will help them understand the text better. While your primary goal is exploration and questioning, make sure that others can understand your response. While it is OK to be informal in your response, make every effort to write in a clear, error-free language. Involve your audience in the discussion of the reading by asking questions, expressing opinions, and connecting to responses made by others. USE READING FOR INVENTION Use reading and your responses to start your own formal writing projects. Reading is a powerful invention tool. While preparing to start a new writing project, go back to the readings you have completed and your responses to those readings in search for possible topics and ideas. Also look through responses your classmates gave to your ideas about the text. Another excellent way to start your own writing projects and to begin research for them is to look through the list of references and sources at the end of the reading that you are working with. They can provide excellent topic-generating and research leads. KEEP A DOUBLE-ENTRY JOURNAL Many writers like double-entry journals because they allow us to make that leap from summary of a source to interpretation and persuasion. To start a double-entry journal, divide a page into two columns. As you read, in the left column write down interesting and important words, sentences, quotations, and passages from the text. In the right column, right your reaction and responses to them. Be as formal or informal as you want. Record words, passages, and ideas from the text that you find useful for your paper, interesting, or, in any, way striking or unusual. Quote or summarize in full, accurately, and fairly. In the right-hand side column, ask the kinds of questions and provide the kinds of responses that will later enable you to create an original reading of the text you are working with and use that reading to create your own paper. DON’T GIVE UP If the text you are reading seems too complicated or “boring,” that might mean that you have not attacked it aggressively and critically enough. Complex texts are the ones worth pursuing and investigating because they present the most interesting ideas. Critical reading is a liberating practice because you do not have to worry about “getting it right.” As long as you make an effort to engage with the text and as long as you are willing to work hard on creating a meaning out of what you read, the interpretation of the text you are working with will be valid. IMPORTANT: So far, we have established that no pre-existing meaning is possible in written texts and that critical and active readers work hard to create such meaning. We have also established that interpretations differ from reader to reader and that there is no “right” or “wrong” during the critical reading process. So, you may ask, does this mean that any reading of a text that I create will be a valid and persuasive one? With the exception of the most outlandish and purposely-irrelevant readings that have nothing to do with the sources text, the answer is “yes.” However, remember that reading and interpreting texts, as well as sharing your interpretations with others are rhetorical acts. First of all, in order to learn something from your critical reading experience, you, the reader, need to be persuaded by your own reading of the text. Secondly, for your reading to be accepted by others, they need to be persuaded by it, too. It does not mean, however, that in order to make your reading of a text persuasive, you simply have to find “proof” in the text for your point of view. Doing that would mean reverting to reading “for the main point,” reading as consumption. Critical reading, on the other hand, requires a different approach. One of the components of this approach is the use of personal experiences, examples, stories, and knowledge for interpretive and persuasive purposes. This is the subject of the next section of this chapter. ONE CRITICAL READER’S PATH TO CREATING A MEANING: A CASE STUDY Earlier on in this chapter, we discussed the importance of using your existing knowledge and prior experience to create new meaning out of unfamiliar and difficult texts. In this section, I’d like to offer you one student writer’s account of his meaning- making process. Before I do that, however, it is important for me to tell you a little about the class and the kinds of reading and writing assignments that its members worked on. All the writing projects offered to the members of the class were promoted by readings, and students were expected to actively develop their own ideas and provide their own readings of assigned texts in their essays. The main text for the class was the anthology Ways of Reading edited by David Bartholomae and Anthony Petrosky that contains challenging and complex texts. Like for most of his classmates, this approach to reading and writing was new to Alex who had told me earlier that he was used to reading “for information” or “for the main point”. In preparation for the first writing project, the class read Adrienne Rich’s essay “When We Dead Awaken: Writing as Revision.” In her essay, Rich offers a moving account of her journey to becoming a writer. She makes the case for constantly “revising” one’s life in the light of all new events and experiences. Rich blends voices and genres throughout the essay, using personal narrative, academic argument, and even poetry. As a result, Rich creates the kind of personal-public argument which, on the one hand, highlights her own life, and on the other, illustrates that Rich’s life is typical for her time and her environment and that her readers can also learn from her experiences. To many beginning readers and writers, who are used to a neat separation of “personal” and “academic” argument, such a blend of genres and styles may seem odd. In fact, on of the challenges that many of the students in the class faced was understanding why Rich chooses to blend personal writing with academic and what rhetorical effects she achieves by doing so. After writing informal responses to the essay and discussing it in class, the students were offered the following writing assignment: Although Rich tells a story of her own, she does so to provide an illustration of an even larger story—one about what it means to be a woman and a writer. Tell a story of your own about the ways you might be said to have been named or shaped or positioned by an established or powerful culture. Like Rich (and perhaps with similar hesitation), use your own experience as an illustration of both your own situation and the situation of people like you. You should imagine that the assignment is a way for you to use (and put to the test) some of Rich’s terms, words like “re-vision,” “renaming,” and “structure.” (Bartholomae and Petrosky 648). Notice that this assignment does not ask students to simply analyze Rich’s essay, to dissect its argument or “main points.” Instead, writers are asked to work with their own experiences and events of their own lives in order to provide a reading of Rich which is affected and informed by the writers’ own lives and own knowledge of life. This is critical reading in action when a reader creates his or her one’s own meaning of a complex text by reflecting on the relationship between the content of that text and one’s own life. In response to the assignment, one of the class members, Alex Cimino-Hurt, wrote a paper that re-examined and re- evaluated his upbringing and how those factors have influenced his political and social views. In particular, Alex was trying to reconcile his own and his parents’ anti-war views with the fact than a close relative of his was fighting in the war in Iraq as he worked on the paper. Alex used such terms as “revision” and “hesitation” to develop his piece. Like most other writers in the class, initially Alex seemed a little puzzled, even confused by the requirement to read someone else’s text through the prism of his own life and his own experiences. However, as he drafted, revised, and discussed his writing with his classmates and his instructor, the new approach to reading and writing became clearer to him. After finishing the paper, Alex commented on his reading strategies and techniques and on what he learned about critical reading during the project: ON PREVIOUS READING HABITS AND TECHNIQUES Previously when working on any project whether it be for a History, English, or any other class that involved reading and research, there was a certain amount of minimalism. As a student I tried to balance the least amount of effort with the best grade. I distinctly remember that before, being taught to skim over writing and reading so that I found “main” points and highlighted them. The value of thoroughly reading a piece was not taught because all that was needed was a shallow interpretation of whatever information that was provided followed by a regurgitation. [Critical reading] provided a dramatic difference in perspective and helped me learn to not only dissect the meaning of a piece, but also to see why the writer is using certain techniques or how the reading applies to my life. ON DEVELOPING CRITICAL READING STRATEGIES When reading critically I found that the most important thing for me was to set aside a block of time in which I would’t have to hurry my reading or skip parts to “Get the gist of it.” Developing an eye for…detail came in two ways. The first method is to read the text several times, and the second is to discuss it with my classmates and my teacher. It quickly became clear to me that the more I read a certain piece, the more I got from it as I became more comfortable with the prose and writing style. With respect to the second way, there is always something that you can miss and there is always a different perspective that can be brought to the table by either the teacher or a classmate. ON READING RICH’S ESSAY In reading Adrienne Rich’s essay, the problem for me was’t necessarily relating to her work but instead just finding the right perspective from which to read it. I was raised in a very open family so being able to relate to others was learned early in my life. Once I was able to parallel my perspective to hers, it was just a matter of composing my own story. Mine was my liberalism in conservative environments—the fact that frustrates me sometimes. I felt that her struggle frustrated her, too. By using quotations from her work, I was able to show my own situation to my readers. ON WRITING THE PAPER The process that I went through to write an essay consisted of three stages. During the first stage, I wrote down every coherent idea I had for the essay as well as a few incoherent ones. This helped me create a lot of material to work with. While this initial material doesn’t always have direction it provides a foundation for writing. The second stage involved rereading Rich’s essay and deciding which parts of it might be relevant to my own story. Looking at my own life and at Rich’s work together helped me consolidate my paper. The third and final stage involved taking what is left and refining the style of the paper and taking care of the mechanics. ADVICE FOR CRITICAL READERS The first key to being a critical and active reader is to find something in the piece that interests, bothers, encourages, or just confuses you. Use this to drive your analysis. Remember there is no such thing as a boring essay, only a boring reader. - Reading something once is never enough so reading it quickly before class just won’t cut it. Read it once to get your brain comfortable with the work, then read it again and actually try to understand what’s going on in it. You can’t read it too many times. - Ask questions. It seems like a simple suggestion but if you never ask questions you’ll never get any answers. So, while you’re reading, think of questions and just write them down on a piece of paper lest you forget them after about a line and a half of reading. CONCLUSION Reading and writing are rhetorical processes, and one does not exist without the other. The goal of a good writer is to engage his or her readers into a dialog presented in the piece of writing. Similarly, the goal of a critical and active reader is to participate in that dialog and to have something to say back to the writer and to others. Writing leads to reading and reading leads to writing. We write because we have something to say and we read because we are interested in ideas of others. Reading what others have to say and responding to them help us make that all-important transition from simply having opinions about something to having ideas. Opinions are often over-simplified and fixed. They are not very useful because, if different people have different opinions that they are not willing to change or adjust, such people cannot work or think together. Ideas, on the other hand, are ever evolving, fluid, and flexible. Our ideas are informed and shaped by our interactions with others, both in person and through written texts. In a world where thought and action count, it is not enough to simply “agree to disagree.” Reading and writing, used together, allow us to discuss complex and difficult issues with others, to persuade and be persuaded, and, most importantly, to act. Reading and writing are inextricably connected, and I hope that this chapter has shown you ways to use reading to inform and enrich you writing and your learning in general. The key to becoming an active, critical, and interested reader is the development of varied and effective reading techniques and strategies. I’d like to close this chapter with the words from the writer Alex Cimino-Hurt: “Being able to read critically is important no matter what you plan on doing with your career or life because it allows you to understand the world around you.” References - Pavel Zemilansky - Bartholomae, David and Anthony Petrosky, Eds. Introduction. Ways of Reading . 8th Ed. New York: Bedford/St. Martin’s, 2008. - Brent, Douglas. 1992. Reading as Rhetorical Invention. NCTE , Urbana, Illinois. Cimino-Hurt, Alex. Personal Interview. 2003. - Martin, Janette. 2004. “Developing ‘Interesting Thoughts:’ Reading for Research.” In Research Writing Revisited: A Sourcebook for Teachers , eds. Pavel Zemliansky and Wendy Bishop, Heinemann, Portsmouth, NH. (3-13). - Rich, Adrienne. 2002. “When We Dead Awaken: Writing as Re-vision.” In Ways of Reading , 6th ed. Eds. Bartholomae, David and Anthony Petrosky. Bedford/St. Martin’s Boston, (627-645).	libretexts	2025-03-17T22:27:24.277188	2019-09-23T22:10:42	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://socialsci.libretexts.org/Courses/Sacramento_City_College/LIBR_324%3A_Critical_Thinking_and_Information_Literacy/3%3A_SLO_3/3.6%3A_Research_and_Critical_Reading_(Part_2)", "book_url": "https://commons.libretexts.org/book/socialsci-23897", "title": "3.6: Research and Critical Reading (Part 2)", "author": null }
https://socialsci.libretexts.org/Courses/Sacramento_City_College/LIBR_324%3A_Critical_Thinking_and_Information_Literacy/4%3A_SLO_4/4.3%3A_Quoting%2C_Paraphrasing%2C_and_Avoiding_Plagiarism	4.3: Quoting, Paraphrasing, and Avoiding Plagiarism Learning Objectives - Summarize, quote, and paraphrase accurately from readings. - Smoothly incorporate summary, paraphrase, and quotations in your writing. - Understand when summary, quotation, or paraphrase are appropriate in a research paper. - Avoid plagiarism. Learning how to effectively quote and paraphrase research can be difficult and it certainly takes practice. Hopefully, your abilities to make good use of your research will improve as you work through the exercises in part two and three ofThe Process of Research Writing, not to mention as you take on other research writing experiences beyond this class. The goal of this chapter is to introduce some basic strategies for summarizing, quoting and paraphrasing research in your writing and to explain how to avoid plagiarizing your research. How to Summarize: An Overview A summary is a brief explanation of a longer text. Some summaries, such as the ones that accompany annotated bibliographies, are very short, just a sentence or two. Others are much longer, though summaries are always much shorter than the text being summarized in the first place. Summaries of different lengths are useful in research writing because you often need to provide your readers with an explanation of the text you are discussing. This is especially true when you are going to quote or paraphrase from a source. Of course, the first step in writing a good summary is to do a thorough reading of the text you are going to summarize in the first place. Beyond that important start, there are a few basic guidelines you should follow when you write summary material: - Stay “neutral” in your summarizing. Summaries provide “just the facts” and are not the place where you offer your opinions about the text you are summarizing. Save your opinions and evaluation of the evidence you are summarizing for other parts of your writing. - Don’t quote from what you are summarizing. Summaries will be more useful to you and your colleagues if you write them in your own words. - Don’t “cut and paste” from database abstracts. Many of the periodical indexes that are available as part of your library’s computer system include abstracts of articles. Do no “cut” this abstract material and then “paste” it into your own annotated bibliography. For one thing, this is plagiarism. Second, “cutting and pasting” from the abstract defeats one of the purposes of writing summaries and creating an annotated bibliography in the first place, which is to help you understand and explain your research. How to Quote and Paraphrase: An Overview Writers quote and paraphrase from research in order to support their points and to persuade their readers. A quote or a paraphrase from a piece of evidence in support of a point answers the reader’s question, “says who?” This is especially true in academic writing since scholarly readers are most persuaded by effective research and evidence. For example, readers of an article about a new cancer medication published in a medical journal will be most interested in the scholar’s research and statistics that demonstrate the effectiveness of the treatment. Conversely, they will not be as persuaded by emotional stories from individual patients about how a new cancer medication improved the quality of their lives. While this appeal to emotion can be effective and is common in popular sources, these individual anecdotes do not carry the same sort of “scholarly” or scientific value as well-reasoned research and evidence. Of course, your instructor is not expecting you to be an expert on the topic of your research paper. While you might conduct some primary research, it’s a good bet that you’ll be relying on secondary sources such as books, articles, and Web sites to inform and persuade your readers. You’ll present this research to your readers in the form of quotes and paraphrases. A “quote” is a direct restatement of the exact words from the original source. The general rule of thumb is any time you use three or more words as they appeared in the original source, you should treat it as a quote. A “paraphrase” is a restatement of the information or point of the original source in your own words. While quotes and paraphrases are different and should be used in different ways in your research writing (as the examples in this section suggest), they do have a number of things in common. Both quotes and paraphrases should: - be “introduced” to the reader, particularly the first time you mention a source; - include an explanation of the evidence which explains to the reader why you think the evidence is important, especially if it is not apparent from the context of the quote or paraphrase; and - include a proper citation of the source. The method you should follow to properly quote or paraphrase depends on the style guide you are following in your academic writing. The two most common style guides used in academic writing are the Modern Language Association (MLA), and the American Psychological Association (APA). Your instructor will probably assign one of these styles before you begin working on your project, however, if he/she doesn’t mention this, be sure to ask. When to Quote, When to Paraphrase The real “art” to research writing is using quotes and paraphrases from evidence effectively in order to support your point. There are certain “rules,” dictated by the rules of style you are following, such as the ones presented by the MLA or the ones presented by the APA. There are certain “guidelines” and suggestions, like the ones I offer in the previous section and the ones you will learn from your teacher and colleagues. But when all is said and done, the question of when to quote and when to paraphrase depends a great deal on the specific context of the writing and the effect you are trying to achieve. Learning the best times to quote and paraphrase takes practice and experience. In general, it is best to use a quote when: - The exact words of your source are important for the point you are trying to make. This is especially true if you are quoting technical language, terms, or very specific word choices. - You want to highlight your agreement with the author’s words. If you agree with the point the author of the evidence makes and you like their exact words, use them as a quote. - You want to highlight your disagreement with the author’s words. In other words, you may sometimes want to use a direct quote to indicate exactly what it is you disagree about. This might be particularly true when you are considering the antithetical positions in your research writing projects. In general, it is best to paraphrase when: - There is no good reason to use a quote to refer to your evidence. If the author’s exact words are not especially important to the point you are trying to make, you are usually better off paraphrasing the evidence. - You are trying to explain a particular a piece of evidence in order to explain or interpret it in more detail. This might be particularly true in writing projects like critiques. - You need to balance a direct quote in your writing. You need to be careful about directly quoting your research too much because it can sometimes make for awkward and difficult to read prose. So, one of the reasons to use a paraphrase instead of a quote is to create balance within your writing. Tips for Quoting and Paraphrasing - Introduce your quotes and paraphrases to your reader, especially on first reference. - Explain the significance of the quote or paraphrase to your reader. - Cite your quote or paraphrase properly according to the rules of style you are following in your essay. - Quote when the exact words are important, when you want to highlight your agreement or your disagreement. - Paraphrase when the exact words aren’t important, when you want to explain the point of your evidence, or when you need to balance the direct quotes in your writing. Four Examples of Quotes and Paraphrases Here are four examples of what I mean about properly quoting and paraphrasing evidence in your research essays. In each case, I begin with a BAD example, or the way NOT to quote or paraphrase. Quoting in MLA Style Here’s the first BAD example, where the writer is trying to follow the rules of MLA style: There are many positive effects for advertising prescription drugs on television. “African-American physicians regard direct-to-consumer advertising of prescription medicines as one way to educate minority patients about needed treatment and healthcare options” (Wechsler, Internet). This is a potentially good piece of information to support a research writer’s claim, but the researcher hasn’t done any of the necessary work to explain where this quote comes from or to explain why it is important for supporting her point. Rather, she has simply “dropped in” the quote, leaving the interpretation of its significance up to the reader. Now consider this revised GOOD (or at least BETTER) example of how this quote might be better introduced into the essay: In her Pharmaceutical Executive article available through the Wilson Select Internet database, Jill Wechsler writes about one of the positive effects of advertising prescription drugs on television. “African-American physicians regard direct-to-consumer advertising of prescription medicines as one way to educate minority patients about needed treatment and healthcare options.” In this revision, it’s much more clear what point the writer is trying to make with this evidence and where this evidence comes from. In this particular example, the passage is from a traditional print journal called Pharmaceutical Executive . However, the writer needs to indicate that she actually found and read this article through Wilson Select, an Internet database which reproduces the “full text” of articles from periodicals without any graphics, charts, or page numbers. When you use a direct quote in your research, you need to the indicate page number of that direct quote or you need to indicate that the evidence has no specific page numbers. While it can be a bit awkward to indicate within the text how the writer found this information if it’s from the Internet, it’s important to do so on the first reference of a piece of evidence in your writing. On references to this piece of evidence after the first reference, you can use just the last name of the writer. For example: Wechsler also reports on the positive effects of advertising prescription drugs on television. She writes… Paraphrasing in MLA Style In this example, the writer is using MLA style to write a research essay for a Literature class. Here is a BAD example of a paraphrase: While Gatsby is deeply in love with Daisy in The Great Gatsby, his love for her is indistinguishable from his love of his possessions (Callahan). There are two problems with this paraphrase. First, if this is the first or only reference to this particular piece of evidence in the research essay, the writer should include more information about the source of this paraphrase in order to properly introduce it. Second, this paraphrase is actually not of the entire article but rather of a specific passage. The writer has neglected to note the page number within the parenthetical citation. A GOOD or at least BETTER revision of this paraphrase might look like this: John F. Callahan suggests in his article “F. Scott Fitzgerald’s Evolving American Dream” that while Gatsby is deeply in love with Daisy in The Great Gatsby, his love for her is indistinguishable from his love of his possessions (381). By incorporating the name of the author of the evidence the research writer is referring to here, the source of this paraphrase is now clear to the reader. Furthermore, because there is a page number at the end of this sentence, the reader understands that this passage is a paraphrase of a particular part of Callahan’s essay and not a summary of the entire essay. Again, if the research writer had introduced this source to his readers earlier, he could have started with a phrase like “Callahan suggests…” and then continued on with his paraphrase. If the research writer were offering a brief summary of the entire essay following MLA style, he wouldn’t include a page number in parentheses. For example: John F. Callahan’s article “F. Scott Fitzgerald’s Evolving American Dream” examines Fitzgerald’s fascination with the elusiveness of the American Dream in the novels The Great Gatsby, Tender is the Night, and The Last Tycoon. Quoting in APA Style Consider this BAD example in APA style, of what NOT to do when quoting evidence: “If the U.S. scallop fishery were a business, its management would surely be fired, because its revenues could readily be increased by at least 50 percent while its costs were being reduced by an equal percentage.” (Repetto, 2001, p. 84). Again, this is a potentially valuable piece of evidence, but it simply isn’t clear what point the research writer is trying to make with it. Further, it doesn’t follow the preferred method of citation with APA style. Here is a revision that is a GOOD or at least BETTER example: Repetto (2001) concludes that in the case of the scallop industry, those running the industry should be held responsible for not considering methods that would curtail the problems of over-fishing. “If the U.S. scallop fishery were a business, its management would surely be fired, because its revenues could readily be increased by at least 50 percent while its costs were being reduced by an equal percentage” (p. 84). This revision is improved because the research writer has introduced and explained the point of the evidence with the addition of a clarifying sentence. It also follows the rules of APA style. Generally, APA style prefers that the research writer refer to the author only by last name followed immediately by the year of publication. Whenever possible, you should begin your citation with the author’s last name and the year of publication, and, in the case of a direct quote like this passage, the page number (including the “p.”) in parentheses at the end. Paraphrasing in APA Style Paraphrasing in APA style is slightly different from MLA style as well. Consider first this BAD example of what NOT to do in paraphrasing from a source in APA style: Computer criminals have lots of ways to get away with credit card fraud (Cameron, 2002). The main problem with this paraphrase is there isn’t enough here to adequately explain to the reader what the point of the evidence really is. Remember: your readers have no way of automatically knowing why you as a research writer think that a particular piece of evidence is useful in supporting your point. This is why it is key that you introduce and explain your evidence. Here is a revision that is GOOD or at least BETTER: Cameron (2002) points out that computer criminals intent on committing credit card fraud are able to take advantage of the fact that there aren’t enough officials working to enforce computer crimes. Criminals are also able to use the technology to their advantage by communicating via email and chat rooms with other criminals. Again, this revision is better because the additional information introduces and explains the point of the evidence. In this particular example, the author’s name is also incorporated into the explanation of the evidence as well. In APA, it is preferable to weave in the author’s name into your essay, usually at the beginning of a sentence. However, it would also have been acceptable to end an improved paraphrase with just the author’s last name and the date of publication in parentheses. How to Avoid Plagiarism in the Research Process Plagiarism is the unauthorized or uncredited use of the writings or ideas of another in your writing. While it might not be as tangible as auto theft or burglary, plagiarism is still a form of theft. In the academic world, plagiarism is a serious matter because ideas in the forms of research, creative work, and original thought are highly valued. Chances are, your school has strict rules about what happens when someone is caught plagiarizing. The penalty for plagiarism is severe, everything from a failing grade for the plagiarized work, a failing grade for the class, or expulsion from the institution. You might not be aware that plagiarism can take several different forms. The most well known, purposeful plagiarism , is handing in an essay written by someone else and representing it as your own, copying your essay word for word from a magazine or journal, or downloading an essay from the Internet. A much more common and less understood phenomenon is what I call accidental plagiarism. Accidental plagiarism is the result of improperly paraphrasing, summarizing, quoting, or citing your evidence in your academic writing. Generally, writers accidentally plagiarize because they simply don’t know or they fail to follow the rules for giving credit to the ideas of others in their writing. Both purposeful and accidental plagiarism are wrong, against the rules, and can result in harsh punishments. Ignoring or not knowing the rules of how to not plagiarize and properly cite evidence might be an explanation, but it is not anexcuse. To exemplify what I’m getting at, consider the examples below that use quotations and paraphrases from this brief passage: Those who denounce cyberculture today strangely resemble those who criticized rock music during the fifties and sixties. Rock started out as an Anglo-American phenomenon and has become an industry. Nonetheless, it was able to capture the hopes of young people around the world and provided enjoyment to those of us who listened to or played rock. Sixties pop was the conscience of one or two generations that helped bring the war in Vietnam to a close. Obviously, neither rock nor pop has solved global poverty or hunger. But is this a reason to be “against” them? (ix). And just to make it clear that I’m not plagiarizing this passage, here is the citation in MLA style: Works cited Lévy, Pierre. Cyberculture . Trans. Robert Bononno. Minneapolis: U of Minnesota P, 2001. Here’s an obvious example of plagiarism: Those who denounce cyberculture today strangely resemble those who criticized rock music during the fifties and sixties. In this case, the writer has literally taken one of Lévy’s sentences and represented it as her own. That’s clearly against the rules. Here’s another example of plagiarism, perhaps less obvious: The same kind of people who criticize cyberculture are the same kind of people who criticized rock and roll music back in the fifties and sixties. But both cyberculture and rock music inspire and entertain young people. While these aren’t Lévy’s exact words, they are certainly close enough to constitute a form of plagiarism. And again, even though you might think that this is a “lesser” form of plagiarism, it’s still plagiarism. Both of these passages can easily be corrected to make them acceptable quotations or paraphrases. In the introduction of his book Cyberculture, Pierre Lévy observes that “Those who denounce cyberculture today strangely resemble those who criticized rock music during the fifties and sixties” (ix). Pierre Lévy suggests that the same kind of people who criticize cyberculture are the same kind of people who criticized rock and roll music back in the fifties and sixties. But both cyberculture and rock music inspire and entertain young people (ix). Note that changing these passages from examples of plagiarism to acceptable examples of a quotation and a paraphrase is extremely easy: properly cite your sources. This leads to the “golden rule” of avoiding plagiarism: The Golden Rule of Avoiding Plagiarism - Always cite your sources. If you are unsure as to whether you should or should not cite a particular claim or reference, you should probably cite your source. Often, students are unclear as to whether or not they need to cite a piece of evidence because they believe it to be “common knowledge” or because they are not sure about the source of information. When in doubt about whether or not to cite evidence in order to give credit to a source (“common knowledge” or not), you should cite the evidence. Plagiarism and the Internet Sometimes, I think the ease of finding and retrieving information on the World Wide Web makes readers think that this information does not need to be cited. After all, it isn’t a traditional source like a book or a journal; it is available for “free.” All a research writer needs to do with a web site is “cut and paste” whatever he needs into his essay, right? Wrong! You need to cite the evidence you find from the Internet or the World Wide Web the same way you cite evidence from other sources. To not do this is plagiarism, or, more bluntly, cheating. Just because the information is “freely” available on the Internet does not mean you can use this information in your academic writing without properly citing it, much in the same way that the information from library journals and books “freely” available to you needs to be cited in order to give credit where credit is due. It is also not acceptable to simply download graphics from the World Wide Web. Images found on the Internet are protected by copyright laws. Quite literally, taking images from the Web (particularly from commercial sources) is an offense that could lead to legal action. There are places where you can find graphics and clip art that Web publishers have made publicly available for anyone to use, but be sure that the Web site where you find the graphics makes this explicit before you take graphics as your own. In short, you can use evidence from the Web as long as you don’t plagiarize and as long as you properly cite it; don’t take graphics from the Web unless you know the images are in the public domain. References - Steven D. Krause - This piece was originally Chapter 3 from The Process of Research Writing .	libretexts	2025-03-17T22:27:24.600703	2019-09-23T22:10:46	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://socialsci.libretexts.org/Courses/Sacramento_City_College/LIBR_324%3A_Critical_Thinking_and_Information_Literacy/4%3A_SLO_4/4.3%3A_Quoting%2C_Paraphrasing%2C_and_Avoiding_Plagiarism", "book_url": "https://commons.libretexts.org/book/socialsci-23897", "title": "4.3: Quoting, Paraphrasing, and Avoiding Plagiarism", "author": null }
https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/01%3A_Chemistry_to_Chromosomes/1.01%3A__The_Structure_of_DNA	1.1: The Structure of DNA Learning Objectives - Identify the sugar, phosphate, nitrogenous base, 5' and 3' carbons in a nucleotide and the key difference between DNA and RNA. - Explain the structure of the double helix, including the role of hydrogen bonds and covalent (phosphodiester) bonds. - Explain why the abundance of A is roughly equal to T and G is roughly equal to C in DNA. - Using the rules of base pairing, predict the complementary (or anti-parallel) strand of DNA for a given sequence. Nucleic acids are the most important macromolecules for the continuity of life. They carry the genetic blueprint of a cell and carry instructions for the functioning of the cell. DNA and RNA The two main types of nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) . DNA is the genetic material found in all living organisms, ranging from single-celled bacteria to multicellular mammals. DNA is found in the nucleus of eukaryotes and in the organelles, chloroplasts, and mitochondria. In prokaryotes, the DNA is not enclosed in a membranous envelope. The entire genetic content of a cell is known as its genome, and the study of genomes is genomics. In eukaryotic cells but not in prokaryotes, DNA forms a complex with histone proteins to form chromatin, the substance of eukaryotic chromosomes. A chromosome may contain tens of thousands of genes. Many genes contain the information to make protein products; other genes code for RNA products. DNA controls all of the cellular activities by turning the genes “on” or “off.” The other type of nucleic acid, RNA, is mostly involved in protein synthesis. The DNA molecules never leave the nucleus but instead use an intermediary to communicate with the rest of the cell. This intermediary is the messenger RNA (mRNA) . Other types of RNA—like rRNA, tRNA, and microRNA—are involved in protein synthesis and its regulation. DNA and RNA are made up of monomers known as nucleotides . The nucleotides combine with each other to form a polynucleotide , DNA or RNA. Each nucleotide is made up of three components: a nitrogenous base, a pentose (five-carbon) sugar, and a phosphate group (Figure $\PageIndex{1}$). Each nitrogenous base in a nucleotide is attached to a sugar molecule, which is attached to one or more phosphate groups. The nitrogenous bases, important components of nucleotides, are organic molecules and are so named because they contain carbon and nitrogen. They are bases because they contain an amino group that has the potential of binding an extra hydrogen, and thus, decreasing the hydrogen ion concentration in the environment, making it more basic. Each nucleotide in DNA contains one of four possible nitrogenous bases: adenine (A), guanine (G) cytosine (C), and thymine (T). Adenine and guanine are classified as purines . The primary structure of a purine is two carbon-nitrogen rings. Cytosine, thymine, and uracil are classified as pyrimidines which have a single carbon-nitrogen ring as their primary structure (Figure $\PageIndex{1}$). Each of these basic carbon-nitrogen rings has different functional groups attached to it. In molecular biology shorthand, the nitrogenous bases are simply known by their symbols A, T, G, C, and U. DNA contains A, T, G, and C whereas RNA contains A, U, G, and C. The pentose sugar in DNA is deoxyribose, and in RNA, the sugar is ribose (Figure $\PageIndex{1}$). The difference between the sugars is the presence of the hydroxyl group on the second carbon of the ribose and hydrogen on the second carbon of the deoxyribose (so deoxyribose is "missing" an -OH group). The carbon atoms of the sugar molecule are numbered as 1′, 2′, 3′, 4′, and 5′ (1′ is read as “one prime”). The phosphate residue is attached to the hydroxyl group of the 5′ carbon of one sugar and the hydroxyl group of the 3′ carbon of the sugar of the next nucleotide, which forms a 5′–3′ phosphodiester linkage. The phosphodiester linkage is not formed by simple dehydration reaction like the other linkages connecting monomers in macromolecules: its formation involves the removal of two phosphate groups. A polynucleotide may have thousands of such phosphodiester linkages. Chargaff’s Rules When Watson and Crick set out in the 1940’s to determine the structure of DNA, it was already known that DNA is made up of a series four different types of molecules, called bases or nucleotides: adenine (A), cytosine (C), thymine (T), guanine (G). Watson and Crick also knew of Chargaff’s Rules , which were a set of observations about the relative amount of each nucleotide that was present in almost any extract of DNA. Chargaff had observed that for any given species, the abundance of A was the same as T, and G was the same as C. This was essential to Watson & Crick’s model. Example $\PageIndex{1}$ Chargaff determined the composition of nucleic acids in samples from a variety of species, including prokaryotes and eukaryotes. In one bacterial sample, the proportion of adenine was 15.5% (data adapted from Vischer et al, 1949). What proportion of guanine would have been present in this sample and why? Solution Because A pairs with T, the amount of T should be roughly equal to A, or approximately 15.5% percent. Thus, A + T = 15.5 + 15.5 = 31%. The percent of G + C = 100% - 31% = 69%. Because G pairs with C, the amount of each of these should be roughly equal, so approximately 34.5% each. Query $\PageIndex{1}$ DNA Double-Helix Structure Using proportional metal models of the individual nucleotides, Watson and Crick deduced a structure for DNA that was consistent with Chargaff’s Rules and with x-ray crystallography data that was obtained (with some controversy) from another researcher named Rosalind Franklin. In Watson and Crick’s famous double helix , each of the two strands contains DNA bases connected through covalent (phosphodiester) bonds to a sugar-phosphate backbone. Because one side of each sugar molecule is always connected to the opposite side of the next sugar molecule, each strand of DNA has polarity: these are called the 5’ (5-prime) end and the 3’ (3-prime) end, in accordance with the nomenclature of the carbons in the sugars. The two strands of the double helix run in anti-parallel (i.e. opposite) directions, with the 5’ end of one strand adjacent to the 3’ end of the other strand. The double helix has a right-handed twist, (rather than the left-handed twist that is often represented incorrectly in popular media). The DNA bases extend from the backbone towards the center of the helix, with a pair of bases from each strand forming hydrogen bonds that help to hold the two strands together. Under most conditions, the two strands are slightly offset, which creates a major groove on one face of the double helix, and a minor groove on the other. Rules of base pairing - A pairs with T (or U if RNA) - G pairs with C Because of the structure of the bases, A can only form hydrogen bonds with T, and G can only form hydrogen bonds with C (remember Chargaff’s Rules). Each strand is therefore said to be complementary to the other, and so each strand also contains enough information to act as a template for the synthesis of the other. This complementary redundancy is important in DNA replication and repair. If the sequence of one strand is AATTGGCC, the complementary strand would have the sequence TTAACCGG. During DNA replication, each strand is copied, resulting in a daughter DNA double helix containing one parental DNA strand and a newly synthesized strand. 3D structure of a DNA double helix Spin the double helix to see the orientation of the sugars and phosphates in the backbone (ribbon in the model), the base pairs, major and minor grooves! (PDB ID = 1bna https://www.rcsb.org/3d-view/1BNA ) Implications of DNA structure As for most biological molecules, the structure is important to the function, and the function of DNA is to contain information. Important properties that are derived from the DNA structure are: - A complementary strand can always be synthesized from a single strand, due to the arrangement of hydrogen bonds between GC and AT bases. - Hydrogen bonds stabilize the double helix, but can be broken when DNA needs to be accessed. - The order of bases contains the information needed to code for amino acids in proteins during translation. - Even sequences of DNA that do not encode amino acids can still provide information by interacting with proteins that function in DNA packaging and regulation. The major and minor grooves of DNA may determine which sequences are visible to DNA interacting proteins. Thinking ahead exercise $\PageIndex{1}$ A mutation occurs, and cytosine is replaced with adenine. What impact do you think this will have on the DNA structure? - Answer - Adenine is larger than cytosine and will not be able to base pair properly with the guanine on the opposing strand, causing the double helix to bulge at that position. DNA repair enzymes may recognize the bulge and replace the incorrect nucleotide. Thinking ahead exercise $\PageIndex{2}$ If the two DNA strands were connected by covalent bonds rather than hydrogen bonds, what problems might occur, if any? - Answer - Covalent bonds are much stronger than hydrogen bonds and not as easily broken. If the DNA strands were connected by covalent bonds, then it would be much more difficult to unwind the double helix. This would be problematic for processes that require unwinding of the DNA molecule, such as replication and transcription. Contributors and Attributions - Connie Rye (East Mississippi Community College), Robert Wise (University of Wisconsin, Oshkosh), Vladimir Jurukovski (Suffolk County Community College), Jean DeSaix (University of North Carolina at Chapel Hill), Jung Choi (Georgia Institute of Technology), Yael Avissar (Rhode Island College) among other contributing authors. Original content by OpenStax (CC BY 4.0; Download for free at http://cnx.org/contents/[email protected] ). Access for free at https://openstax.org/books/biology/pages/1-introduction - Dr. Todd Nickle and Isabelle Barrette-Ng (Mount Royal University) The content on this page is licensed under CC SA 3.0 licensing guidelines. References VISCHER E, ZAMENHOF S, CHARGAFF E. Microbial nucleic acids; the desoxypentose nucleic acids of avian tubercle bacilli and yeast. J Biol Chem . 1949;177(1):429-438.	libretexts	2025-03-17T22:27:28.805581	2019-10-01T21:21:00	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/", "url": "https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/01%3A_Chemistry_to_Chromosomes/1.01%3A__The_Structure_of_DNA", "book_url": "https://commons.libretexts.org/book/bio-25718", "title": "1.1: The Structure of DNA", "author": "Stefanie West Leacock" }
https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/01%3A_Chemistry_to_Chromosomes/1.02%3A_Chromosomes_and_chromatin	1.2: Chromosomes and chromatin Learning Objectives - Understand that chromosomes contain genes, which are DNA sequences that encode products and describe how the positions of individual genes on a given chromosome are related to their positions on the homolog of that chromosome. - Discuss how DNA is packaged in the chromosomes in terms of histones, nucleosomes, and chromatin (heterochromatin and euchromatin). - Explain the meaning of ploidy (haploid, diploid) and how it relates to the number of homologues of each chromosome. - Compare prokaryotic and eukaryotic chromosomes. - Interpret a karyotype. What is a chromosome? Chromosomes are units of DNA stored within cells. In prokaryotes, these units are most often circular, whereas in eukaryotes the units are typically linear. Genes are sequences within chromosomes that contain information in the sequence of nucleotide bases that encodes a product (RNA or a protein). Features and Compaction of Circular Chromosomes The bacterial chromosome is typically one molecule of double-stranded, helical DNA. In most bacteria, the two ends of the double-stranded DNA covalently bond together to form both a physical and genetic circle. The chromosome is generally around 1000 µm long and frequently contains as many as 3,500 gen es. E . coli , a bacterium that is 2-3 µm in length, has a chromosome approximately 1400 µm long. What does it mean to be haploid? Prokaryotic cells usually have one only copy of their chromosome(s) and therefore one copy of each gene on the chromosome. To enable a macromolecule this large to fit within the bacterium, proteins bind to the DNA, segregating the DNA molecule into around 50 chromosomal domains and making it more compact. Prokaryotes primarily compact chromosomes by supercoiling, the process of twisting a piece of DNA that causes it to "fold up" on itself. Think of an old-fashioned phone cord or piece of string that you keep twisting. Supercoiling is not random, but is controlled by enzymes (topoisomerases) that can add or remove "twists" in the double helix to loosen or tighten the chromosome compaction. Video: Representing Bacterial DNA Features of Linear Chromosomes Linear chromosomes contain structural features such as centromeres and telomeres. In most cases, each chromosome contains one centromere . These sequences are bound by proteins that will link the centromere to microtubules that facilitate chromosome movement during cell division. Under the microscope, centromeres of metaphase chromosomes can sometimes appear as constrictions in the body of the chromosome. If a centromere is located near the middle of a chromosome, it is metacentric , whereas a telocentric centromere is at, or near, the very end of the chromosome. Some organisms also do not have a single centromere but are holocentric . Telomeres are repetitive sequences near the ends of linear chromosomes, and are important in maintaining the length of the chromosomes during replication, and protecting the ends of the chromosomes from alterations. Homologous chromosomes are typically pairs of similar, but non-identical, chromosomes in which one member of the pair comes from the male parent, and the other comes from the female parent. Homologs contain the same gene loci in the same order. Non-homologous chromosomes contain different gene loci, and are usually distinguishable based on cytological features such as length, centromere position, and banding patterns produced by staining. What does it mean to be diploid? Most eukaryotic organisms are diploid , meaning they have two sets of chromosomes. The diploid number is represented by 2n . Remember this means that each cell has: - two homologs of each chromosome - two copies of each gene The number of non-homologous chromosomes varies by organism. Levels of compaction in eukaryotes If stretched to its full length, the DNA molecule of the largest human chromosome would be 85mm. Yet during mitosis and meiosis, this DNA molecule is compacted into a chromosome approximately 5µm long (17,000 times smaller!). Although this compaction makes it easier to transport DNA within a dividing cell, it also makes DNA less accessible for other cellular functions such as DNA synthesis and transcription. Thus, chromosomes vary in how tightly DNA is packaged, depending on the stage of the cell cycle and also on the level of gene activity required in any particular region of the chromosome. There are several different levels of structural organization in eukaryotic chromosomes, with each successive level contributing to the further compaction of DNA. The compaction of DNA requires proteins and the combination of proteins and DNA is chromatin . For more loosely compacted DNA, only the first few levels of organization may apply. Each level involves a specific set of proteins that associate with the DNA to compact it. First, proteins called the core histones act as spool around which DNA is coiled twice to form a structure called the nucleosome , which is composed of eight polypeptides, two copies of histone proteins H2A, H2B, H3, and H4. Nucleosomes are formed at regular intervals along the DNA strand, giving the molecule the appearance of “beads on a string”. At the next level of organization, histone H1 helps to compact the DNA strand and its nucleosomes into a 30nm fiber . Subsequent levels of organization involve the addition of scaffold proteins that wind the 30nm fiber into coils, which are in turn wound around other scaffold proteins. Chromatin Packaging Varies Within a Chromosome: Euchromatin & Heterochromatin Classically, there are two major types of chromatin, but these are more the ends of a continuous and varied spectrum. Euchromatin is more loosely packed, and tends to contain genes that are being transcribed (or actively being utilized by the cell). For example, there might be widely-spaced nucleosomes within a euchromatin region, leaving more of the DNA accessible for proteins that interact with that region. In contrast, heterochromatin usually contains densely-packed nucleosomes, is often rich in repetitive sequences, and tends not to contain genes that are actively being transcribed. Within these regions, nucleosomes might be close together, restricting protein access to DNA. Both the centromeres and telomeres are usually heterochromatin, whereas other regions of chromosomes can be switched from heterochromatin to euchromatin or vice versa, often by proteins that modify histones and nucleosomes. Keeping chromosomes organized in nuclei During interphase, the decondensed chromosomes often have specific locations within the nucleus and relative to one another, which has been studied using a technique called FISH, fluorescent in situ hybridization. In FISH, fluorescently-tagged probes (pieces of single-stranded DNA) recognize complementary sequences specific to each chromosome and allow visualization of specific chromosome locations within a nucleus full of DNA. Breaking down terms to understand FISH: F = fluorescent because a small molecule that is excited by a certain wavelength of light is attached to the probe IS = in situ is a Latin term that means "in the original place" because the experiment examines something within a cell H = hybridization because the probe and the cellular DNA are complementary and therefore bind each other (or hybridize) Exercise $\PageIndex{1}$ How do DNA probes recognize sequences of DNA in cells in this experiment? - Answer - The rules of base pairing A-T and G-C and anti-parallel orientation! The probes are complementary to sequences of DNA specific to each chromosome. Karyotypes Chromosomes stain with some types of dyes, which is how they got their name (chromosome means “colored body”). Certain dyes stain some regions along a chromosome more intensely than others, giving some chromosomes a banded appearance when stained. A karyotype is a representation of a complete set of chromosomes. Karyotypes are usually determined by isolating mitotic chromosomes to view them as a karyogram. Chemicals that arrest the cells in metaphase of mitosis are used and then the chromosomes are released from nuclei, usually onto a slide. Chromosomes at this stage are at the most compact state. The images of these chromosomes can be captured digitally and arranged into pairs based on size, centromere position, and banding pattern to examine the complete set of chromosomes in the cell. Exercise $\PageIndex{1}$ Based on the above karyotype from the spiny frog, what is the diploid (2n) number of this species? - Answer - 2n = 26 Thinking ahead $\PageIndex{2}$ A karyotype is a representation of a complete set of chromosomes. These chromosomes are typically extracted from a cell arrested in metaphase of mitosis. Why do you think the chromosomes are extracted during the mitotic phase instead of interphase? - Answer - During interphase, chromosomes are in a decondensed configuration. However, during mitosis, chromosomes become highly condensed (17,000 times smaller than during interphase). It is much easier to extract chromosomes from a cell when they are tightly packaged as opposed to being in a loose configuration. Video: Performing Cytogenetic Test for Chromosomal Study Additional resources For additional information about karyotyping: https://www.nature.com/scitable/topicpage/karyotyping-for-chromosomal-abnormalities-298/ References Bolzer A, Kreth G, Solovei I, Koehler D, Saracoglu K, Fauth C, et al. (2005) Three-Dimensional Maps of All Chromosomes in Human Male Fibroblast Nuclei and Prometaphase Rosettes. PLoS Biol 3(5): e157. https://doi.org/10.1371/journal.pbio.0030157 Duan J, Jiang W, Cheng Z, Heikkila JJ, Glick BR (2013) The Complete Genome Sequence of the Plant Growth-Promoting Bacterium Pseudomonas sp. UW4. PLoS ONE 8(3): e58640. https://doi.org/10.1371/journal.pone.0058640 Qing L, Xia Y, Zheng Y, Zeng X (2012) A De Novo Case of Floating Chromosomal Polymorphisms by Translocation in Quasipaa boulengeri (Anura, Dicroglossidae). PLoS ONE 7(10): e46163. https://doi.org/10.1371/journal.pone.0046163 Contributors and Attributions - Dr. Todd Nickle and Isabelle Barrette-Ng (Mount Royal University) The content on this page is licensed under CC SA 3.0 licensing guidelines. - Chromsome-DNA-gene image By Thomas Splettstoesser - This file has been altered from the original under a creative commons license. Original can be found at https://commons.wikimedia.org/wiki/F...e-DNA-gene.png , CC BY-SA 4.0, https://commons.wikimedia.org/w/inde...curid=76810117	libretexts	2025-03-17T22:27:28.890699	2019-10-01T21:21:00	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/", "url": "https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/01%3A_Chemistry_to_Chromosomes/1.02%3A_Chromosomes_and_chromatin", "book_url": "https://commons.libretexts.org/book/bio-25718", "title": "1.2: Chromosomes and chromatin", "author": "Stefanie West Leacock" }
https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/02%3A_Central_Dogma/2.01%3A_Overview_of_Transcription	2.1: Overview of Transcription Learning Objectives - Identify the key steps of transcription, the function of the promoter and the function of RNA polymerase. - Distinguish between coding (RNA-like) and non-coding (template) strands of DNA. Understand that within a single piece of DNA, either strand can be used as the template for different genes, but the RNA will still be produced from 5’ → 3’. - Draw a line diagram showing a segment of DNA from a gene and its RNA transcript, indicating which DNA strand is the template, the direction of transcription and the polarities of all DNA and RNA strands. - Give examples of non-coding RNA molecules. What is transcription? Consider that all of the cells in a multicellular organism have arisen by division from a single fertilized egg and therefore, all have the same DNA. Division of that original fertilized egg produces, in the case of humans, over a trillion cells, by the time a baby is produced from that egg (that's a lot of DNA replication!). Yet, we also know that a baby is not a giant ball of a trillion identical cells, but has the many different kinds of cells that make up tissues like skin and muscle and bone and nerves. How did cells that have identical DNA turn out so different? The answer lies in gene expression, which is the process by which the information in DNA is used. Although all the cells in a baby have the same DNA, each different cell type uses a different subset of the genes in that DNA to direct the synthesis of a distinctive set of RNAs and proteins. The first step in gene expression is transcription, the process of copying information from DNA sequences into RNA sequences. This process is also known as DNA-dependent RNA synthesis. When a sequence of DNA is transcribed, only one of the two DNA strands is copied into RNA, when this RNA encodes a protein is it known as messenger RNA (mRNA). Important features of transcription - All RNA, mRNA as well as tRNA, rRNA, microRNA and more, is produced by transcription. - Only one strand of DNA is used as a template by enzymes called RNA polymerases - RNA is synthesized from 5' to 3'. - RNA polymerases do not need primers to begin transcription. - The four ribonucleotide triphosphates (rNTPs) are ATP, GTP, UTP, and CTP. - RNA polymerases begin transcription at DNA sequences called promoters. - RNA polymerases end transcription at sequences called terminators. In transcription, an RNA polymerase uses only one strand of DNA, called the template strand, of a gene to catalyze synthesis of a complementary, antiparallel RNA strand. RNA polymerases use ribose nucleotide triphosphate (NTP) precursors, in contrast to DNA polymerases, which use deoxyribose nucleotide (dNTP) precursors (compared on page 1.1: The Structure of DNA ). In addition, RNAs incorporate uracil (U) nucleotides into RNA strands instead of the thymine (T) nucleotides used in DNA. RNA polymerases differ from DNA polymerases in that they do not require primers. With the help of transcription initiation factors, RNA polymerase locates the transcription start site of a gene and begins synthesis of a new RNA strand from scratch by joining the two ribonucleotides that are complementary to the first two bases of the template strand. Overview of the Stages of Transcription The basic steps of transcription are initiation, elongation, and termination. Here we can identify several of the DNA sequences that characterize a gene. The promoter is the binding site for RNA polymerase. It usually lies 5’ to, or upstream of the transcription start site. Binding of the RNA polymerase positions the enzyme to near the transcription start site, where it will start unwinding the double helix and begin synthesizing new RNA. The transcribed grey DNA region in each of the three panels are the transcription unit of the gene. Termination sites are typically 3’ to, or downstream from the transcribed region of the gene. By convention, upstream refers to DNA 5’ to a given reference point on the DNA (e.g., the transcription start-site of a gene). Downstream then, refers to DNA 3’ to a given reference point on the DNA. RNA polymerase Building an RNA strand is very similar to building a DNA strand. This is not surprising, knowing that DNA and RNA are very similar molecules. What enzyme carries out transcription? Transcription is catalyzed by the enzyme RNA Polymerase. "RNA polymerase" is a general term for an enzyme that makes RNA. There are many different RNA polymerases. Like DNA polymerases, RNA polymerases synthesize new strands only in the 5' to 3' direction, but because they are making RNA, they use ribonucleotides (i.e., RNA nucleotides) rather than deoxyribonucleotides. Ribonucleotides are joined in exactly the same way as deoxyribonucleotides, which is to say that the 3'OH of the last nucleotide on the growing chain is joined to the 5' phosphate on the incoming nucleotide. One important difference between DNA polymerases and RNA polymerases is that the latter do not require a primer to start making RNA. Once RNA polymerases are in the right place to start copying DNA, they just begin making RNA by stringing together RNA nucleotides complementary to the DNA template. This, of course, brings us to an obvious question- how do RNA polymerases "know" where to start copying on the DNA. Unlike the situation in replication, where every nucleotide of the parental DNA must eventually be copied, transcription, as we have already noted, only copies selected genes into RNA at any given time.What indicates to an RNA polymerase where to start copying DNA to make a transcript? Signals in DNA indicate to RNA polymerase where it should start (and end) transcription. These signals are special sequences in DNA that are recognized by the RNA polymerase or by proteins that help RNA polymerase determine where it should bind the DNA to start transcription. A DNA sequence at which the RNA polymerase binds to start transcription is called a promoter. A promoter is generally situated upstream of the gene that it controls. What this means is that on the DNA strand that the gene is on, the promoter sequence is "before" the gene. Remember that, by convention, DNA sequences are read from 5' to 3'. So the promoter lies 5' to the start point of transcription. Also notice that the promoter is said to "control" the gene it is associated with. This is because expression of the gene is dependent on the binding of RNA polymerase to the promoter sequence to begin transcription. If the RNA polymerase and its helper proteins do not bind the promoter, the gene cannot be transcribed and it will therefore, not be expressed. What is special about a promoter sequence? In an effort to answer this question, scientists looked at many genes and their surrounding sequences. It makes sense that because the same RNA polymerase has to bind to many different promoters, the promoters should have some similarities in their sequences. Sure enough, common sequence patterns were seen to be present in many promoters. We will first take a look at prokaryotic promoters. When prokaryotic genes were examined, the following features commonly emerged: - A transcription start site (this the base in the DNA across from which the first RNA nucleotide is paired). - A -10 sequence: this is a 6 bp region centered about 10 bp upstream of the start site. The consensus sequence at this position is TATAAT. In other words, if you count back from the transcription start site, which by convention, is called the +1, the sequence found at -10 in the majority of promoters studied is TATAAT). - A -35 sequence: this is a sequence at about 35 basepairs upstream from the start of transcription. The consensus sequence at this position is TTGACA. What is the significance of these sequences? It turns out that the sequences at -10 and -35 are recognized and bound by a subunit of prokaryotic RNA polymerase before transcription can begin. The RNA polymerase of E. coli, for example, has a subunit called the sigma (σ) subunit (or sigma factor) in addition to the core polymerase, which is the part of the enzyme that actually makes RNA. Together, the sigma subunit and core polymerase make up what is termed the RNA polymerase holoenzyme . The sigma subunit of the polymerase can recognize and bind to the -10 and -35 sequences in the promoter, thus positioning the RNA polymerase at the right place to initiate transcription. Once transcription begins, the core polymerase and the sigma subunit separate, with the core polymerase continuing RNA synthesis and the sigma subunit wandering off to escort another core polymerase molecule to a promoter. The sigma subunit can be thought of as a sort of usher that leads the polymerase to its "seat" on the promoter. As already mentioned, an RNA chain, complementary to the DNA template, is built by the RNA polymerase by the joining of the 5' phosphate of an incoming ribonucleotide to the 3'OH on the last nucleotide of the growing RNA strand. How does the polymerase know where to stop? A sequence of nucleotides called the terminator is the signal to the RNA polymerase to stop transcription and dissociate from the template. Although the process of RNA synthesis is the same in eukaryotes as in prokaryotes, there are some additional issues to keep in mind in eukaryotes. One is that in eukaryotes, the DNA template exists as chromatin, where the DNA is tightly associated with histones and other proteins. The "packaging" of the DNA must therefore be opened up to allow the RNA polymerase access to the template in the region to be transcribed. A second difference is that eukaryotes have multiple RNA polymerases, not one as in bacterial cells. The different polymerases transcribe different genes. For example, RNA polymerase I transcribes the ribosomal RNA genes, while RNA polymerase III copies tRNA genes. The RNA polymerase we will focus on most is RNA polymerase II, which transcribes protein-coding genes to make mRNAs. All three eukaryotic RNA polymerases need additional proteins to help them get transcription started. In prokaryotes, RNA polymerase by itself can initiate transcription (remember that the sigma subunit is a subunit of the prokaryotic RNA polymerase). The additional proteins needed by eukaryotic RNA polymerases are referred to as transcription factors. Finally, in eukaryotic cells, transcription is separated in space and time from translation. Transcription happens in the nucleus, and the mRNAs produced are processed further before they are sent into the cytoplasm. Protein synthesis (translation) happens in the cytoplasm. In prokaryotic cells, mRNAs can be translated as they are coming off the DNA template, and because there is no nucleus, transcription and protein synthesis occur in a single cellular compartment. Like genes in prokaryotes, eukaryotic genes also have promoters. Eukaryotic promoters commonly have a TATA box, a sequence about 25 base pairs upstream of the start of transcription that is recognized and bound by proteins that help the RNA polymerase to position itself correctly to begin transcription. (Some eukaryotic promoters lack TATA boxes, and have, instead, other recognition sequences to help the RNA polymerase find the spot on the DNA where it spot on the DNA where it binds and initiates transcription.) We noted earlier that eukaryotic RNA polymerases need additional proteins to bind promoters and start transcription. What are these additional proteins that are needed to start transcription? General transcription factors are proteins that help eukaryotic RNA polymerases find transcription start sites and initiate RNA synthesis. We will focus on the transcription factors that assist RNA polymerase II. These transcription factors are named TFIIA, TFIIB and so on (TF= transcription factor, II=RNA polymerase II, and the letters distinguish individual transcription factors). Transcription in eukaryotes requires the general transcription factors and the RNA polymerase to form a complex at the TATA box called the basal transcription complex or transcription initiation complex. This is the minimum requirement for any gene to be transcribed. The first step in the formation of this complex is the binding of the TATA box by a transcription factor called the TATA Binding Protein or TBP. Binding of the TBP causes the DNA to bend at this spot and take on a structure that is suitable for the binding of additional transcription factors and RNA polymerase. As shown in the figure at left, a number of different general transcription factors, together with RNA polymerase (Pol II) form a complex at the TATA box. The final step in the assembly of the basal transcription complex is the binding of a general transcription factor called TFIIH. TFIIH is a multifunctional protein that has helicase activity (i.e., it is capable of opening up a DNA double helix) as well as kinase activity. The kinase activity of TFIIH adds a phosphate onto the C-terminal domain (CTD) of the RNA polymerase. This phosphorylation appears to be the signal that releases the RNA polymerase from the basal transcription complex and allows it to move forward and begin transcription. Either DNA strand can be a template The promoter is the sequence of DNA that encodes the information about where to begin transcription for each gene. Depending on the promoter, either strand of DNA can be used as the template strand. Watch this video to see how either strand of DNA can be used as a template for different genes on the same chromosome. template vs. non-template strands summary - The template strand is the one that RNA polymerase uses as the basis to build the RNA. This strand is also called the non-coding strand or the antisense strand. - The non-template strand has the identical sequence of the RNA (except for the substituion of U for T). This strand is also called the coding strand or sense strand . Major Types of Cellular RNA Cells make several different kinds of RNA: - mRNAs that code for proteins - rRNAS that form part of ribosomes - tRNAs that serve as adaptors between mRNA and amino acids during translation - MicroRNAs that regulate gene expression - Other small RNAs that have a variety of functions.	libretexts	2025-03-17T22:27:29.166389	2020-06-03T20:55:55	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/02%3A_Central_Dogma/2.01%3A_Overview_of_Transcription", "book_url": "https://commons.libretexts.org/book/bio-25718", "title": "2.1: Overview of Transcription", "author": "Stefanie West Leacock" }
https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/03%3A_Mutation/3.01%3A_Origins_of_Mutations	3.1: Origins of Mutations Learning Objectives - Identify some causes and types of mutations. - Define mutagen and predict the effects of mutagens on DNA sequences. Introduction to mutations Mutations have a bad reputation, BUT they are ESSENTIAL to life! Watch the video for an introduction to why understanding mutations is so important for us. Types of mutations Mutations may involve the loss ( deletion ), gain ( insertion ) of one or more base pairs, or else the substitution of one or more base pairs with another DNA sequence of equal length. These changes in DNA sequence can arise in many ways, some of which are spontaneous and due to natural processes, while others are induced by humans intentionally (or unintentionally) using mutagens. There are many ways to classify mutagens , which are the agents or processes that cause mutation or increase the frequency of mutations. We will classify mutagens here as being (1) biological, (2) chemical, or (3) physical. Mutations of biological origin A major source of spontaneous mutation is errors that arise during DNA replication. DNA polymerases are usually very accurate in adding a base to the growing strand that is the exact complement of the base on the template strand. However, occasionally, an incorrect base is inserted. Usually, the DNA polymerase proofreading activity will recognize and repair mispaired bases, but nevertheless, some errors become permanently incorporated in a daughter strand, and so become mutations that will be inherited by the cell’s descendents. Figure $\PageIndex{1}$: Mispairing of bases (e.g. G with T) can occur due to tautomerism, alkylating agents, or other effects. As a result, in this example the AT base pair in the original DNA strand will become permanently substituted by a GC based pair in some progeny. The mispaired GT pair will likely be repaired or eliminated before further rounds of replication. (Original-Deyholos-CC:AN) The most common insult to the DNA of living organisms is depurination , in which the bond between an adenine or guanine and the deoxyribose is hydrolyzed. Depyrimidination of cytosine and thymine residues can also occur, but do so at a much slower rate than depurination. Despite the high rate of loss of these bases, they are generally identified and remediated by base excision repair (BER). Therefore, it is rare for depurination or depyrimidination to lead to mutation. Three of the four DNA bases, adenine, guanine, and cytosine, contain amine (-NH 2 ) groups that can be lost in a variety of pH and temperature-dependent reactions that convert the bases to hypoxanthine, xanthine, and uracil, respectively. This deamination can sometimes lead to permanent mutations during replication. Hypoxanthine base pairs with C (cytosine); xanthine base pairs with cytosine; uracil base pairs with adenine. If hypoxanthine is present when DNA polymerase is replicating DNA, a C would be incorporated instead of T; similarly if uracil is present an A would be incorporated instead of G. Query $\PageIndex{1}$ Another deamination, of the modified base methylcytosine, can also lead to a mutation upon replication. Some cytosines may be methylated as part of a regulatory process to inactivate certain genes in eukaryotes, or in prokaryotes as protection against restriction endonucleases. When the methylated cytosine is deaminated, it produces a thymine, which changes the complementary nucleotide (upon replication) from a guanine to an adenine. Deamination of cytosines occurs at nearly the same rate as depurination, but deamination of other bases are not as pervasive: deamination of adenines, for example, is 50 times less likely than deamination of cytosine. Query $\PageIndex{2}$ Another type of error introduced during replication is caused by a rare, temporary misalignment of a few bases between the template strand and daughter strand (Figure $\PageIndex{5}$ ). This strand-slippage causes one or more bases on either strand to be temporarily displaced in a loop that is not paired with the opposite strand. If this loop forms on the template strand, the bases in the loop may not be replicated, and a deletion will be introduced in the growing daughter strand. Conversely, if a region of the daughter strand that has just been replicated becomes displaced in a loop, this region may be replicated again, leading to an insertion of additional sequence in the daughter strand, as compared to the template strand. Regions of DNA that have several repeats of the same few nucleotides in a row are especially prone to this type of error during replication. Thus regions with short tanderm repeats ( STRs or short sequence repeats SSRs) are tend to be highly polymorphic, and are therefore particularly useful in genetics. They are also called microsatellites . Mutations can also be caused by the insertion of viruses, transposable elements (transposons), see below, and other types of DNA that are naturally added at more or less random positions in chromosomes. The insertion may disrupt the coding or regulatory sequence of a gene, including the fusion of part of one gene with another. These insertions can occur spontaneously, or they may also be intentionally stimulated in the laboratory as a method of mutagenesis called transposon-tagging . For example, a type of transposable element called a P element is widely used in Drosophila as a biological mutagen. T-DNA , which is an insertional element modified from a bacterial pathogen, is used as a mutagen in some plant species. Mutations due to Transposable Elements Transposable elements (TEs) are also known as mobile genetic elements , or more informally as jumping genes . They are present throughout the chromosomes of almost all organisms. These DNA sequences have a unique ability to be cut or copied from their original location and inserted into new locations in the genome. This is called transposition. These insert locations are not entirely random, but TEs can, in principle, be inserted into almost any region of the genome. TEs can therefore insert into genes, disrupting its function and causing a mutation. Researchers have developed methods of artificially increasing the rate of transposition, which makes some TEs a useful type of mutagen. However, the biological importance of TEs extends far beyond their use in mutant screening. TEs are also important causes of disease and phenotypic instability, and they are a major mutational force in evolution. There are two major classes of TEs in eukaryotes: - Class I elements include retrotransposons ; these transpose by means of an RNA intermediate. The TE transcript is reverse transcribed into DNA before being inserted elsewhere in the genome through the action of enzymes such as integrase . - Class II elements are known also as transposons . They do not use reverse transcriptase or an RNA intermediate for transposition. Instead, they use an enzyme called transposase to cut DNA from the original location and then this excised dsDNA fragment is inserted into a new location. TEs are relatively short DNA sequences (100-10,000 bp), and encode no more than a few proteins (if any). Normally, the protein-coding genes within a TE are all related to the TE’s own transposition functions. These proteins may include reverse transcriptase , transposase, and integrase. However, some TEs (of either Class I or II) do not encode any proteins at all. These non-autonomous TEs can only transpose if they are supplied with enzymes produced by other, autonomous TEs located elsewhere in the genome. In all cases, enzymes for transposition recognize conserved nucleotide sequences within the TE, which dictate where the enzymes begin cutting or copying. The human genome consists of nearly 45% TEs, the vast majority of which are families of Class I elements called LINEs (long intersperse nuclear elements) and SINEs (short interspersed nuclear elements) . The short, Alu type of SINE occurs in more than one million copies in the human genome (compare this to the approximately 21,000, non-TE, protein-coding genes in humans). Indeed, TEs make up a significant portion of the genomes of almost all eukaryotes. Class I elements, which usually transpose via an RNA copy-and-paste mechanism, tend to be more abundant than Class II elements, which mostly use a cut-and-paste mechanism. But even the cut-paste mechanism can lead to an increase in TE copy number. For example, if the site vacated by an excised transposon is repaired with a DNA template from a homologous chromosome that itself contains a copy of a transposon, then the total number of transposons in the genome will increase. Besides greatly expanding the overall DNA content of genomes, TEs contribute to genome evolution in many other ways. As already mentioned, they may disrupt gene function by insertion into a gene’s coding region or regulatory region. More interestingly adjacent regions of chromosomal DNA are sometimes mistakenly transposed along with the TE; this can lead to gene duplication. The duplicated genes are then free to evolve independently, leading in some cases to the development of new functions. The breakage of strands by TE excision and integration can disrupt genes, and can lead to chromosome rearrangement or deletion if errors are made during strand rejoining. Furthermore, having so many similar TE sequences distributed throughout a chromosome sometimes allows mispairing of regions of homologous chromosomes at meiosis, which can cause unequal crossing-over, resulting in deletion or duplication of large segments of chromosomes. Thus, TEs are a potentially important evolutionary force, and may not be included as merely “junk DNA”, as they once were. Are my genes always jumping?! If the human genome is 45% transposable elements and these elements can move around, then how does our genome stay relatively constant? Some factors that help keep the genome (mostly) intact include: - Many of the TE elements have acquired mutations that render them unable to transpose. - Regulatory RNAs called piRNAs (piwi-related RNAs) appear to suppress transposition. - In which tissue(s) would you expect piRNAs to be most active? Mutations of Chemical Origins Many chemical compounds, whether natural or synthetic, can react with DNA to cause mutations. In some of these reactions the chemical structure of particular bases may change, so that they are misread during replication. In others the chemical mutagens distort the double helix causing it to be replicated inaccurately, while still others may cause breaks in chromosomes that lead to deletions and other types of aberrations. The following are examples of two classes of chemical mutagens that are important in genetics and medicine: alkylating agents , and intercalating agents . What is an alkyl group? An alkyl group is formed by removing one hydrogen from an alkane chain (C n H 2n +2 ) and is described by the formula C n H 2n +1 . The removal of this hydrogen results in a stem change from -ane to -yl . Take a look at the following examples. Ethane methyl sulfonate ( EMS ) is an example of an alkylating agent that is commonly used by geneticists to induce mutations in a wide range of both prokaryotes and eukaryotes. The organism is fed or otherwise exposed to a solution of EMS. It reacts with some of the G bases it encounters in a process called alkylation, where the addition of an alkyl group to G changes the base pairing properties so that the next time the alkylated DNA strand is replicated, a T instead of a C will be inserted opposite to the alkylated-G in the daughter strand. The new strand therefore bears a mutation (base substitution), which will be inherited in all the strands that are subsequently replicated from it. Figure $\PageIndex{7}$: Alkylation of G (shown in red) allows G to bond with T, rather than with C. (Original-Deyholos-CC:AN) Intercalating agents are another type of chemical mutagen. They tend to be flat, planar molecules like benzo[ a ]pyrene , a component of wood and tobacco smoke, and induce mutations by inserting between the stacked bases at the center of the DNA helix. This intercalation distorts the shape of the DNA helix, which can cause the wrong bases to be added to a growing DNA strand during DNA synthesis. There are a large number of chemicals that act as intercalating agents, can mutate DNA, and are carcinogenic (can cause cancer). Many of these are also used to treat cancer, as they preferentially kill actively dividing cells. Another important intercalating agent is ethidium bromide , the fluorescent dye that stains DNA in laboratory assays. For this reason, molecular biologists are trained to handle this chemical carefully. Exercise $\PageIndex{1}$ The chemical EMS adds an ethyl group to G, which then pairs with T instead of C. What would be the effect of exposing the DNA sequence ...CATGTCA... to EMS? - Answer - When DNA is exposed to a mutagen it is unlikely that all the Gs in a given sequence will be affected. We will assume that only the G in the above sequence is affected (not the two Gs that will be on the complementary strand). I will write the modified sequence as ...CATG m TCA... - When the methylated strand is used as a template for DNA replication, the newly synthesized strand will be ...GTATAGT... - If the cell undergoes another round of cell division, using the new strand as a template will create the strand ...CATATCA... and the former GC base pair is now an AT base pair. The cell has no way of knowing that this base pair is incorrect or whether or not it will affect the cell. Mutations of physical origin Anything that damages DNA by transferring energy to it can be considered a physical mutagen. Usually this involves radioactive particles, x-rays, or UV light. The smaller, fast moving particles may cause base substitutions or delete a single bases, while larger, slightly slower particles may induce larger deletions by breaking the double stranded helix of the chromosome. Physical mutagens can also create unusual structures in DNA, such as the thymine dimers formed by UV light (Figure $\PageIndex{9}$). Thymine dimers disrupt normal base-pairing in the double helix, and may block replication altogether if not repaired by the cell’s DNA repair enzymes. Summary Table of Mutagenic Agents \| Mutagenic Agents \| Mode of Action \| Effect on DNA \| Resulting Type of Mutation \| \|---\|---\|---\|---\| \| Nucleoside analogs \| \|\|\| \| 2-aminopurine \| Is inserted in place of A but base pairs with C \| Converts AT to GC base pair \| Point \| \| 5-bromouracil \| Is inserted in place of T but base pairs with G \| Converts AT to GC base pair \| Point \| \| Nucleotide-modifying agent \| \|\|\| \| Nitrous oxide \| Deaminates C to U \| Converts GC to AT base pair \| Point \| \| Ethane methyl sulfonate (EMS) \| Alkylates G (which pairs with T) \| \|\| \| Intercalating agents \| \|\|\| \| Acridine orange, ethidium bromide, polycyclic aromatic hydrocarbons \| Distorts double helix, creates unusual spacing between nucleotides \| Introduces small deletions and insertions \| Frameshift \| \| Ionizing radiation \| \|\|\| \| X-rays, γ-rays \| Forms hydroxyl radicals \| Causes single- and double-strand DNA breaks \| Repair mechanisms may introduce mutations \| \| X-rays, γ-rays \| Modifies bases (e.g., deaminating C to U) \| Converts GC to AT base pair \| Point \| \| Nonionizing radiation \| \|\|\| \| Ultraviolet \| Forms pyrimidine (usually thymine) dimers \| Causes DNA replication errors \| Frameshift or point \| Query $\PageIndex{3}$ Contributors and Attributions - Dr. Todd Nickle and Isabelle Barrette-Ng (Mount Royal University) The content on this page is licensed under CC SA 3.0 licensing guidelines. - E. V. Wong Axolotl Academica Publishing (Biology) at Axolotl Academica Publishin g - Mutations. (2021, January 3). Retrieved August 6, 2021, from https://bio.libretexts.org/@go/page/5184 " Mutations" by OpenStax, LibreTexts is licensed under CC BY ( 11.5: Mutations ).	libretexts	2025-03-17T22:27:29.464136	2019-10-01T21:21:10	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/", "url": "https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/03%3A_Mutation/3.01%3A_Origins_of_Mutations", "book_url": "https://commons.libretexts.org/book/bio-25718", "title": "3.1: Origins of Mutations", "author": "Stefanie West Leacock" }
https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/03%3A_Mutation/3.03%3A_Mutations_reveal_functions_of_genes/3.3.01%3A_Example_-_Mutations_and_Cystic_Fibrosis	3.3.1: Example - Mutations and Cystic Fibrosis Cystic Fibrosis (CF) Cystic fibrosis (CF) is one of many diseases that geneticists have shown to be caused by mutation of a single, well-characterized gene. Cystic fibrosis is the most common (1/2,500) life-limiting autosomal recessive disease among people of European heritage, with ~ 1 in 25 people being carriers. The frequency varies in different populations. Most of the deaths caused by CF are the result of lung disease, but many CF patients also suffer from other disorders including infertility and gastrointestinal disease. The disease is due to a mutation in the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene, which was identified by Lap-chee Tsui’s group at the University of Toronto. Epithelial tissues in some organs rely on the CFTR protein to transport ions (especially Cl - ) across their cell membranes. The passage of ions through a six-sided channel is gated by another part of the CFTR protein, which binds to ATP. If there is insufficient activity of CFTR, an imbalance in ion concentration results, which disrupts the properties of the liquid layer that normally forms at the epithelial surface. In the lungs, this causes mucus to accumulate and can lead to infection. Defects in CFTR also affect pancreas, liver, intestines, and sweat glands, all of which need this ion transport. CFTR is also expressed at high levels in the salivary gland and bladder, but defects in CFTR function do not cause problems in these organs, probably because other ion transporters are able to compensate. Over one thousand different mutant alleles of CFTR have been described. Any mutation that prevents CFTR from sufficiently transporting ions can lead to cystic fibrosis (CF). Worldwide, the most common CFTR allele among CF patients is called ΔF508 (delta-F508 or PHE508DEL), which is a deletion of three nucleotides that eliminates a phenylalanine (F) from position 508 of the 1480 amino acid wild-type protein. Mutation ΔF508 causes CFTR to be folded improperly in the endoplasmic reticulum (ER) and preventing CFTR from reaching the cell membrane. The allele ΔF508 accounts for approximately 70% of CF cases in North America, with ~1/25 people of European descent being carriers. The high frequency of the ΔF508 allele around the world has led to speculation that it may confer some selective advantage to heterozygotes, perhaps by reducing dehydration during cholera epidemics or by reducing susceptibility to certain pathogens that bind to epithelial membranes, although little data has been uncovered to support these hypotheses. However, recent mining of health data and genetic data from over 19,000 CFTR mutant allele heterozygotes suggests that heterozygotes exhibit an increased incidence of a variety of conditions including infertility, pancreatitis, diabetes, short stature, failure to thrive, constipation and scoliosis (Miller et al., 2020). Query $\PageIndex{1}$ CFTR is also notable because it is one of the well-characterized genetic diseases for which a drug has been developed that compensates for the effects of a specific mutation. The drug, Kalydeco® (ivacaftor) made by Vertex Pharmaceuticals, was approved by the FDA and Health Canada in 2012, decades after the CFTR gene was first mapped to DNA markers (Tsui et al. 1985; Wainwright et al. 1985; White et al., 1985) and cloned (Kerem et al. 1989; Riordan et al. 1989; Rommens et al. 1989). Kalydeco is effective on only some CFTR mutations, for example G551D (i.e. where glycine is substituted by aspartic acid at position 551 of the protein; GLY551ASP). This mutation is found in less than 5% of CF patients. The G551D mutation affects the ability of ATP to bind to CFTR and open the channel it for transport. Kalydeco® compensates for the mutation by binding to CFTR and holding it in an open conformation. Kalydeco® is priced at approximately $300,000 per patient per year, but costs may be covered by insurance. In 2015 an additional drug, Orkambi® was approved. This therapy combines ivacaftor with a second drug lumacaftor, which increases the trafficking of the ΔF508 protein to the cell surface. Like Kalydeco, Orkambi costs about $300,000 per year. Watch this video about Cystic Fibrosis Mechanism and Treatment (from HHMI BioInteractive). Query $\PageIndex{2}$ Contributors and Attributions - Dr. Todd Nickle and Isabelle Barrette-Ng (Mount Royal University) The content on this page is licensed under CC SA 3.0 licensing guidelines. References Kerem B, Rommens JM, Buchanan JA, Markiewicz D, Cox TK, Chakravarti A, Buchwald M, Tsui LC 1989. Identification of the cystic fibrosis gene: genetic analysis. Science. Sep 8; 245(4922):1073-80. Miller AC, Comellas AP, Hornick DB, Stoltz DA, Cavanaugh JE, Gerke AK, Welsh MJ, Zabner J, Polgreen PM. Cystic fibrosis carriers are at increased risk for a wide range of cystic fibrosis-related conditions. Proceedings of the National Academy of Sciences Jan 2020, 117 (3) 1621-1627; DOI: 10.1073/pnas.1914912117 ( https://www.pnas.org/content/117/3/1621 ) Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, Grzelczak Z, Zielenski J, Lok S, Plavsic N, Chou JL. 1989. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science. Sep 8; 245(4922):1066-73. Rommens JM, Iannuzzi MC, Kerem B, Drumm ML, Melmer G, Dean M, Rozmahel R, Cole JL, Kennedy D, Hidaka N. 1989. Identification of the cystic fibrosis gene: chromosome walking and jumping. Science. Sep 8; 245(4922):1059-65. Tsui LC, Buchwald M, Barker D, Braman JC, Knowlton R, Schumm JW, Eiberg H, Mohr J, Kennedy D, Plavsic N, et al. 1985. Cystic fibrosis locus defined by a genetically linked polymorphic DNA marker. Science 230: 1054–1057. Wainwright BJ, Scambler PJ, Schmidtke J, Watson EA, Law HY, Farrall M, Cooke HJ, Eiberg H, Williamson R 1985. Localization of cystic fibrosis locus to human chromosome 7cen-q22. Nature 318: 384–385. White R, Woodward S, Leppert M, O’Connell P, Hoff M, Herbst J, Lalouel JM, Dean M, Vande Woude G 1985. A closely linked genetic marker for cystic fibrosis. Nature 318: 382–384.	libretexts	2025-03-17T22:27:29.581067	2020-06-03T20:56:00	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/", "url": "https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/03%3A_Mutation/3.03%3A_Mutations_reveal_functions_of_genes/3.3.01%3A_Example_-_Mutations_and_Cystic_Fibrosis", "book_url": "https://commons.libretexts.org/book/bio-25718", "title": "3.3.1: Example - Mutations and Cystic Fibrosis", "author": "Stefanie West Leacock" }
https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/04%3A_Inheritance/4.01%3A_Meiosis	4.1: Meiosis Learning Objectives - Describe and draw the key events and stages of meiosis that lead to haploid gametes. - Recall that homologous chromosomes separate during meiosis I (a reductional division) and that sister chromatids separate during meiosis II (an equational division). - Compare mitosis and meiosis. - Compare the processes of oogenesis and spermatogenesis in humans, including the chromosome complements of the gametes. Most eukaryotes replicate sexually - a cell from one individual joins with a cell from another to create the next generation. For this to be successful, the cells that fuse must contain half the number of chromosomes as in the adult organism. Otherwise, the number of chromosomes would double with each generation! The reduction in chromosome number is achieved by the process of meiosis . In meiosis, there are usually two steps, Meiosis I and II. In Meiosis I homologous chromosomes segregate, while in Meiosis II sister chromatids segregate. Most multicellular organisms use meiosis to produce gametes , the cells that fuse to make offspring. Some single celled eukaryotes such as yeast also use meiosis. Meiosis begins similarly to mitosis (a cell has replicated its chromosomes and grown large enough to divide), but requires two rounds of division. In the first, known as meiosis I, the homologous chromosomes separate and segregate. During meiosis II the sister chromatids separate and segregate. Note how meosis I and II are both divided into prophase, metaphase, anaphase, and telophase. After two rounds of cytokinesis, four cells will be produced, each with a single copy of each chromosome. Meiosis is divided into two stages designated by the roman numerals I (one) and II (two). Meiosis I is called a reductional division, because it reduces the number of chromosomes inherited by each of the daughter cells. Meiosis I is further divided into Prophase I, Metaphase I, Anaphase I, and Telophase I, which are roughly similar to the corresponding stages of mitosis, except that in Prophase I and Metaphase I, homologous chromosomes pair with each other, or synapse , and are called bivalents . This is an important difference between mitosis and meiosis, because it affects the segregation of alleles, and also allows for recombination to occur through crossing-over, as described later. During Anaphase I, one member of each pair of homologous chromosomes migrates to each daughter cell (1N). Meiosis II resembles mitosis, with one sister chromatid from each chromosome separating to produce two daughter cells. Because Meiosis II, like mitosis, results in the segregation of sister chromatids, Meiosis II is called an equational division. Meiosis I In meiosis I replicated, homologous chromosomes pair up, or synapse, during the pachytene stage of prophase I, line up in the middle of the cell during metaphase I, and separate during anaphase I. For this to happen the homologous chromosomes need to be brought together while they condense during prophase I. These attachments are formed in two ways. Proteins bind to both homologous chromosomes along their entire length and form the synaptonemal complex (synapse means junction). These proteins hold the chromosomes in a transient structure called a bivalent . The proteins are released when the cell enters anaphase I, so that the homologous chromosomes can be separated. Query $\PageIndex{1}$ Chromosome condensation during meiosis As meiosis proceeds, chromatin becomes increasingly condensed. In some organisms, the DNA becomes so condensed that it appears as a spot of DNA instead of a line under the microscope. As you might expect from condensed chromatin, little transcriptional activity occurs during these stages of meiosis, so cells must produce the needed mRNAs in advance of meiosis. Homologous Recombination Within the synaptonemal complex during prophase 1, homologous recombination, or crossing over, occurs. These are places where DNA endonucleases break two non-sister chromatids in similar locations and then covalently reattach non-sister chromatids together to create a crossover between non-sister chromatids ( 4.1.1: Homologous recombination ). This reorganization of chromatids will persist for the remainder of meiosis and result in recombination of alleles in the gametes. Crossovers function to hold homologous chromosomes together during meiosis I so that they segregate successfully; they also cause the reshuffling of allele combinations to create genetic diversity, which can have an important effect on evolution. Meiosis II At the completion of meiosis I there are two haploid cells, each with one, replicated copy of each chromosome (1n). Because only one copy of each homolog is present, bivalents are not formed. In metaphase of meiosis II, the chromosomes will once again be brought to the middle of the cell, but this time it is the sister chromatids that will segregate during anaphase II. After cytokinesis there will be four cells, each containing only one unreplicated chromosome of each type. Meiosis II resembles mitosis in that the number of chromosomes per cell is unchanged - both are equational cell divisions – but in meiosis II all four cells have different genetic composition. There will be allelic differences among the gametes. Query $\PageIndex{2}$ Outcomes of meiosis The outcome of meiosis is a cell or cells with half the number of chromosomes as the starting cell. But what combinations of chromosomes are possible? The homologous chromosomes separate during meiosis I, but the separation of the pairs of homologs is independent of other homologs. As an example in the figure below, for a cell with two pairs of chromosomes (2n=4), there can be four possible combinations of the four chromosomes. When we add recombination and additional pairs of chromosomes, there are almost infinite combinations of chromosomes in gametes. Watch the video below to follow how 4 pair of chromosomes are passed during meiosis I and II. Gamete maturation In animals and plants, the cells produced at the end of meiosis need to mature before they become functional gametes. Spermatogenesis In most male animals, the four products of meiosis are called spermatids . They change morphology to develop tails and become functional sperm cells. In the meiotic steps of spermatogenesis, the cell divisions are equal, with the meiotic spindle aligned with the center of the cell, and the cells have equal amounts of cytoplasm, much like an average cell that has undergone mitosis. The streamlined, minimal-cytoplasm mature sperm is a product of post-meiotic differentiation, in which it gains the flagellar tail, and ejects most of its cytoplasmic material, keeping only some mitochondria to power the flagella and an acrosomal vesicle that contains the enzymes and other molecules needed to reach and fuse with (i.e. fertilize) a mature egg. Oogenesis In female animals, the gametes are oocytes . Each mature ovum (egg) will need to be as large possible to contain the maximum amount of cytoplasm including organelles, proteins, mRNAs, and nutrients to support the embryo after fertilization. To create large oocytes, only one of the four products of meiosis becomes an egg. The other three cells end up as tiny "disposable" cells called polar bodies , essentially little "packages" of extra DNA and very little cytoplasm. These cells are not viable and will eventually be degraded. How can you make a really small cell? The asymmetric distribution of cytoplasm in the first meiotic division for oocytes is due to the position of the meiotic spindle in the periphery of the cell rather than centered. During oogenesis, chromosomes do not line up in the middle of the cell during metaphase I or II. Because the center of the spindle determines the position of the contractile ring for cytokinesis, one small and one large cell are produced. Timing of Spermatogenesis and Oogenesis In addition to the differences in gamete size and number, in mammals the timing of meiosis differs between males and females. In males, germ cells are pre-meiotic at birth and do not enter meiosis until the onset of puberty. Mitosis maintains a population of precursor cells, so that sperm production can continue throughout adulthood. In females, germ cells enter meiosis I during embryonic development. These primary oocytes remain arrested ("stuck") in meiosis I until puberty. After this time, one primary oocyte per month (roughly for humans, depends upon cycle length for other mammals) completes meiosis I, enters meiosis II and is ovulated. Actually, meiosis II is only completed if the oocyte is successfully fertilized! The timing of meiotic arrest can differ between different species. For example, in the nematode C. elegans, oocytes are arrested in late prophase of meiosis I and only complete meiosis I and II rapidly after fertilization. A note about plants In plants, the products of meiosis reproduce a few times using mitosis as they develop into functional male or female gametes. Fertilization The purpose of gametes is to allow reproduction generation after generation. By uniting two gametes with half the number of chromosomes, the full chromosome number is restored each generation. Remember that homologous recombination and assortment of chromosomes create a genetically diverse population of gametes. Fertilization restores the diploid number oocyte (n) + sperm (n) = zygote (2n) Errors in meiosis Like any biological process, errors can occur during meiosis. If homologous chromosomes or sister chromatids are not correctly distributed during meiosis (known as nondisjunction), gametes can have too many or too few chromosomes ( 5.1: Changes in Chromosome Number ). Contributors and Attributions - Dr. Todd Nickle and Isabelle Barrette-Ng (Mount Royal University) The content on this page is licensed under CC SA 3.0 licensing guidelines. - 15.8: Meiosis - Stefanie Leacock, University of Arkansas-Little Rock	libretexts	2025-03-17T22:27:29.780370	2019-10-01T21:21:04	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/", "url": "https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/04%3A_Inheritance/4.01%3A_Meiosis", "book_url": "https://commons.libretexts.org/book/bio-25718", "title": "4.1: Meiosis", "author": "Stefanie West Leacock" }
https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/04%3A_Inheritance/4.02%3A__Mendelian_Genetics	4.2: Mendelian Genetics Learning Objectives - Define and identify examples of: homozygous, heterozygous, allele, gene, locus, dominant, recessive, genotype, phenotype. - Construct genetic crosses for Po, F1, and F2 generations and predict the genotypes and phenotypes of offspring. - Explain Mendel’s laws of segregation and independent assortment, and how they predict the 3:1 dominant-to-recessive phenotypic ratio among the F2 of a monohybrid cross, or the 9:3:3:1 phenotypic ratio in a dihybrid cross, respectively. Relate the key events of meiosis that explain Mendel’s first and second laws. - Be able to draw chromosomes during meiosis with alleles labeled. - Interpret phenotypic ratios of progeny in experimental organisms to infer how particular traits are inherited. - Predict genotypic and phenotypic ratios or probabilities of outcomes among progeny of single factor and multifactor crosses using simple rules of probability (sum rule and product rule). - Cite the most common molecular explanations for dominant and recessive alleles. Introduction to Gregor Mendel and his Work Mendel Studied Visible Character Traits in Pea Plants Through careful study of patterns of inheritance, Mendel recognized that a single trait could exist in different versions, or alleles , even within an individual plant or animal. Recalling that genes contain information needed to make proteins, we now understand that alleles are differences in gene sequence. If these differences alter the production, structure, or function of the protein, an observable or measurable change in the organism may occur. For example, Mendel identified two forms of a gene for seed color: one allele gave green seeds and the other gave yellow seeds. Representing genes and alleles Alleles are forms of genes, if genes are DNA sequences, then alleles are variations in the sequence of a gene. In genetics, you may encounter different ways of representing alleles. Traditionally, genes are represented as letters when studying Mendelian genetics. This representation is usually easy to follo w in problems involving crosses ; h owever , using letters does not reflect our modern understanding of the genetic dif ferences between alleles, which often involves knowing whether or not a product is functional or how the allele was identified. - A plus can be used to indicate that the gene product of an allele is functional. - A minus can be used to indicate that the gene product is not functional. - If sequence information is known, the nucleotide or amino acid change can be identified. - In model organisms, alleles are often given numbers when they are identified as mutants and these numbers can be used to identify different alleles of the gene. The table shows some examples of how we might represent genes and alleles. \| Examples of ways genes can be represented \| Examples of representing wild-type alleles \| Examples of representing the mutant alleles \| \| C representing a hypothetical gene \| C or C + \| c or C - or C 1 \| \| white or w gene in Drosophila \| white or white + or w + \| w - or w 1118 \| \| ced-1 gene in C. elegans \| ced-1 or ced-1(+) or ced-1 + \| ced-1(-) or ced-1(e1754) \| \| CFTR gene in humans Cftr gene in mouse \| CFTR or CFTR + \| CFTR( delF 508 ) or CFTR( 482G-A) \| Heterozygous and homozygous Mendel’s work and discoveries are especially remarkable because he made his observations and conclusions (in 1865) without knowing about the relationships between genes, chromosomes, and DNA. We now know the reason why more than one allele of a gene can be present in an individual: most eukaryotic organisms have at least two sets of homologous chromosomes. For organisms that are predominantly diploid, such as humans or Mendel’s peas, chromosomes exist as pairs, with one homolog inherited from each parent. Diploid cells therefore contain two different alleles of each gene, with one allele on each member of a pair of homologous chromosomes. If both alleles of a particular gene are identical, the individual is said to be homozygous for that gene. On the other hand, if the alleles are different from each other, the genotype is heterozygous . Although a typical diploid individual can have at most two different alleles of a particular gene, many more than two different alleles can exist in a population of individuals. In a natural population the most common allelic form is usually called the wild-type allele. However, in many populations there can be multiple variants at the DNA sequence level that are visibly indistinguishable because all produce a normal, wild-type appearance. There can also be various mutant alleles (in wild populations and in lab strains) that vary from wild type in their appearance, each with a different change at the DNA sequence level. Such collections of mutations are known as an allelic series . wild type vs wild-type The noun wild type (without a hyphen) means the the most common form of a gene, phenotype, or organism under standard conditions. - Example: The mutant worms produce fewer offspring than wild type . The adjective wild-type (with a hyphen) can be used to describe a gene, allele, organism, phenotype, or trait as what is most commonly present among a population or under standard conditions. - Example: A wild-type worm produces about 300 offspring. Contributors and Attributions - Dr. Todd Nickle and Isabelle Barrette-Ng (Mount Royal University) The content on this page is licensed under CC SA 3.0 licensing guidelines.	libretexts	2025-03-17T22:27:29.914068	2019-10-01T21:21:04	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/", "url": "https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/04%3A_Inheritance/4.02%3A__Mendelian_Genetics", "book_url": "https://commons.libretexts.org/book/bio-25718", "title": "4.2: Mendelian Genetics", "author": "Stefanie West Leacock" }
https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/04%3A_Inheritance/4.02%3A__Mendelian_Genetics/4.2.01%3A_Monohybrid_Crosses_and_Segregation	4.2.1: Monohybrid Crosses and Segregation True Breeding Lines Mendel used true-breeding lines of pea plants, which are in-bred populations of plants or animals in which all parents and their offspring (over many generations) have the same phenotypes with respect to a particular trait. True breeding lines are useful, because they are typically assumed to be homozygous for the alleles that affect the trait of interest. When two individuals that are homozygous for the same alleles are crossed, all of their offspring will all also be homozygous. The continuation of such crosses constitutes a true breeding line or strain. A large variety of different strains, each with a different, true breeding character, can be collected and maintained for genetic research. Monohybrid Crosses A monohybrid cross is one in which both parents are heterozygous (or a hybrid) for a single (mono) trait. The trait might be petal color in pea plants. When conducting crosses, the first generation is called P (or P 0 ), the second generation is F 1 (F is for filial), and the next generation is F 2 . Using monohybrid crosses, Mendel observed that although different alleles could influence a single trait, they remained indivisible and could be inherited separately. Additionally, the allele could be present but invisible in one generation, only to reappear in the next generation. Definition: Mendel's First Law The Law of Segregation states that during gamete formation, the two alleles at a gene locus segregate from each other; each gamete has an equal probability of containing either allele. Punnett Squares Given the genotypes of any two parents, we can predict the genotypes of gametes that will be produced during meiosis. Using that information, we can predict all of the possible genotypes of the offspring. Furthermore, if we also know the dominance relationships for all of the alleles, we can predict the phenotypes of the offspring. A convenient method for calculating the expected genotype and phenotype ratios from a cross was invented by Reginald Punnett. A Punnett square is a matrix in which all of the possible gametes produced by one parent are listed along one axis, and the gametes from the other parent are listed along the other axis. Each possible combination of gametes is listed at the intersection of each row and column. Punnett squares can also be used to calculate the frequency of types offspring that are expected. Query $\PageIndex{1}$ Test Crosses Knowing the genotypes of an individual is usually an important part of a genetic experiment. However, genotypes cannot be observed directly; they must be inferred based on phenotypes. Because of dominance, it is often not possible to distinguish between a heterozygote and a homozgyote based on phenotype alone. To determine the genotype of a specific individual, a test cross can be performed, in which the individual with an uncertain genotype is crossed with an individual that is homozygous recessive for all of the loci being tested. For example, if you were given a pea plant with purple flowers it might be a homozygote ( AA ) or a heterozygote ( Aa ). You could cross this purple-flowered plant to a white-flowered plant, because you know the genotype of the plant is homozygous recessive aa . Depending on the genotype of the purple-flowered parent, you will observe different phenotypic ratios in the F 1 generation. If the purple-flowered parent was a homozygote, all of the F 1 progeny will be purple. If the purple-flowered parent was a heterozygote, the F 1 progeny should segregate purple-flowered and white-flowered plants in a 1:1 ratio. Query $\PageIndex{2}$ Contributors and Attributions - Dr. Todd Nickle and Isabelle Barrette-Ng (Mount Royal University) The content on this page is licensed under CC SA 3.0 licensing guidelines. - Stefanie Leacock, UA-Little Rock Molecular basis of dominant and recessive alleles What determines whether alleles are dominant or recessive? The alleles have different DNA sequences. Because the sequence of DNA contains information to make products, different sequences can lead to different products. One advantage of diploid species is that there are two copies of every sequence. If one sequence makes a "faulty" or non-functional product, it would be called a loss-of-function allele. However, there is likely another sequence that produces a "correct" or functional product. For most genes a single wild-type (usually the normal / functional) allele is capable of producing enough product for the cell, resulting in a dominant phenotype. However, if both copies of the gene are loss-of-function alleles, there will not be any functional protein and the recessive phenotype will be observed. An example from pea plants Mendel studied pea plants in which the peas could be round or wrinkled, with wrinkled being the recessive characteristic. The gene that determines this trait (originally represented as R) has since been identified and named SBE1 (for s tarch- b ranching e nzyme). The wild-type sequence encodes an enzyme that catalyzes a chemical reaction of carbohydrates to form branched chains of starch in plants. When SBE1 protein is present and functional, the peas produce branched starch and exhibit a round shape. When SBE1 protein is absent, the amount of branched starch is reduced, but the levels of disaccharides are higher, resulting in increased water absorption. Later, this water will be lost and the pea will become wrinkled. Molecular studies of DNA in round (RR) and wrinkled (rr) plants revealed an insertion of about 800 base pairs in an exon of the SBE1 gene in the rr plants (Bhattacharyya et al, 1990). The insertion is derived from a transposon and disrupts the SBE1 protein-coding sequence. Therefore, the RR plants have two copies of a functional SBE1 allele (SBE1+/+) to make functional SBE1 enzyme, while rr plants have two copies of the SBE1 gene in which the sequence is disrupted (SBE1-/-) and cannot produce any functional SBE1 enzyme. So what happens in a heterozygous Rr (SBE1+/-) plant? These plants have one chromosome with the SBE1+ allele and one chromosome with the SBE1- allele. The SBE1- allele is still transcribed, but does not code for the functional protein during translation. The SBE1+ allele is transcribed and translated into a functional enzyme, starch-branching occurs, and the peas have the round phenotype. Although the total amount of SBE1 enzyme is less than homozygous dominant peas, it is sufficient for the round phenotype and therefore dominant. In this case, molecular discoveries 125 years after Mendel's work reveal the reason for the dominance of the R allele over the r allele. References Bhattacharyya MK, Smith AM, Ellis TH, Hedley C, Martin C. The wrinkled-seed character of pea described by Mendel is caused by a transposon-like insertion in a gene encoding starch-branching enzyme. Cell. 1990 Jan 12;60(1):115-22. doi: 10.1016/0092-8674(90)90721-p. PMID: 2153053.	libretexts	2025-03-17T22:27:29.983785	2019-10-01T21:21:06	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/", "url": "https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/04%3A_Inheritance/4.02%3A__Mendelian_Genetics/4.2.01%3A_Monohybrid_Crosses_and_Segregation", "book_url": "https://commons.libretexts.org/book/bio-25718", "title": "4.2.1: Monohybrid Crosses and Segregation", "author": "Stefanie West Leacock" }
https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/04%3A_Inheritance/4.02%3A__Mendelian_Genetics/4.2.02%3A_Dihybrid_Crosses_and_Independent_Assortment	4.2.2: Dihybrid Crosses and Independent Assortment Mendel’s Second Law Before Mendel, it had not yet been established that heritable traits were controlled by discrete factors. Therefore an important question was whether distinct traits were controlled by separate factors that were inherited independently of one another. To answer this, Mendel took two apparently unrelated traits, such as seed shape and seed color, and studied their inheritance together in one individual. He studied two variants of each trait: seed color was either green or yellow, and seed shape was either round or wrinkled. When either of these traits was studied individually, the phenotypes segregated in the classical 3:1 ratio among the progeny of a monohybrid cross, with ¾ of the seeds green and ¼ yellow in one cross, and ¾ round and ¼ wrinkled in the other cross. Would this be true when both were in the same individual? To analyze the segregation of both traits at the same time in the same individual, Mendel crossed a pure breeding line of green, wrinkled peas with a pure breeding line of yellow, round peas to produce F 1 progeny that were all yellow and round, and which were also dihybrids ; they carried two alleles at each of two loci. If the alleles for the two genes for pea shape and pea color cannot be separated from each other, then in the F 2 generation, the offspring should be only green, round pea plants or yellow, wrinkled plants, like the P generation plants. If the genes controlling shape and color can be inherited independently, then what is the probability of phenotypes in the F 2 generation? Using the product rule, we can multiply the individual probabilities of obtaining a round phenotype (¾) with the probability of obtaining a yellow phenotype (¾), then ¾ × ¾ = 9/16 of the progeny would be both round and green. Likewise, ¾ × ¼ = 3/16 of the progeny would be both round and yellow, and so on. By applying the product rule to all combinations of phenotypes, we can predict a 9:3:3:1 phenotypic ratio among the progeny of a dihybrid cross, if certain conditions are met, including the independent segregation of the alleles at each locus. Definition: Mendel's Second Law The Law of Independent Assortment states that two loci assort independently of each other during gamete formation. \| Frequency of phenotypic crosses within separate monohybrid crosses: seed shape: ¾ round ¼ wrinkled seed color: ¾ yellow ¼ green Frequency of phenotypic crosses within a dihybrid cross: ¾ round × ¾ yellow = 9/16 round & yellow ¾ round × ¼ green = 3/16 round & green ¼ wrinkled × ¾ yellow = 3/16 wrinkled & yellow ¼ wrinkled × ¼ green = 1/16 wrinkled & green \| The 9:3:3:1 phenotypic ratio calculated using the product rule can also be obtained using Punnett Square. First, we list the genotypes of the possible gametes along each axis of the Punnett Square. In a diploid with two heterozygous genes of interest, there are up to four combinations of alleles in the gametes of each parent. The gametes from the respective rows and column are then combined in the each cell of the array. When working with two loci, genotypes are written with the symbols for both alleles of one locus, followed by both alleles of the next locus (e.g. AaBb , not ABab ). Note that the order in which the loci are written does not imply anything about the actual position of the loci on the chromosomes. To calculate the expected phenotypic ratios, we assign a phenotype to each of the 16 genotypes in the Punnett Square, based on our knowledge of the alleles and their dominance relationships. In the case of Mendel’s seeds, any genotype with at least one R allele and one Y allele will be round and yellow; these genotypes are shown in the nine, green-shaded cells. We can represent all of four of the different genotypes shown in these cells with the notation ( R_Y _), where the blank line (__), means “any allele”. The three offspring that have at least one R allele and are homozygous recessive for y (i.e. R_yy ) will have a round, green phenotype. Conversely the three progeny that are homozygous recessive r , but have at least one Y allele ( rrY_ ) will have wrinkled, yellow seeds. Finally, the rarest phenotypic class of wrinkled, yellow seeds is produced by the doubly homozygous recessive genotype, rryy , which is expected to occur in only one of the sixteen possible offspring represented in the square. Assumptions of the 9:3:3:1 ratio Both the product rule and the Punnett Square approaches showed that a 9:3:3:1 phenotypic ratio is expected among the progeny of a dihybrid cross such as Mendel’s RrYy × RrYy . In making these calculations, we assumed that: - both loci assort independently; - one allele at each locus is completely dominant; and - each of four possible phenotypes can be distinguished unambiguously, with no interactions between the two genes that would alter the phenotypes. Deviations from the 9:3:3:1 phenotypic ratio may indicate that one or more of the above conditions has not been met. Modified ratios in the progeny of a dihybrid cross can therefore reveal useful information about the genes involved. Query $\PageIndex{1}$ Applying the product rule Mendel's conclusions about the segregation of alleles and independent of assortment of genes continue to hold true for inheritance in diploid organisms, in which meiosis produces gametes that have one copy of thousands genes - not just one or two. However, making a Punnett Square for more than two genes becomes tricky and cumbersome. The Product Rule can be used to predict outcomes when considering more than two genes at a time. Find the probability of each genotype or phenotype and multiple each probability. Exercise $\PageIndex{1}$ If two organisms with genotype GgHhJi are crossed, what percent of offspring are expected to be homozygous dominant for all three genes? Hint: GG and HH and JJ - Answer - (Probability GG: 1/4) x (Probability HH: 1/4) x (Probability JJ: 1/4) = 1/64 Contributors and Attributions - Dr. Todd Nickle and Isabelle Barrette-Ng (Mount Royal University) The content on this page is licensed under CC SA 3.0 licensing guidelines. - Stefanie Leacock, UA-Little Rock	libretexts	2025-03-17T22:27:30.056502	2019-10-01T21:21:06	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/", "url": "https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/04%3A_Inheritance/4.02%3A__Mendelian_Genetics/4.2.02%3A_Dihybrid_Crosses_and_Independent_Assortment", "book_url": "https://commons.libretexts.org/book/bio-25718", "title": "4.2.2: Dihybrid Crosses and Independent Assortment", "author": "Stefanie West Leacock" }
https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/04%3A_Inheritance/4.04%3A_Exceptions_to_autosomal_inheritance	4.4: Exceptions to autosomal inheritance Learning Objectives - Compare different modes of sex determination, such as mammalian XY, fruit flies, insects, or environmental. - Explain why dosage compensation is needed and the role of the non-coding RNA Xist in mammalian dosage compensation. - Predict the number of Barr bodies in a particular cell. - Predict offspring phenotypes for X-linked genes. For loci on autosomes, in which all individuals have two copies of the chromosome and either copy is equally likely to be passed to offspring, the alleles follow the normal Mendelian pattern of inheritance. However, for loci on the allosomes (also called sex chromosomes) individuals may have zero, one, or two copies of a chromosome. Because homologous chromosomes contain the same genes, individuals may have zero, one, or two copies of some genes. In humans, the allosomes are the X and Y chromosomes, which contain different genes, even though they act as a homologous pair during meiosis. Instead, genes on the X chromosome will follow an X-linked pattern of inheritance, while genes on the Y chromosome will follow a Y-linked pattern of inheritance. X-Linked inheritance example: the white gene in Drosophila melanogaster A well-studied sex-linked gene is the white gene on the X chromosome of Drosophila melanogaster . Normally flies have red eyes but flies with a mutant allele of this gene called white - ( w - ) have white eyes because the red pigments are absent. Because this mutation is recessive to the wild type w + allele females that are heterozygous have normal red eyes. Female flies that are homozygous for the mutant allele have white eyes. Because there is no white gene on the Y chromosome, male flies can only be hemizygous for the wild type allele or the mutant allele. Note about nomenclature In genetics, genes are often named after their mutant (frequently, but not always, loss-of-function) phenotypes. Remember, genetics has its origin in observing phenotypes. The white allele described here was identified in 1910 (Morgan, T.H.) long before gene sequences could be determined, in fact, even before DNA had been identified as the unit of heredity. The DNA sequence of this locus was not published until 1984 (O'Hare et al). We know now that this gene encodes a transporter protein that deposits pigment and that the mutant allele does not produce a functional transporter, but the gene name white continues to be used. The process of identifying mutant phenotypes and then later determining which gene is involved is called forward genetics . The alternative, to identify a candidate gene and manipulate it to observe the phenotype, is called reverse genetics , and was only made possible by the advent of modern molecular biology tools. A researcher may not know beforehand whether a novel mutation is sex-linked. The definitive method to test for sex-linkage is reciprocal crosses , in which a male and a female that have different phenotypes are crossed. In a second cross, the phenotypes are reversed relative to the sex of the parents in the first cross. Whenever reciprocal crosses give different results in the F 1 and F 2 , and whenever the male and female offspring have different phenotypes, the usual explanation is sex-linkage. Remember, if the locus were autosomal, the F 1 and F 2 progeny are expected to show the same genotypes and phenotypes from either of these crosses. A similar pattern of sex-linked inheritance is seen for X-chromosome loci in other species with an XX-XY sex chromosome system, including mammals and humans. The ZZ-ZW system is similar, but reversed (see below). Diversity in Sex Determination Sex determination refers to the process by which reproductive organs are specified to become ovaries or testes. Mechanisms for sex determination in plants and animals are remarkably diverse, and include sex chromosomes, chromosome dosage, and environment among others. Some organisms used chromosomal sex determination. For example in humans and other mammals XY embryos commonly develop testes while XX embryos develop ovaries. This difference in development is usually initiated by the presence of a single gene, the SRY gene ( s ex-determining r egion on Y ), on the Y-chromosome. SRY encodes is a transcription factor that activates expression of genes that promote testes differentiation. Without SRY, these genes are not activated and genes that promote ovary development are transcribed. Notably, SRY initiates the process of testes development, but a series of genes are required to complete testes and reproductive organ development. If the downstream genes are not fully functional, development of these structures may not occur despite the presence of the Y chromosome and SRY. Query $\PageIndex{1}$ Although Drosophila melanogaster also has a Y chromosome, there is not an SRY gene and the X to Autosome (X:A) ratio determines male or female differentiation. Individuals with two autosome sets and two X chromosomes (2A:2X) will develop ovaries, while those with only one X chromosome (2A:1X) will develop testes. The presence/absence of the Y-chromosome and its genes are not significant. For example, both an XO and XY fruit fly will develop testes and other male phenotypes, if they both have two sets of autosomes. In other animal species, the number of chromosome sets can determine sex. For example the haploid-diploid system is used in bees, ants, and wasps. Typically haploid individuals, which develop from unfertilized eggs, develop testes, while diploid individuals, which develop from fertilized eggs, form ovaries. For this system to work, meiosis does not occur during sperm production in haploids; instead sperm are identical copies produced by mitosis because chromosome number does not need to be reduced by half. In other species, the environment can determine an individuals sex. In alligators (and some other reptiles) the temperature of development dictates the sex, while in many reef fish, the population sex ratio can produce environmental signals that cause some individuals to change sex. Dosage Compensation Mammals and Drosophila both have XX - XY sex determination systems. However, because these systems evolved independently they work differently with regard to compensating for the difference in gene dosage. Remember, in most cases the sex chromosomes act as a homologous pair even though the Y chromosome has lost most of the loci when compared to the X chromosome. Comparison of X and Y chromosomes had led to the view that Y chromosomes have evolved from autosomes that degenerated, slowly mutating and losing loci. In modern day mammals, the Y chromosomes have very few genes, while the X chromosomes remain as they were. This is a general feature of all organisms that use chromosome based sex determination systems. Chromosomes found in both sexes (the X or the Z) have retained their genes while the chromosome found in only one sex (the Y or the W) have lost most of their genes. Human X and Y Compared \| X chromosome \| Y chromosome \| \| \| Number of base pairs \| 156,040,895 \| 57,227,415 \| \| Number of coding genes \| 857 \| 64 \| \| Contains essential genes for viability? \| Yes \| No \| Ensembl 2021. (Release 104) Nucleic Acids Res. 2021, vol. 49(1):884–891 PubMed PMID: 33137190. doi:10.1093/nar/gkaa942 Chromosomal sex determination requires dealing with the gene dosage difference between individuals with different chromosome complements: e.g. XX females have two copies ("doses") of X-chromosome genes while XY males only have one copy of these genes. For all the autosomes, XX and XY individuals have the same number of copies of genes. Producing too much of some proteins can be problematic, even lethal, for organisms. This difference in gene dosage needs to be compensated in a process called dosage compensation . In Drosophila and many other insects, individuals with single X chromosome express the genes on it at twice the normal rate. This mechanism of dosage compensation restores a balance of proteins encoded by X-linked genes and those made by autosomal genes. X-chromosome Inactivation in Mammals In mammals the dosage compensation system operates in any cell with more than one X chromosome. In XX embryos, one X in each cell is randomly chosen and marked for inactivation by expressing a long non-coding RNA Xist ( X i nactive s pecific t ranscript). The Xist RNA stays in the nucleus and "coats" the X chromosome, recruiting proteins that affect chromosome packing. This X chromosome becomes highly compact heterochromatin. The expression of Xist is repressed on the other chromosome, so it remains more loosely packed euchromatin. The X i is replicated during S phase and transmitted during mitosis the same as any other chromosome, but most of its genes are never allowed to turn on and the chemical modifications that regulate chromatin are passed on following each round of cell division. Make a prediction What would happen to XY or XX mouse embryos that lack the Xist gene? - Answer - XY mouse embryos with a deletion of the Xist gene were viable and fully fertile. The XX mouse embryos died during embryonic development. (Marahrens Y, Panning B, Dausman J, Strauss W, Jaenisch R. Xist-deficient mice are defective in dosage compensation but not spermatogenesis. Genes Dev. 1997 Jan 15;11(2):156-66. doi: 10.1101/gad.11.2.156. PMID: 9009199.) From this point forward this chromosome will be inactive, abbreviated X inactive (X i ). The chromosome appears as a condensed mass within interphase nuclei called the Barr body . With the inactivation of genes on one X chromosome, XX individuals have the same number of functioning X-linked genes as XY individuals. The other X chromosome, the X active (X a ), is unaffected. The mechanisms that determine X inactivation can also be used in other situations, such as XXX, in which cells inactivate two X chromosomes, or XXY individuals, in which cells inactivate one X chromosome. This remarkable system always leaves one X chromosome active, such that every mammalian cell expresses the correct dose of X chromosome genes. Counting chromosomes The exact mechanism cells use to "count" their X chromosomes is not entirely clear. Essentially all mammalian cells leave only one X chromosome active, even when abnormal numbers of X chromosomes are present. Examples: - Females with an extra X (XXX genotype) have 2 Barr bodies and 1 active X. - Males with an extra X (XXY) have 1 Barr body and 1 active X. This random inactivation of one X-chromosome leads to a commonly observe phenomenon in cats. A familiar X-linked gene is the Orange gene ( O ) in cats. The O O allele encodes an enzyme that results in orange pigment for the hair. The O B allele causes the hairs to be black. Heterozygous females have an orange and black mottled phenotype known as tortoiseshell. This is due to patches of skin cells having different X-chromosomes inactivated. In each orange hair the X i chromosome carrying the O B allele is inactivated. The O O allele on the X a is functional and orange pigments are made. In black hairs the reverse is true, the X i chromosome with the O O allele is inactive and the X a chromosome with the O B allele is active. Because the inactivation decision happens early during embryogenesis, the cells continue to divide to make large patches on the adult cat skin where one or the other X is inactivated. The Orange gene in cats is a good demonstration of how the mammalian dosage compensation system affects gene expression. However, most X-linked genes do not produce such dramatic mosaic phenotypes in heterozygous females. Another example is the F8 gene in humans that encodes Factor VIII, a protein needed for blood clotting that is produced in liver cells. If an XY individual is hemizygous for a mutant allele ( F8 – /F8 – ) the result is hemophilia type A. An XX individual homozygous for mutant alleles will also have hemophilia. Heterozygous F8 + / F8 – individuals do not have hemophilia because even though half of their liver cells do not make Factor VIII (because the X with the F8 + allele is inactive) the other 50% can. Because some of their liver cells are exporting Factor VIII proteins into the blood stream, they have the ability to form blood clots throughout their bodies. The genetic mosaicism in the cells of their bodies does not produce a visible mosaic phenotype. Example $\PageIndex{1}$ Predicting genetic crosses with X-linked genes follows that of autosomal genes, remembering that each gamete contains only one sex chromosome, unless otherwise specified. A tortoiseshell female cat and a black male cat have 8 offspring. How many are expected to be tortoiseshell? Solution Using a Punnett square reveals that 25% (or two) offspring are predicted to be tortoiseshell. Exercise $\PageIndex{1}$ If you were able to clone a tortoiseshell cat, would the pattern of orange and black markings be identical in the clone? Why or why not? - Answer - The pattern is not likely to be identical because X chromosome inactivation happens during embryogenesis. When the cloned cat is in the early stages of development, the X chromosomes with the orange and black alleles will be randomly inactivated and passed on during each mitosis as more cells are formed. Exercise $\PageIndex{2}$ A heterozygous individual (genotype F8 + / F8 — ) is a carrier for the hemophilia allele. Her partner is an XY male with normal blood clotting. What is the probability their son will have hemophilia? What about a daughter? - Answer - A son will be either F8 + Y or F8 — Y (50% with hemophilia). A daughter will be either F8 + / F8 — or F8 + / F8 + (0% with hemophilia). Z-linked genes The ZW system is distinguished from the XY system based on which sex has two different sex chromosomes, also called the heterogametic sex. In this case, male birds commonly have ZZ chromosomes, whereas female birds commonly have ZW chromosomes. Thus, female birds may have only one copy of Z-linked genes. For example, a Z-linked gene influences feather color in turkeys. Turkeys are birds, which use the ZZ-ZW sex chromosome system. The E allele makes the feathers bronze and the e allele makes the feathers brown (Figure $\PageIndex{5}$). Only turkeys with two Z chromosomes, commonly males, can be heterozygous for this locus. Heterozygous turkeys ( Ee ) are uniformly bronze because the E allele is completely dominant to the e allele and birds use a dosage compensation system similar to Drosophila and not mammals. Reciprocal crosses between turkeys from pure-breeding bronze and brown breeds would reveal that this gene is in fact Z-linked. Contributors and Attributions - Dr. Todd Nickle and Isabelle Barrette-Ng (Mount Royal University) The content on this page is licensed under CC SA 3.0 licensing guidelines. References Bachtrog D, Mank JE, Peichel CL, Kirkpatrick M, Otto SP, Ashman T-L, et al. (2014) Sex Determination: Why So Many Ways of Doing It? PLoS Biol 12(7): e1001899. https://doi.org/10.1371/journal.pbio.1001899	libretexts	2025-03-17T22:27:30.257976	2019-10-01T21:21:08	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/", "url": "https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/04%3A_Inheritance/4.04%3A_Exceptions_to_autosomal_inheritance", "book_url": "https://commons.libretexts.org/book/bio-25718", "title": "4.4: Exceptions to autosomal inheritance", "author": "Stefanie West Leacock" }
https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/04%3A_Inheritance/4.04%3A_Exceptions_to_autosomal_inheritance/4.4.02%3A_Organellar_Inheritance	4.4.2: Organellar Inheritance Learning Objectives - Explain why genetic information in organelles is passed independently of nuclear DNA. In eukaryotes, DNA and genes also exist outside of the nuclear chromosomes. Both the chloroplast and mitochondrion have circular chromosomes (Figure $\PageIndex{1}$). These organellar genomes are often present in multiple copies within each organelle. In most sexually-reproducing species, organellar chromosomes are inherited from only one parent, usually the one that produces the largest gamete. Thus, in mammals, angiosperms, and many other organisms, mitochondria and chloroplasts are inherited only through the oocyte. These organelles are likely the remnants of prokaryotic endosymbionts that entered the cytoplasm of ancient progenitors of today’s eukaryotes ( endosymbiont theory ). These endosymbionts had their own, circular chromosomes, like most bacteria that exist today. Chloroplasts and mitochondria typically have circular chromosomes that behave more like bacterial chromosomes than eukaryotic chromosomes, i.e. these organellar genomes do not undergo mitosis or meiosis. Implications of mitochondrial inheritance As with nuclear DNA, organellar DNA can be mutated. Cells can have a mixture of hundreds to thousands of organelles with different alleles for genes. Because there are not simply one or two organelles in a cell, terms like heterozygous and homozygous do not apply to this situation and patterns of inheritance can be unpredictable. Some patterns of inheritance that are usually observed for mitochondrial inheritance are: - Traits can be passed via egg to offspring - Traits are not passed via sperm to offspring - Variable penetrance and expressivity are often observed are often due to different proportions of wild type and mutant organelles in the organism of even differing proportions between different tissues in the same organism. Mitochondrial DNA polymorphisms are also used to investigate evolutionary lineages, both ancient and recent. Current Research in Plant Genetics: Although organelles are most often inherited through oocytes, exceptions have been identified. Recent studies of cucumber plants ( Cucumis sativus var. sativus) identified SNPs in true-breeding lines and performed reciprocal crosses. The results showed that the chloroplasts were inherited from the maternal parent but that mitochondria are inherited from the male parent. Reference: Park, HS., Lee, W.K., Lee, SC. et al. Inheritance of chloroplast and mitochondrial genomes in cucumber revealed by four reciprocal F 1 hybrid combinations. Sci Rep 11, 2506 (2021). https://doi.org/10.1038/s41598-021-81988-w Contributors and Attributions - Dr. Todd Nickle and Isabelle Barrette-Ng (Mount Royal University) The content on this page is licensed under CC SA 3.0 licensing guidelines.	libretexts	2025-03-17T22:27:30.349138	2019-10-01T21:21:01	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/", "url": "https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/04%3A_Inheritance/4.04%3A_Exceptions_to_autosomal_inheritance/4.4.02%3A_Organellar_Inheritance", "book_url": "https://commons.libretexts.org/book/bio-25718", "title": "4.4.2: Organellar Inheritance", "author": "Stefanie West Leacock" }
https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/04%3A_Inheritance/4.05%3A__Linkage	4.5: Linkage Mendel reported that the pairs of loci he observed behaved independently of each other; for example, the segregation of seed color alleles was independent from the segregation of alleles for seed shape. This observation was the basis for his Second Law (Independent Assortment), and contributed greatly to our understanding of heredity. However, further research showed that Mendel’s Second Law did not apply to every pair of genes that could be studied. In fact, we now know that alleles of loci that are located close together on the same chromosome tend to be inherited together. This phenomenon is called linkage , and is a major exception to Mendel’s Law of Independent Assortment. Researchers use linkage to determine the location of genes along chromosomes in a process called genetic mapping. The concept of gene linkage is important to the natural processes of heredity and evolution and current efforts to find loci that contribute to complex traits. Contributors and Attributions - Dr. Todd Nickle and Isabelle Barrette-Ng (Mount Royal University) The content on this page is licensed under CC SA 3.0 licensing guidelines.	libretexts	2025-03-17T22:27:30.403007	2019-10-01T21:21:07	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/", "url": "https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/04%3A_Inheritance/4.05%3A__Linkage", "book_url": "https://commons.libretexts.org/book/bio-25718", "title": "4.5: Linkage", "author": "Stefanie West Leacock" }
https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/04%3A_Inheritance/4.06%3A__Exceptions_to_simple_dominance	4.6: Exceptions to simple dominance Learning Objectives - Categorize interactions between alleles of single genes as completely dominant, incompletely dominant, or codominant and explain the biochemical basis of these relationships. Predict phenotypes of offspring using each mode of inheritance. - Understand that multiple alleles (not just two) may exist for a gene. - Recognize examples of incomplete penetrance, variable expressivity, and pleiotropy . - Predict ratios of offspring when certain genotypes are lethal alleles . Incomplete Dominance During Mendel’s time, people believed in a concept of blending inheritance whereby offspring demonstrated intermediate phenotypes between those of the parental generation. This was refuted by Mendel’s pea experiments that illustrated a Law of Dominance. Despite this, non-Mendelian inheritance can be observed in sex-linkage and co-dominance where the expected ratios of phenotypes are not observed clearly. Incomplete dominance superficially resembles the idea of blending inheritance, but can still be explained using Mendel’s laws with modification. In this case, alleles do not exert full dominance and the offspring resemble a mixture of the two phenotypes. The most obvious case of a two allele system that exhibits incomplete dominance is in the snapdragon flower. The alleles that give rise to flower coloration (Red or White) both express and the heterozygous genotype yields pink flowers. There are different ways to denote this. In this case, the superscripts of R or W refer to the red or white alleles, respectively. Since no clear dominance is in effect, using a shared letter to denote the common trait with the superscripts (or subscripts) permit for a clearer denotation of the ultimate genotype to phenotype translations. Exercise $\PageIndex{1}$ If pink flowers arose from blending inheritance, then subsequent crosses of pink flowers with either parental strain would continue to change the phenotype. Using a Punnett Square, perform a cross between a heterozygous plant and a parental to predict the phenotypes of the offspring. - Answer - A cross between a heterozygote F R F W and a red parental F R F R would produce 1/2 red-flowered and 1/2 pink-flowered offspring. F R F R F R F R F R (red) F R F R (red) F W F R F W (pink) F R F W (pink) Thinking about the biochemical basis of incomplete dominance Refer back to the biochemical basis of dominant and recessive alleles. Why do many loss-of-function alleles act as recessive? What must be different about incompletely dominant alleles? How would you test your idea? The alleles that determine curly or straight hair in dogs provide another example of incomplete dominance. Using GWAS, the KRT71 gene, which encodes a keratin protein, was identified as the locus that determines this phenotype (Cadieu et al, 2010). Dogs with C alleles for a SNP in the KRT71 gene exhibit straight hair, while dogs with curly hair have two T alleles for the SNP that alter an amino acid, which is a missense mutation. When curly and straight haired dogs are bred, such as the cross of poodles and Labrador Retrivers that create Labradoodles, the offspring are heterozygous for the SNP (C/T) and have an intermediately wavy hair. Query $\PageIndex{1}$ Codominance and Multiple Alleles Codominance occurs when phenotypes associated with two dominant alleles occur simultaneously. The prototypical case for this is the human ABO blood grouping. The ABO gene (also known as the I locus) on human chromosome 9 encodes a glycosyltransferase enzyme that attaches a carbohydrate molecule to proteins present on the surface of red blood cells. The I A and I B alleles have several DNA sequence differences that produce amino acid changes. As a result, the I A allele produces an enzyme that adds an acetylated galactosamine (GalNac) to a surface protein. The I B allele produces an enzyme that adds galactose (Gal) instead. These alleles are codominant to each other because each the enzymes catalyze reactions that occur independently of the other enzymes that are present. On the surface of red blood cells in heterozygous individuals ( I A I B ) , a mixture of proteins with GalNAc and Gal will be present. A third allele of the gene, designated i , also exists but contains a single nucleotide deletion that causes a frameshift, completing disrupting the protein (Yamamoto et al, 1990). The protein produced by the i allele does not catalyze the attachment of any sugar. This allele is recessive to I A and I B because the protein has no activity and does not influence the activity of the proteins encoded by the dominant alleles. If an I A or I B allele is also present, those enzymes will still function, so the A or B antigens will be present, producing the dominant phenotype. Query $\PageIndex{2}$ Query $\PageIndex{3}$ Importance of ABO Blood Types The alleles of the ABO gene determine features that are present on the surface of blood cells that are used when cells identify foreign cells. An individual with blood type A has proteins with the GalNac modification, but does not have proteins with the Gal modification produced by the I B allele. The individual will produce antibodies that recognize the Gal modification that is present in Type B blood. Therefore, if Type B blood should not be introduced into a Type A individual. Blood type influences blood donors and recipients The table shows how blood type influences blood donations. If an individual receives a blood type that they make antibodies against, the blood will be recognized as foreign and the immune system will attack the donated cells. Because individuals will Type O blood have cells without any sugar modifications, their blood can be donated to any other blood type; they are sometimes called universal donors. In contrast, individuals with Type AB blood recognize either the GalNac or Gal modification and can receive any type of blood; they are sometimes called universal recipient. \| Blood type \| Antibodies against: \| Can donate blood to: \| Can receive blood from: \| \| A \| B \| A or AB \| A or O \| \| B \| A \| B or AB \| B or O \| \| AB \| none \| AB \| A, B, AB, or O \| \| O \| A and B \| A, B, AB, or O \| O \| There are additional genes that influence human blood type, for example, blood type might also be described as O positive or AB negative. Whether blood type is positive or negative is determined by a second locus, the RHD gene, which encodes a surface protein called the Rh Blood Group D Antigen. As Mendelian inheritance predicts, the two genes, ABO on chromosome 9 and RHD on chromosome 1, are inherited independently during meiosis. Penetrance and Expressivity The terms penetrance and expressivity are also useful to describe the relationship between certain genotypes and their phenotypes. - Penetrance is the proportion of individuals (usually expressed as a percentage) with a particular genotype that display a corresponding phenotype. For example, all pea plants that are homozygous for the allele for white flowers actually have white flowers, this genotype is completely penetrant. In contrast, many human genetic diseases are incompletely penetrant, because not all individuals with the disease genotype actually develop symptoms associated with the disease. - Expressivity describes the variability in mutant phenotypes observed in individuals with a particular phenotype. Many human genetic diseases provide examples of broad expressivity, because individuals with the same genotypes may vary greatly in the severity of their symptoms. Incomplete penetrance and broad expressivity are due to random chance, non-genetic (environmental), and genetic factors (mutations in other genes), although often it remains unknown which of these cause(s) are most responsible for individual phenotypes. Example of variable penetrance and expressivity from nematodes Mutations in the gene meg-1 in C. elegans provide an example of variable penetrance as well as broad expressivity. This gene encodes a protein (MEG-1) that functions to establish the reproductive system in cells during early embryonic development. Mutant worms that lack meg-1 therefore produce sterile offspring. However, the sterile phenotype is not fully penetrant, approximately 20-25% of mutant offspring will be fertile, despite the lack of MEG-1. These sterile adults also exhibit variable expressivity; some sterile adults have severely-reduced germ-line development with few identifiable germ cells, whereas other sterile adults have an identifiable germ line but lack gametes. Data summarized from Leacock and Reinke, 2008 Pleiotropy While some genes are associated with a single trait, many genes encode proteins that function in multiple ways or are the basis for other traits. Pleiotropy occurs when a single genotype affects multiple phenotypes. For example, the human gene CFTR is associated with the disease cystic fibrosis. However, variations in this gene are also associated with other phenotypes, such as congenital bilateral absence of the vas deferens (a feature that causes male infertility) and pancreatitis. Pleiotropy is often observed when a gene is expressed in more than one tissue or organ. The CFTR protein plays a role in ion balance in the lungs, where lack of the protein is associated with the thick lung mucus characteristic of cystic fibrosis. However, CFTR is expressed and functions in ion balance in other tissues as well. Experiments that assay transcripts in multiple tissues, show that the CFTR mRNA is present in a number of tissues, including pancreas and testis. This expression pattern is consistent with some of the observed phenotypes that occur when CFTR is mutated. Lethal Alleles Mutations that cause loss-of-function of a protein that is essential for organismal viability could result in lethality; often these alleles are recessive because the second copy of the gene will produce sufficient protein for survival. When two heterozygotes are crossed, 25% of offspring are expected to be homozygous for the lethal allele. Thus, the ratio of homozygous wild type to heterozygotes among the surviving offspring will be 2:1 and the offspring may all have the same phenotype, depending on the relationship between the two alleles. For some alleles, the heterozygote may have a different phenotype than the homozygote, in which case the phenotypic ratios of the progeny may differ from the expected ratios. The agouti yellow (A y ) allele in mice is one example of an allele that produces a dominant yellow phenotype in heterozygotes, as well as pleiotropic effects including obesity and increased tumor incidence. Mammals typically produce two types of pigments, pheomelanin (a yellow pigment) and eumelanin (a black-brown pigment). The protein encoded by agouti is a signaling protein that suppresses a receptor controlling eumelanin production. When agouti protein is present transiently during hair production, individual hairs have eumelanin at the base and tip with a central band of pheomelanin, giving an overall brown fur appearance. In A/A y heterozygotes agouti is constantly expressed, suppressing eumelanin completely and the phenotype is yellow. The pleiotropic effects observed are likely due to interactions of the Agouti signaling protein with similar receptors that control weight and feeding behavior. The A y allele is lethal when homozygous due to the nature of the mutation. The A y allele is a large (170kb) deletion that removes a part of an upstream gene Raly, which stands for R NA-binding protein a ssociated with the l ethal y ellow mutation , and the a gouti promoter (Michaud et al 1993; Duhl et al 1994). Now the Raly promoter controls the transcription of agouti, such that it is overproduced, suppressing melanin production and leading to yellow fur. Due to the size and position of the deletion, the coding sequences of the Raly protein are absent in the A y allele. In heterozygous embryos a functional copy of Raly remains, but Raly protein, which is normally expressed in early mouse embryos (preimplantation), is absent in homozygous A y / A y embryos. When translation of the Raly mRNA was blocked in wild-type mouse embryos, only 4% of embryos developed to the blastocyst stage, consistent with the deletion of Raly , and not the effect of agouti , as the cause of the embryonic lethality (Duhl et al. 1994). Example $\PageIndex{1}$ The mouse gene Agouti (A) influences the pattern of pigment in mouse fur. The mutant allele A y produces fur that is entirely yellow, instead of the wild-type pattern of black hairs with a central band of yellow pigment. However, A y A y homozygous embryos die in early stages of development. You cross two yellow mice. What ratio of offspring genotypes and phenotypes do you expect? Solution The yellow mice must have the heterozygous genotype AA y . The fertilized embryos will have the genotype ratio 1 A y A y : 2 AA y : 1 AA. However the A y A y embryos will not survive. The ratio of the surviving offspring will be 2 AA y yellow : 1 AA agouti. References: Cadieu E, Neff MW, Quignon P, Walsh K, Chase K, Parker HG, Vonholdt BM, Rhue A, Boyko A, Byers A, Wong A, Mosher DS, Elkahloun AG, Spady TC, André C, Lark KG, Cargill M, Bustamante CD, Wayne RK, Ostrander EA. Coat variation in the domestic dog is governed by variants in three genes. Science. 2009 Oct 2;326(5949):150-3. doi: 10.1126/science.1177808. Epub 2009 Aug 27. PMID: 19713490; PMCID: PMC2897713. Duhl DM, Stevens ME, Vrieling H, Saxon PJ, Miller MW, Epstein CJ, Barsh GS. Pleiotropic effects of the mouse lethal yellow (Ay) mutation explained by deletion of a maternally expressed gene and the simultaneous production of agouti fusion RNAs. Development. 1994 Jun;120(6):1695-708. PMID: 8050375. Howe K, et al Ensembl 2021. Nucleic Acids Res. 2021, vol. 49(1):884–891. PubMed PMID: 33137190. Leacock SW, Reinke V. MEG-1 and MEG-2 are embryo-specific P-granule components required for germline development in Caenorhabditis elegans. Genetics. 2008 Jan;178(1):295-306. doi: 10.1534/genetics.107.080218. PMID: 18202375; PMCID: PMC2206079. ( https://doi.org/10.1534/genetics.107.080218 ) Lin S, Lin Y, Nery JR, Urich MA, Breschi A, Davis CA, Dobin A, Zaleski C, Beer MA, Chapman WC, Gingeras TR, Ecker JR, Snyder MP. Comparison of the transcriptional landscapes between human and mouse tissues. Proc Natl Acad Sci U S A. 2014 Dec 2;111(48):17224-9. doi: 10.1073/pnas.1413624111. Epub 2014 Nov 20. PMID: 25413365; PMCID: PMC4260565. Michaud EJ, Bultman SJ, Klebig ML, et al. A molecular model for the genetic and phenotypic characteristics of the mouse lethal yellow (Ay) mutation. Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2562-6. doi: 10.1073/pnas.91.7.2562. PMID: 8146154; PMCID: PMC43409. ( https://www.pnas.org/content/91/7/2562.long ) Papatheodorou I, Moreno P, Manning J, et al. Expression Atlas update: from tissues to single cells. Nucleic Acids Res. 2020 Jan 8;48(D1):D77-D83. doi: 10.1093/nar/gkz947. PMID: 31665515; PMCID: PMC7145605. Yamamoto et al. Molecular genetic basis of the histo-blood group ABO system. Nature 345, 229–233 (1990). https://doi.org/10.1038/345229a0 Contributors and Attributions - Dr. Todd Nickle and Isabelle Barrette-Ng (Mount Royal University) The content on this page is licensed under CC SA 3.0 licensing guidelines.	libretexts	2025-03-17T22:27:30.564214	2019-10-01T21:21:08	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/", "url": "https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/04%3A_Inheritance/4.06%3A__Exceptions_to_simple_dominance", "book_url": "https://commons.libretexts.org/book/bio-25718", "title": "4.6: Exceptions to simple dominance", "author": "Stefanie West Leacock" }
https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/04%3A_Inheritance/4.07%3A_Gene_Interactions	4.7: Gene Interactions Learning Objectives - Describe the results of crosses involving two genes that affect the same trait as exhibiting complementation, epistasis, or redundancy . - Predict phenotypes of offspring of crosses involving two genes, given information about biohemical pathways or functions of the genes. Some dihybrid crosses produce a phenotypic ratio that differs from 9:3:3:1, such as 9:3:4, 12:3:1, 9:7, or 15:1. Note that each of these modified ratios can be obtained by summing one or more of the 9:3:3:1 classes expected from our original dihybrid cross. In the following sections, we will look at some modified phenotypic ratios obtained from dihybrid crosses and what they might tell us about interactions between genes. Recessive epistasis Epistasis (which means “standing upon”) occurs when the phenotype of one locus masks, or prevents, the phenotype of another locus. Thus, following a dihybrid cross fewer than the typical four phenotypic classes will be observed with epistasis. As we have already discussed, in the absence of epistasis, there are four phenotypic classes among the progeny of a dihybrid cross. The four phenotypic classes correspond to the genotypes: A_B_, A_bb, aaB_, and aabb . If either of the singly homozygous recessive genotypes (i.e. A_bb or aaB_ ) has the same phenotype as the double homozygous recessive ( aabb ), then a 9:3:4 phenotypic ratio will be obtained. For example, in the Labrador Retriever breed of dogs, the B locus encodes a gene for an important step in the production of melanin. The dominant allele, B is more efficient at pigment production than the recessive b allele, thus B _ hair appears black, and bb hair appears brown. A second locus, which we will call E , controls the deposition of melanin in the hairs. At least one functional E allele is required to deposit any pigment, whether it is black or brown. Thus, all retrievers that are ee fail to deposit any melanin (and so appear pale yellow), regardless of the genotype at the B locus. The ee genotype is therefore said to be epistatic to both the B and b alleles, since the homozygous ee phenotype masks the phenotype of the B locus. The B/b locus is said to be hypostatic to the ee genotype. Because the masking allele is in this case is recessive, this is called recessive epistasis . Biochemical Basis for the genetic interaction between E and B in dog coat color Why is this genetic interaction observed? The B locus encodes the protein TYRP1 (Tyrosinase-Related Protein 1). The enzyme catalyzes a step in the production of melanin within melanocytes. Predicted loss-of-function alleles of TYRP1 were identified in brown dogs, for example a premature stop codon (Schmutz et al. TYRP1 and MC1R genotypes and their effects on coat color in dogs. Mamm Genome . 2002;13(7):380-387). Dogs homozygous or heterozygous for functional TYRP1 produce sufficient melanin to have a black coat. The E locus encodes the protein MC1R (melanocortin receptor 1), which is a membrane bound receptor for melanocyte-stimulating hormone (MSH) on the surface of the melanocytes. Activation of this receptor is transduced into a transcriptional response to produce proteins needed for eumelanin production. Recessive loss-of-function alleles of MC1R result in reduced or absent receptor activity and therefore loss of the intracellular response of proteins proteins needed for eumelanin production. When MC1R is not activated, cells can still produce the yellow pheomelanin, resulting in a yellow coat. In the Punnett square above, any genotype that includes a homozygous recessive for MC1R ( ee ) results in a yellow coat, even when the dog has a dominant allele B that is predicted to produce black fur. Without a functional MC1R receptor, the downstream events needed to produce TYRP1 will not be activated, so the ee genotype "masks" the production of brown of black fur. Dominant epistasis In some cases, a dominant allele at one locus may mask the phenotype of a second locus. This is called dominant epistasis , which produces a segregation ratio such as 12:3:1 , which can be viewed as a modification of the 9:3:3:1 ratio in which the A_B_ class is combined with one of the other genotypic classes that contains a dominant allele. One of the best known examples of a 12:3:1 segregation ratio is fruit color in some types of squash. Alleles of a locus that we will call B produce either yellow ( B _) or green ( bb ) fruit. However, in the presence of a dominant allele at a second locus that we call A , no pigment is produced at all, and fruit are white. The dominant A allele is therefore epistatic to both B and bb combinations. One possible biological interpretation of this segregation pattern is that the function of the A allele somehow blocks an early stage of pigment synthesis, before neither yellow or green pigments are produced. Figure $\PageIndex{4}$: Genotypes and phenotypes among the progeny of a dihybrid cross of squash plants heterozygous for two loci affecting fruit color. (Original-Deyholos-CC:AN) Redundant genes When a dihybrid cross produces progeny in two phenotypic classes in a 15:1 ratio, this can be because the two loci’s gene products have the same ( redundant ) functions within the same biological pathway. Yet another pigmentation pathway, in this case in wheat, provides an example of gene redundancy, sometimes known as duplicate gene action. The biosynthesis of red pigment near the surface of wheat seeds involves many genes, two of which we will label A and B . Normal, red coloration of the wheat seeds is maintained if function of either of these genes is lost in a homozygous mutant (e.g. in either aaB_ or A_bb ). Only the doubly recessive mutant ( aabb ), which lacks function of both genes, shows a phenotype that differs from that produced by any of the other genotypes. A reasonable interpretation of this result is that both genes encode the same biological function, and either one alone is sufficient for the normal activity of that pathway. Complementary gene action The progeny of a dihybrid cross may produce just two phenotypic classes, in an approximately 9:7 ratio. An interpretation of this ratio is that the loss of function of either A or B gene function has the same phenotype as the loss of function of both genes, due to complementary gene action (meaning that the functions of both genes work together to produce a final product). For example, consider a simple biochemical pathway in which a colorless substrate is converted by the action of gene A to another colorless product, which is then converted by the action of gene B to a visible pigment. Loss of function of either A or B, or both, will have the same result: no pigment production. Thus A_bb , aaB_ , and aabb will all be colorless, while only A_B_ genotypes will produce pigmented product. The modified 9:7 ratio may therefore be obtained when two genes act together in the same biochemical pathway, and when their loss of function phenotypes are indistinguishable from each other or from the loss of both genes. Contributors and Attributions - Dr. Todd Nickle and Isabelle Barrette-Ng (Mount Royal University) The content on this page is licensed under CC SA 3.0 licensing guidelines.	libretexts	2025-03-17T22:27:30.634037	2019-10-01T21:21:08	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/", "url": "https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/04%3A_Inheritance/4.07%3A_Gene_Interactions", "book_url": "https://commons.libretexts.org/book/bio-25718", "title": "4.7: Gene Interactions", "author": "Stefanie West Leacock" }
https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/05%3A_Chromosome_variation/5.01%3A__Changes_in_Chromosome_Number	5.1: Changes in Chromosome Number Learning Objectives - Describe nondisjunction and its outcomes. - Distinguish between aneuploidy (example: nullisomy or trisomy) and polyploidy. - Give an example of a human trisomy and explain why trisomies of some chromosomes are not tolerated (viable in offspring). - Explain why organisms with an odd number of chromosome sets are usually sterile or not even viable. - Recall that polyploidy is common in plants but rare in animals. Give an example of an advantage of polyploidy. Single Chromosomes can be Lost or Gained by Nondisjunction If something goes wrong during cell division, an entire chromosome may be lost and the cell will lack all of these genes. The causes behind these chromosome abnormalites and the consequences they have for the cell and the organism is the subject of this section. Cause: Nondisjunction During Mitosis or Meiosis Segregation occurs in anaphase. In mitosis and meiosis II, sister chromatids (of replicated chromosomes) are normally pulled to opposite ends of the cell. In Meiosis I, it is homologous chromosomes, which are synapsed at that time, that segregate and move apart. In rare cases, the sister chromatids (or paired chromosomes in Meiosis I) fail to separate. This failure to segregate properly is called nondisjunction and it can happen during mitosis, meiosis I, or meiosis II. This nondisjunction results in both chromatids (or chromosomes) moving to one pole and none at the other. One cell will have an extra copy and the other will lack a copy. Thus failure to segregate properly leads to unbalanced products. Consequence: Decreased Viability The result of a non-disjunction event is daughter cells that have an abnormal number of chromosomes. Cells, such as the parent cell in Figure 5.1.1, which have the proper number of chromosomes, are said to be euploid . The daughter cells have one too many or one too few chromosomes and are thus aneuploid . Even though both product cells have at least one copy of all genes, both cells will probably die. The reason is due to the loss or gain of a large number of genes. Genes produce an standard amount of product - either functional RNAs or proteins. The parent cell shown has a balanced genotype because it has two copies of all of its genes. Each of its genes produces half of the products needed by the cell. But if one of these cells suddenly had only one copy (or three copies) of an important gene, the amount of product would be either 50% (or 150%) of what was required. Such a change for a single gene could probably be tolerated by the cell and it would probably survive. But the sudden change to one copy (or three copies) of the hundreds or thousands of genes on an entire chromosome the results would be more than tolerable and be catastrophic for the daughter cells. They have what’s called an unbalanced genotype, which usually decreases their viability. If a nondisjunction event occurs during either meiosis I or meiosis II, the result is an unbalanced gamete. The gamete will often be functional, but after fertilization the embryo will be genetically unbalanced. This usually leads to the death of the embryo. There are some exceptions to this in humans and these will be presented later in this chapter. Changes in sets of chromosomes Humans, like most animals and most eukaryotic genetic model organisms, are diploids because they have two copies of each autosome. This means that most of their cells have two homologous copies of each chromosome. In contrast, many plant species and even a few animal species are polyploids . This means they have more than two chromosome sets, and so have more than two homologs of each chromosome in each cell. When the nuclear content changes by a whole chromosome set we call it a change in ploidy. Gametes are haploid (n) and thus most animals are diploid (2n), formed by the fusion of two haploid gametes. However, some species can exist as monoploid (1x), triploid (3x), tetraploid (4x), pentaploid (5x), hexaploid (6x), or higher. Notation of ploidy As we have already seen, the letter n is the number of chromosomes in a gamete, and 2n is the number of chromosomes following fertilization. To distinguish polyploids, the letter x is used to represent chromosome sets. For example: - For a tetraploid 2n=4x and a gamete has n=2x chromosomes. - For a hexaploid 2n=6x and a gamete has n=3x chromosomes. Query $\PageIndex{1}$ Male Bees are Monoploid Monoploids, with only one set of chromosomes, are usually inviable in most species; however, in many species of Hymenoptera (bees, wasps, ants) the males are monoploid and develop from unfertilized eggs. These males don’t undergo meiosis for gametes; mitosis produces sperm. Females are diploid (from fertilized eggs) and produce eggs via meiosis. Female bees are diploid (2n=32) and are formed when an egg (n=16) is fertilized by a sperm (n=16). If an egg is not fertilized, it can still develop and the result is a n=16 male drone. Males are described as haploid (because they have the same number of chromosomes as a gamete) or monoploid (because they have only one chromosome set). Females produce eggs by meiosis while males produce sperm by mitosis. This form of sex determination produces more females, workers, which do the work than males, who are only needed for reproduction. Polyploids can be stable or sterile Like diploids (2n=2x), stable polyploids generally have an even number of copies of each chromosome: tetraploid (2n=4x), hexaploid (2n=6x), and so on. The reason for this is clear from a consideration of meiosis. Remembering that the purpose of meiosis is to reduce the sum of the genetic material by half, meiosis can equally divide an even number of chromosome sets, but not an odd number. Thus, polyploids with an odd number of chromosomes (e.g. triploids, 2n=3x) tend to be sterile, even if they are otherwise healthy. The mechanism of meiosis in stable polyploids is essentially the same as in diploids: during metaphase I, homologous chromosomes pair with each other. Depending on the species, all of the homologs may be aligned together at metaphase, or in multiple separate pairs. For example, in a tetraploid, some species may form tetravalents in which the four homologs from each chromosome align together, or alternatively, two pairs of homologs may form two bivalents. Note that because that mitosis does not involve any pairing of homologous chromosomes, mitosis is equally effective in diploids, even-number polyploids, and odd-number polyploids. Many Crop Plants are Hexaploid or Octoploid Polyploid plants tend to be larger and healthier than their diploid counterparts. The strawberries sold in grocery stores come from octoploid (8x) strains and are much larger than the strawberries formed by wild diploid strains. Bread wheat is a hexaploid (2n=6x) strain. This species is derived from the combination of three other wheat species, T. monococcum , T. searsii , and T. tauschii . Each of these chromosome sets has 7 chromosomes so the diploid species are 2n=2x=14 and bread wheat is 2n=6x=42 and has 14 chromosomes from each species. Bread wheat is viable because each chromosome behaves independently during mitosis. The species is also fertile because during meiosis I the equivalent chromosomes from each species can pair each other during meiosis I. Thus, even in a polyploid, homologous chromosomes can segregate equally and gene balance can be maintained. Bananas, Watermelons, and Other Seedless Plants are Triploid The bananas found in grocery stores are a seedless variety called Cavendish. They are a triploid variety (chromosome sets = AAA) of a normally diploid species called Musa acuminata (AA). Cavendish plants are viable because mitosis can occur. However they are sterile because the chromosomes cannot pair properly during meiosis I. During prophase I there are three copies of each chromosome trying to “pair” with each other. Because proper chromosome segregation in meiosis fails, seeds cannot be made and the result is a fruit that is easier to eat because there are no seeds to spit out. Why are triploids sterile? Remember that during meiosis in a diploid there are four sister chromatids for each chromosome and 4 cells (gametes) produced. Each gamete inherits one copy of each chromosome. In a triploid after DNA replication (S phase), there will be six sister chromatids for each chromosome, but meiosis I and II still undergo the same process. - How many chromatids will be in each gamete? - Will the number of copies of each chromosome be the same in a single gamete? For a cell with three types of chromosomes (n=3), draw the chromosomes in a triploid cell. Show how those chromosomes might segregate during meiosis. What possibilities exist for number of chromosomes in each gamete? Exercise $\PageIndex{1}$ Seedless watermelons are another common triploid. The haploid number (n) of standard watermelons is 11. How many chromosomes are present in a single triploid watermelon cell? The thin white seeds in these triploids are the result of unbalanced gametes, but rare black viable seeds are sometimes found in a seedless watermelon. What would have to happen for these viable seeds to be produced? - Answer - A triploid watermelon has 3 sets of 11 chromosomes: 3 x 11 = 33. Although most gametes will have unbalanced chromosomes (example: one copy of five chromosomes but two copies of six chromosomes), rare balanced gametes can be produced. For example, by chance a balanced gamete with one copy of all eleven chromosomes is fertilized by another balanced gamete with two copies of all chromosomes. If triploids cannot make seeds, how do we obtain enough triploid individuals for cultivation? The answer depends on the plant species involved. In some cases, such as banana, it is possible to propagate the plant asexually; new progeny can simply be grown from cuttings from a triploid plant. On the other hand, seeds for seedless watermelon are produced sexually: a tetraploid watermelon plant is crossed with a diploid watermelon plant. Both the tetraploid and the diploid are fully fertile, and produce gametes with two (1n=2x) or one (1n=1x) sets of chromosomes, respectively. These gametes fuse to produce a zygote (2n=3x) that is able to develop normally into an adult plant through multiple rounds of mitosis, but is unable to compete normal meiosis or produce seeds. Polyploids are often larger in size than their diploid relatives. Food plant cultivation takes advantage of this property. For example, most strawberries you eat are not diploid, but octoploid (8x). Polyploidy in Animals Polyploidy in animals is rare, essentially limited to lower forms, although some vertebrates (fish) have been identified as triploid. Cultivated oysters are an example of a viable and commercially relevant triploid animal. In the 1990s, biologists explored ways to alter the number of chromosome sets in the oyster Crassostrea gigas (Guo et al, 1992). Triploid oysters Triploid oysters are cultivated for human consumption ( https://www.theatlantic.com/technology/archive/2014/09/todays-oysters-are-mutants/380858/ ). What are the advantages of triploidy in oysters? How are triploid oysters similar to triploid plants? Based on the observation of rare fertilized seeds in seedless watermelons, would you expect to find rare fertile triploid oysters? Triploidy in humans is not a viable condition, occurring in as much as 20% of miscarriages that show chromosomal abnormalities (reviewed in Kolarski et al, 2017). Triploidy in humans may be caused by abnormal meiosis that produces a gamete with two sets of chromosomes or by fertilization of a single egg by two sperm. References Guo, Ximing, et al. “Genetic Consequences of Blocking Polar Body I with Cytochalasin B in Fertilized Eggs of the Pacific Oyster, Crassostrea Gigas: I. Ploidy of Resultant Embryos.” Biological Bulletin , vol. 183, no. 3, 1992, pp. 381–386. JSTOR , www.jstor.org/stable/1542013. Accessed 7 July 2020. Kolarski M, Ahmetovic B, Beres M, et al. Genetic Counseling and Prenatal Diagnosis of Triploidy During the Second Trimester of Pregnancy. Med Arch . 2017;71(2):144-147. doi:10.5455/medarh.2017.71.144-147 Contributors and Attributions - Dr. Todd Nickle and Isabelle Barrette-Ng (Mount Royal University) The content on this page is licensed under CC SA 3.0 licensing guidelines. - Dr. Todd Nickle and Isabelle Barrette-Ng (Mount Royal University) The content on this page is licensed under CC SA 3.0 licensing guidelines.	libretexts	2025-03-17T22:27:30.825129	2019-10-01T21:21:10	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/", "url": "https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/05%3A_Chromosome_variation/5.01%3A__Changes_in_Chromosome_Number", "book_url": "https://commons.libretexts.org/book/bio-25718", "title": "5.1: Changes in Chromosome Number", "author": "Stefanie West Leacock" }
https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/05%3A_Chromosome_variation/5.02%3A__Changes_in_Chromosome_Structure	5.2: Changes in Chromosome Structure Learning Objectives - Distinguish between the 4 types of chromosomal rearrangements and their effects on gene dosage and meiosis. If the chromosome is altered, but still retains the three critical features of a chromosome (centromeres, telomeres, and origin of replication), it will continue to be inherited during subsequent cell divisions, however the daughter cell may not retain all the genes. For example, if a segment of the chromosome has been lost, the cell may be missing some genes. The causes and consequences of chromosome structural abnormalities are described here. They all involve breaks in the chromosomal DNA backbone. Cause #1: Incorrect Repair of Double Strand DNA Breaks During Interphase A chromosome is a very long but very thin molecule. In the phosphodiester backbone there are only two covalent bonds holding each base pair to the next. If one of these covalent bonds is broken the chromosome will still remain intact, although a DNA ligase will be needed to repair the nick. Problems arise when both strands are broken at or near the same location. This double strand break will cleave the chromosome into two independent pieces. Because these events do occur in cells, there is a repair system called the non-homologous end joining (NHEJ) system to fix them. Proteins bind to each broken end of the DNA and reattach them with new covalent bonds. This system is not perfect and sometimes leads to chromosome rearrangements (see next section). The NHEJ system proteins only function if required. If the chromosomes within an interphase nucleus are all intact, the system is not active. The telomeres at the natural ends of chromosomes prevent the NHEJ system from attempting to join the normal ends of chromosomes together. If there is one double-strand break, the two broken ends can be recognized and joined. However, if there are two (or more) double-strand breaks at the same time there will be four broken ends in total. The NHEJ system proteins recognize the breaks but do not have the ability to identify exactly which ends should be joined to restore the original chromosome. Thus, NHEJ may join the ends together correctly, but if they do not the result is a chromosome rearrangement . The Four Types of Chromosome Rearrangements Errors during the repair of multiple double-strand breaks can cause four types of chromosome rearrangements. The type of chromosome rearrangement is dependent upon where the two breaks were originally and how they are rejoined. First double-strand DNA break occurs in one site; a second DNA break occurs and the NHEJ proteins mend the damage incorrectly by joining the ends. There are four major types of rearrangements: Cause #2: Incorrect Crossovers During Meiosis Meiotic crossovers occur at the beginning of meiosis for two reasons. They help hold the homologous chromosomes together until separation occurs during anaphase I. They also allow recombination to occur between linked genes. The event itself takes place during prophase I when a double strand break on one piece of DNA is joined with a double strand break on another piece of DNA and the ends are put together. Most of the time the breaks are on non-sister chromatids and most of the time the breaks are at the same relative locations. Problems occur when the wrong pieces of DNA are matched up along the chromosomes during crossover events. This can happen if the same or similar DNA sequence is found at multiple sites on the chromosomes. For example, if there are two Alu transposable elements on a chromosome. When the homologous chromosomes pair during prophase I the wrong Alu sequences might line up. A crossover may occur in this region. If so, when the chromosomes separate during anaphase I one of the chromatids will have a duplication and one will have a deletion. Ultimately, of the four cells produced by this meiosis, two will be normal, one will have a chromosome with extra genes, and one will have a chromosome missing some genes. Errors of this type can also cause inversions and translocations. Consequence #1: Abnormal pairing at Meiosis DNA forms loops to achieve pairing when chromosomes are rearranged Homologous regions of chromosomes pair at meiosis I (prophase I). With rearranged chromosomes this can lead to visible abnormalities and segregation abnormalities. Deletion chromosomes will pair up with a normal homolog along the shared regions and at the missing segment, the normal homolog will loop out (nothing to pair with) to form a deletion loop. This feature can be used to locate the deletion cytologically. The deleted region is also pseudo-dominant , in that the cell no longer has two copies of the genes in this region, therefore mutant recessive alleles may produce a phenotypic change. When an inversion chromosome is paired up in meiosis there is an inversion loop formed. If there is a crossover within the loop then abnormal products will result and abnormal, unbalanced gametes will be produced. For example, a crossover event within the loop of a par a centric inversion will lead to a dicentric product that will break into deletion products and produce unbalanced gametes. Similarly, with a per i centric inversion , a crossover event leads to duplicate/deletion products that are unbalanced. Video explanation: Crossing over in inversions Translocated chromosomes pair with more than one homologous chromosome For translocations , a consequence for the two chromosomes involved is that when they pair at meiosis both replicated chromosome pairs will be together. During meiosis I, homologous chromosomes separate. The four chromosomes can segregate in different ways, depending on which centromeres are pulled to each pole during anaphase. This set of paired, replicated chromosomes can segregate as Alternate (balanced) where both non-translocated (N1 and N2) chromosomes go to one pole and both translocated chromosomes go to the other pole. A gamete that contains the two translocated (T1 and T2) chromosome still contains one full copy of both chromosomes. However, the chromosomes may segregate as Adjacent-1 (unbalanced) where a cells gets one normal and one translocation chromosome; each gamete will contain extra copies of some genes and lack copies of other genes. Each of these possibilities occur with approximately equal frequency and thus about half the gametes will be unbalanced. Consequence #2: Decreased Viability All of the chromosome rearrangements shown above produce functional chromosomes. Each has one centromere, two telomeres, and thousands of origins of replication. Because inversions and translocations do not change the number of genes in a cell or organism they are said to be balanced rearrangements. Unless one of the breakpoints occurred in the middle of a gene the cells will not be affected. On the other hand, deletions and duplications are unbalanced rearrangements. The larger they are (more genes involved) the more disruption they cause to the proper functioning of the cell or organism. Note about gene dosage Having too much or too little gene activity for a large number of genes can disrupt the cellular metabolism to generate a phenotype or reduce viability. Consequence #3: Decreased Fertility Recall that during meiosis I homologous chromosomes pair up. If a cell has a chromosome with a rearrangement this chromosome will have to pair with its normal homolog. Cells heterozygous for balanced rearrangements actually have more difficulties in prophase I. There are different ways they might pair during prophase I, but if a crossover occurs in the inverted region the result will be unbalanced gametes. Embryos made with unbalanced gametes rarely survive. The consequence is that the heterozygous organism will have reduced fertility . Figure $\PageIndex{8}$: Meiosis in a cell heterozygous for the chromosomes shown in Figure 9.11. Note that of the four gametes one has a deletion of the A gene and a duplication of the D gene while another gamete has a duplication of A and a deletion of D. (Original-Harrington-CC:AN) Note that an organism homozygous for this inversion chromosome will not be affected in this way because no loops are formed. The chromosomes can pair along their entire length and crossovers will not produce any unbalanced gametes. This is a general property of inversions and translocations. In heterozygotes there are problems during meiosis, that result in a lot many unbalanced gametes and an overall reduction in fertility. In homozygotes the rearranged chromosomes pair with one another just fine and there is no effect on fertility. Consequence #4: Cancer Some chromosome rearrangements have breakpoints within genes leading to the creation of hybrid genes – the first part of one gene with the last part of another. If the hybrid gene inappropriately promotes cell replication, the cell can become cancerous. One example of such a translocation t(9:22) is the cause of chronic myelogenous leukemia (CML). Association of this translocation in cancer was first suggested in a brief letter 1960 (Nowell and Hungerford, Science 132:1497). The translocation creates a fusion gene with the BCR gene promoter and 5' region and the ABL 3' end. When transcribed and translated, this fusion protein is a constitutively active kinase. Consequence #5: Evolution Those chromosome changes that duplicate genes are important for evolution. If an organism has an extra copy of important genes, one gene can be retained for their original function while others can mutate and potentially acquire new functions. For example, multiple copies of the globin genes are found in mammals and have unique functions. Chromosome rearrangements that decrease fertility are also important for the origin of new species. If a rearrangement becomes common in a small isolated population, that population has 100% fertility if they mate within their group, but a reduced fertility if they mate with members of the larger population. As rearrangements accumulate the small population will become more and more reproductively isolated. When members are incapable of forming viable, fertile offspring with the original population the group will have become a new species. Contributors and Attributions - Dr. Todd Nickle and Isabelle Barrette-Ng (Mount Royal University) The content on this page is licensed under CC SA 3.0 licensing guidelines.	libretexts	2025-03-17T22:27:30.900495	2019-10-01T21:21:10	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/", "url": "https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/05%3A_Chromosome_variation/5.02%3A__Changes_in_Chromosome_Structure", "book_url": "https://commons.libretexts.org/book/bio-25718", "title": "5.2: Changes in Chromosome Structure", "author": "Stefanie West Leacock" }
https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/06%3A_Regulation_of_Gene_Expression	6: Regulation of Gene Expression Within most multicellular organisms, every cell contains essentially the same genomic sequence. How then do cells develop and function differently from each other? The answer lies in the regulation of gene expression . Only a subset of all the genes is expressed (i.e. are functionally active) in any given cell participating in a particular biological process. Gene expression is regulated at many different steps along the process that converts DNA information into active proteins. In the first stage, transcript abundance can be controlled by regulating the rate of transcription initiation and processing, as well as the degradation of transcripts. In many cases, higher abundance of a gene’s transcripts is correlated with its increased expression. In this chapter, we will focus on transcriptional regulation . Prokaryotes and other unicellular organisms regulate genes too, changes in temperature or chemicals in the environment can require changing what proteins are produced at certain times. Cells also regulate the overall activity of genes in other ways. For example, by controlling the rate of mRNA translation, processing, and degradation, as well as the post-translational modification of proteins and protein complexes. - - 6.1: Prokaryotic gene regulation - Early insights into mechanisms of transcriptional regulation came from studies of E. coli by researchers Francois Jacob & Jacques Monod. In E. coli, and many other bacteria, genes encoding several different proteins may be located on a single transcription unit called an operon. The genes in an operon share the same transcriptional regulation, but are translated individually. Eukaryotes generally do not group genes together as operons (exception is C. elegans and a few other species). Contributors and Attributions - Dr. Todd Nickle and Isabelle Barrette-Ng (Mount Royal University) The content on this page is licensed under CC SA 3.0 licensing guidelines.	libretexts	2025-03-17T22:27:30.990506	2019-10-01T21:21:13	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/", "url": "https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/06%3A_Regulation_of_Gene_Expression", "book_url": "https://commons.libretexts.org/book/bio-25718", "title": "6: Regulation of Gene Expression", "author": "Stefanie West Leacock" }
https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/06%3A_Regulation_of_Gene_Expression/6.01%3A_Prokaryotic_gene_regulation	6.1: Prokaryotic gene regulation Learning Objectives - Define operon and describe how it differs from genes organized by single proteins. - Identify the functions of regulatory proteins, promoters, operators, cis- and trans- acting factors, and structural genes. - Distinguish between positive vs negative and inducible vs repressible operons. - Identify and understand the role of the structure and components of the lac operon and predict whether or not gene expression will occur under particular conditions. Early insights into mechanisms of transcriptional regulation came from studies of E. coli by researchers Francois Jacob & Jacques Monod. In E. coli, and many other bacteria, genes encoding several different proteins may be located on a single transcription unit called an operon . The genes in an operon share the same transcriptional regulation, but are translated individually. Eukaryotes generally do not group genes together as operons (though there are some exceptions including C. elegans and a few other species). How can an operon encode multiple proteins? Think back to the steps involved in transcription and translation. Which steps will be altered if multiple proteins must be translated from a single transcript? How do you think this works? Types of regulation Operons can be turned "off" (repressible) or "on" (inducible) and be controlled protein activators or repressors. \| Inducible regulation turns transcription ON. \| Repressible regulation turns transcription OFF. \| \| \|---\|---\|---\| \| Positive regulation uses activator proteins. \| An activator protein turns ON transcription in response to a stimulus / condition. \| An activator protein is inactivated in response to a stimulus / condition, turning transcription OFF. \| \| Negative regulation uses repressor proteins. \| A repressor protein is inactivated in response to a stimulus / condition, turning transcription turns ON. \| A repressor protein turns transcription turns OFF in response to a stimulus / condition. \| Basic lac operon structure E. coli encounters many different sugars in its environment. These sugars, such as lactose and glucose, require different enzymes for their metabolism. Three of the enzymes for lactose metabolism are grouped in the lac operon: lacZ , lacY , and lacA . LacZ encodes an enzyme called β-galactosidase, which digests lactose into its two constituent sugars: glucose and galactose. lacY is a permease that helps to transfer lactose into the cell. Finally, lacA is a trans-acetylase. Transcription of the lac operon normally occurs only when lactose is available for it to digest. Presumably, this avoids wasting energy in the synthesis of enzymes for which no substrate is present. A single mRNA transcript includes all three enzyme-coding sequences and is called polycistronic. A cistron is equivalent to a gene. cis- and trans - Regulatory Elements The lac operon includes DNA sequences that do not encode proteins, but are instead binding sites for proteins that regulate transcription of the operon. In the lac operon, these sequences are called P (promoter), O (operator), and CBS (CAP-binding site). Collectively, sequence elements such as these are called cis -elements because they must be located on the same piece of DNA as the genes they regulate. On the other hand, the proteins that bind to these cis -elements are called trans -regulators because (as diffusible molecules) they do not necessarily need to be encoded on the same piece of DNA as the genes they regulate. lacI is an allosterically regulated repressor One of the major trans -regulators of the lac operon is encoded by lacI. Four identical molecules of lacI proteins, encoded by a gene distinct from the operon that encodes lacZ , lacY , and lacA , assemble together to form a homotetramer called a repressor. This repressor binds to two operator sequences adjacent to the promoter of the lac operon. Binding of the repressor prevents RNA polymerase from binding to the promoter. Therefore, the operon will not be transcribed when the operator is occupied by a repressor. Allosteric regulation occurs when a substrate causes a change to the structure and function of a protein. In this example, in addition to domains that bind operator seqeucnes in DNA, lacI protein has sites that bind to allolactose, which is a small molecule produced when lactose is present. When allolactose is bound to lacI , the shape of the protein changes and prevents it from binding to the operator. Therefore, in the presence of lactose, RNA polymerase is able to bind to the promoter and transcribe the lac operon, leading to a moderate level of expression of the lacZ , lacY , and lacA genes. CAP is an allosteric activator of the lac operon A second aspect of lac operon regulation is conferred by a trans -factor called cAMP binding protein (CAP). CAP is another example of an allosterically regulated trans -factor. Only when the CAP protein is bound to cAMP can another part of the protein bind to a specific cis -element within the lac promoter called the CAP binding sequence (CBS). CBS is located very close to the promoter (P). When CAP is bound to at CBS, RNA polymerase is better able to bind to the promoter and initiate transcription. Thus, the presence of cAMP ultimately leads to a further increase in lac operon transcription. Figure $\PageIndex{3}$: CAP, when bound to cAMP, helps RNApol to bind to the lac operon. cAMP is produced only when glucose [Glc] is low. (Origianl-Deyholos-CC:AN) The physiological significance of regulation by cAMP becomes more obvious in the context of the following information. The concentration of cAMP is inversely proportional to the abundance of glucose: when glucose concentrations are low, an enzyme called adenylate cyclase is able to produce cAMP from ATP. Evidently, E. coli prefers glucose over lactose, and so expresses the lac operon at high levels only when glucose is absent and lactose is present. This provides another layer of logical control of lac operon expression: only in the presence of lactose, and in the absence of glucose is the operon expressed at maximal levels. Video Summary of the lac operon Contributors and Attributions - Dr. Todd Nickle and Isabelle Barrette-Ng (Mount Royal University) The content on this page is licensed under CC SA 3.0 licensing guidelines.	libretexts	2025-03-17T22:27:31.061851	2019-10-01T21:21:13	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/", "url": "https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/06%3A_Regulation_of_Gene_Expression/6.01%3A_Prokaryotic_gene_regulation", "book_url": "https://commons.libretexts.org/book/bio-25718", "title": "6.1: Prokaryotic gene regulation", "author": "Stefanie West Leacock" }
https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/06%3A_Regulation_of_Gene_Expression/6.01%3A_Prokaryotic_gene_regulation/6.1.01%3A_The_Use_of_Mutants_to_Study_the_lac_Operon	6.1.1: The Use of Mutants to Study the lac Operon Learning Objectives - Describe how partial diploids can be used to examine genetic relationships in haploid organisms. - Predict whether lac operon genes will be expressed when given mutations are present. Single mutants of the lac operon The lac operon and its regulators were first characterized by studying mutants of E. coli that exhibited various abnormalities in lactose metabolism. Some mutants expressed the lac operon genes constitutively, meaning the operon was expressed whether or not lactose was present in the medium. Such mutant are called constitutive mutants. The operator locus ( lacO ) - One example is O c , for operator constitutive, in which a mutation in an operator sequence and reduces or precludes the repressor (the lacI gene product) from recognizing and binding to the operator sequence. Thus, in O c mutants, lacZ , lacY , and lacA are transcribed whether or not lactose is present. The lacI locus – One type of mutant allele of lacI (callled I – ) prevents either the production of a repressor polypeptide or produces a polypeptide that cannot bind to the operator sequence. This allele is also a constitutive expresser of the lac operon because absence of repressor binding permits transcription. Another type of mutant of lacI, called lacI S for super-repressor, prevents the repressor polypeptide from binding allolactose, and thus will always bind to the operator and be non-inducible. This mutant constitutively represses the lac operon whether lactose is present or not. Two lac operons in a single cell create partial diploids in E.coli The regulation of the lac operon became further understood by using two copies of the operon sequences in one cell. Although E. coli are haploid organisms, scientists use a plasmid called the F-factor to bring an additional copy of gene sequences into the cell, while the other copy is on the genomic E. coli chromosome. This results in a partial diploid in E. coli. The F-factor is extra-chromosomal DNA that is capable of being either a free plasmid or integrated into the host bacterial chromosome. This switching is accomplished by IS elements where unequal crossing over can recombine the F-factor and adjacent DNA sequences (genes) in and out of the host chromosome. Researchers have used this genetic tool to create partial diploids (merozygotes) that allow them to test the regulation with different combinations of different mutations in one cell. For example, the F-factor copy may have a lacI S mutation while the genomic copy might have an O C mutation. How would this cell respond to the presence/absence of lactose (or glucose)? This partial diploid can be used to determine that lacI S is dominant to lacI + , which in turn is dominant to lacI – . It can also be used to show the O C mutation only acts in cis - while the lacI mutation can act in trans - . Example $\PageIndex{1}$ Consider the question posed above: A cell with a copy of the lac operon from an F-factor has the I S mutation, whereas the genomic copy has an O C mutation. How would this cell respond to the presence/absence of lactose (or glucose)? Solution The I S mutation encodes a form of the protein cannot bind allolactose and therefore continues to recognize the operator, even when lactose is present. However, the genomic copy of the operator O C is mutated to a sequence that is no longer recognized by the lac repressor. For this reason, the lacZYA genes in the genomic lac operon will be continuously expressed in this cell, because lacI repressor will not be able to bind this sequence. Query $\PageIndex{1}$ Contributors and Attributions - Dr. Todd Nickle and Isabelle Barrette-Ng (Mount Royal University) The content on this page is licensed under CC SA 3.0 licensing guidelines.	libretexts	2025-03-17T22:27:31.202168	2019-10-01T21:21:14	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/", "url": "https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/06%3A_Regulation_of_Gene_Expression/6.01%3A_Prokaryotic_gene_regulation/6.1.01%3A_The_Use_of_Mutants_to_Study_the_lac_Operon", "book_url": "https://commons.libretexts.org/book/bio-25718", "title": "6.1.1: The Use of Mutants to Study the lac Operon", "author": "Stefanie West Leacock" }
https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/06%3A_Regulation_of_Gene_Expression/6.02%3A_Eukaryotic_Gene_Regulation	6.2: Eukaryotic Gene Regulation Learning Objectives - Describe the cis -acting elements that control tissue-specific transcription of eukaryotic protein encoding genes: promoters, enhancers, insulators and how trans- acting transcription factors can influence gene expression. - Predict the effects of mutations in regulatory sequences on gene expression. Like prokaryotes, transcriptional regulation in eukaryotes involves both cis -elements and trans -factors; however, there are more factors and they interact in more complex ways, many of which are still be elucidated. Regulatory sequences Proximal regulatory sequences As in prokaryotes the RNA polymerase binds to the gene at its promoter to begin transcription. In eukaryotes, however, RNA polymerase is part of a large protein complex that includes additional proteins (transcription initiation factors) that bind to one or more specific cis -elements in the promoter region, including GC-rich boxes, CAAT boxes, and TATA boxes. High levels of transcription require both the presence of this protein complex at the promoter, as well as their interaction with other trans -factors described below. The approximate position of these elements is described relative to the transcription start site (+1), but the distance between any of these elements and the transcription start site can vary. These sites are typically within ~200 base pairs of the start of transcription. Distal regulatory elements Even more variation is observed in the position and orientation of the second major type of cis -regulatory element in eukaryotes, which are DNA sequences called enhancer elements or simply enhancers. Regulatory trans -factor proteins called transcription factors bind to specific enhancer sequences, then, while still bound to DNA, these proteins interact with RNA polymerase and other proteins at the promoter to enhance the rate of transcription. There are a wide variety of different transcription factors and each recognizes a specific DNA sequence (enhancer element) to promote gene expression in the adjacent gene under specific circumstances. Enhancers can be located near (~100s of bp) or far (~10K of bp), and either upstream or downstream, from the promoter. The transcription factors and the proteins associated with transcription initiation complex will bind each other, bending the DNA in the process. Enhancer sequence example Some genes are transcriptionally regulated in response to hormones, such as estrogen. Estrogen receptors are proteins that bind the hormone estrogen and the the protein dimers are transported into the nucleus where they recognize sequences called estrogen response elements (EREs). The consensus ERE sequence is 5′-GGTCAnnnTGACC-3′ (where n is any base) at many locations in a genome and recruit RNA polymerase to induce the transcription of the corresponding genes. This consensus sequence was elucidated by studies of a human cell line to which a Xenopus (frog) estrogen responsive gene had been added, revealing that the roles of estrogen receptors in binding EREs and activating transcription of target genes is conserved across species. Klein-Hitpass L, Schorpp M, Wagner U, Ryffel GU. An estrogen-responsive element derived from the 5' flanking region of the Xenopus vitellogenin A2 gene functions in transfected human cells. Cell. 1986 Sep 26;46(7):1053-61. doi: 10.1016/0092-8674(86)90705-1. PMID: 3463433. Reviewed by Klinge C. Nucleic Acids Res. 2001 Jul 15; 29(14): 2905–2919. Enhancers regulate the Drosophila yellow gene The yellow gene of Drosophila provides an example of the modular nature of enhancers. This gene encodes an enzyme in the pathway that produces a dark pigment in the insect exoskeleton. Mutants have a yellow cuticle rather than the wild type darker pigmented cuticle. (Why call the gene “yellow”? Recall that genes are often named after their mutant phenotype.) Three enhancer elements each bind a different tissue-specific transcription factor in the wing, body, or mouth to enhance transcription of yellow in that tissue and make the dark pigment. So, the wing cells will have a transcription factor that binds to the wing enhancer to drive expression; likewise in the body and mouth cells. Thus, specific combinations of cis -elements and trans -factors control the differential, tissue-specific expression of genes. This type of combinatorial action of enhancers is typical of the transcriptional activation of most eukaryotic genes: specific transcription factors activate the transcription of target genes under specific conditions. While enhancer sequences promote expression, there is an oppositely acting type of element, called silencers. These elements function in much the same manner, with transcription factors that bind to DNA sequences, but they act to repress or reduce transcription from the adjacent gene. A gene’s complete expression profile (transcription level, tissue specific, temporal specific) is a combination of various enhancer and silencer elements Thinking about regulation of transcription factors Understanding the transcription factors that regulate the expression of the yellow gene can prompt us to consider: why are those transcription factors present in different tissues? These transcription factors themselves are regulated by their own sets of transcription factors and so on and so on. Where does it begin? For Drosophila, the answer is in the egg! Specific proteins and mRNAs in the egg define the regions of the future embryo and a cascade of gene expression events lead to each adult cell and tissue type expressing the correct genes for their function. Exercise $\PageIndex{1}$ What is the phenotype of the fruit fly when the yellow gene and enhancers are fully functional? What would be the effect of a loss-of-function mutation in the yellow coding sequence? What would be the effect of a mutation in the wing enhancer? What would be the effect of a mutation in the TATA box region? - Answer - The wild-type fruit fly has a dark black/brown cuticle. A loss-of-function mutation in the yellow gene would lead to flies with yellow appearance, instead of brown/black. A mutation in the wing enhancer would result in yellow wings, but other parts of cuticle should be unaffected. A mutation in the TATA box should result in a phenotype similar to loss-of-function in the yellow gene. Contributors and Attributions - Dr. Todd Nickle and Isabelle Barrette-Ng (Mount Royal University) The content on this page is licensed under CC SA 3.0 licensing guidelines. Using reporter genes to study transcription	libretexts	2025-03-17T22:27:31.300543	2019-10-01T21:21:14	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/", "url": "https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/06%3A_Regulation_of_Gene_Expression/6.02%3A_Eukaryotic_Gene_Regulation", "book_url": "https://commons.libretexts.org/book/bio-25718", "title": "6.2: Eukaryotic Gene Regulation", "author": "Stefanie West Leacock" }
https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/06%3A_Regulation_of_Gene_Expression/6.02%3A_Eukaryotic_Gene_Regulation/6.2.01%3A_Gene_Expression_in_Evolution	6.2.1: Gene Expression in Evolution Learning Objectives - Using examples, describe how changes in gene expression can be association with changes in phenotype and evolution. Mutations can occur in both cis -elements and trans -factors; both can result in altered patterns of gene expression. If an altered pattern of gene expression results in a selective advantage (or at least do not produce a major disadvantage), they may be selected and maintained in future populations. They may even contribute to the evolution of new species. An example of a sequence change in an enhancer is found in the Pitx gene. Pitx expression in Stickleback The three-spined stickleback provides an example of natural selection of a mutation in a cis -regulatory element. This fish occurs in two forms: (1) populations that inhabit deep, open water and have a spiny pelvic fin that deters larger predator fish from feeding on them; (2) populations from shallow water environments and lack this spiny pelvic fin. In shallow water, it appears that a long, spiny pelvic fin would be a disadvantage because it frequently contacts the sediment at the bottom of the pond and allows parasitic insects in the sediment to invade the stickleback. Researchers compared gene sequences of individuals from both deep and shallow water environments. They observed that in embryos from the deep-water population, a gene called Pitx was expressed in several groups of cells, including those that developed into the pelvic fin. Embryos from the shallow-water population expressed Pitx in the same groups of cells as the other population, with an important exception: Pitx was not expressed in the pelvic fin primordium (the cells that will generate the fin) in the shallow-water population. Further genetic analysis showed that the absence of Pitx gene expression from the developing pelvic fin of shallow-water stickleback was due to the absence (mutation) of a particular enhancer element upstream of Pitx . Figure $\PageIndex{1}$: Development of a large, spiny pelvic fin in deep-water stickleback (left) depends on the presence of a particular enhancer element upstream of a gene called Pitx . Mutants lacking this element, and therefore the large pelvic fin (right), have been selected for in shallow-water environments. (Wikipedia-Richard Wheeler-GFDL) Thinking about the mutation Consider the enhancer element mutated in the shallow-water sticklebacks: - What is the function of that DNA sequence? What type of protein would bind there? - Do you think this mutation acts in a dominant or recessive manner? - Which of these processes are affected by the mutation: DNA replication, transcription, splicing, and/or translation? - Would it be possible for another mutation to reverse the effects of this mutation and a shallow-water stickleback with a long fin? Example: Hemoglobin expression in placental mammals. Hemoglobin is the oxygen-carrying component of red blood cells (erythrocytes). Hemoglobin usually exists as tetramers of four non-covalently bound hemoglobin molecules. Each hemoglobin molecule consists of a globin polypeptide with a covalently attached heme molecule. Heme is made through a specialized metabolic pathway and is then bound to globin polypeptide through post-translational modification. The composition of the hemoglobin tetramers changes during development. From early childhood onward, most tetramers are of the type $\alpha$ 2 $\beta$ 2 , which means they contain of two copies of each of two slightly different globin proteins named $\alpha$ and $\beta$. A small amount of adult hemoglobin is $\alpha$ 2 $\delta$ 2 , which has $\delta$ globin instead of the more common $\beta$ globin. Other tetrameric combinations predominate before birth: $\zeta$ 2 $\varepsilon$ 2 is most abundant in embryos, and $\alpha$ 2 $\gamma$ 2 is most abundant in fetuses. Although the six globin proteins ($\alpha$ = alpha, $\beta$ = beta , $\gamma$ = gamma, $\delta$ =delta, $\varepsilon$ =epsilon , $\zeta$ = zeta) are very similar to each other, they do have slightly different functional properties. For example, fetal hemoglobin has a higher oxygen affinity than adult hemoglobin, allowing the fetus to more effectively extract oxygen from maternal blood. The specialized $\gamma$ globin genes that are characteristic of fetal hemoglobin are found only in placental mammals. Figure $\PageIndex{3}$: Expression of globin genes during prenatal and postnatal development in humans. The organs in which globin genes are primarily expressed at each developmental stage are also indicated. (Origianl-Deyholos-CC:AN) Each of these globin polypeptides is encoded by a different gene. In humans, globin genes are located in clusters on two chromosomes. We can infer that these clusters arose through a series of duplications of an ancestral globin gene. Gene duplication events can occur through rare errors in processes such as DNA replication, meiosis, or transposition ( 5.2: Changes in Chromosome Structure ). The duplicated genes can then accumulate mutations independently of each other. Mutations can occur in either the regulatory regions (e.g. promoter regions), or in the coding regions, or both. In this way, the promoters of globin genes have evolved to be expressed at different phases of development, and to produce proteins optimized for the prenatal environment. Of course, not all mutations are beneficial: some mutations can lead to inactivation of one or more of the products of a gene duplication. This can produce what is called a pseudogene. Examples of pseudogenes ($\psi$) are also found in the globin clusters. Pseudogenes have mutations that prevent them from being expressed at all. The globin genes provide an example of how gene duplication and mutation, followed by selection, allows genes to evolve specialized expression patterns and functions. Many genes have evolved as gene families in this way, although they are not always clustered together as are the globins. Exercise $\PageIndex{1}$ Individuals with diseases such as sickle cell disease or $\beta$-thalassemia have mutations that either cause a malformed protein or the absence of the HBB (beta-globin) protein. How could understanding the normal expression of other hemoglobin proteins help develop new therapies for these patients? What tools and processes might be used? - Answer - Recent clinical trials are exploring the possibility of using gene editing to express fetal hemoglobin in sickle cell and beta-thalessemia patients. This process isolates stem cells from patients, edits the DNA in vitro , and then transplants the edited cells back into the patient (reviewed in https://academic.oup.com/hmg/advance-article/doi/10.1093/hmg/ddaa088/5836961 ). A news report about one of these patients is at this link https://www.npr.org/sections/health-shots/2020/06/23/877543610/a-year-in-1st-patient-to-get-gene-editing-for-sickle-cell-disease-is-thriving . Selected references: Ye L, Wang J, Tan Y, et al. (2016) Genome editing using CRISPR-Cas9 to create the HPFH genotype in HSPCs: An approach for treating sickle cell disease and β-thalassemia. Proc Natl Acad Sci U S A . 2016;113(38):10661-10665. doi:10.1073/pnas.1612075113 ( https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5035856/ ) Weber L, Frait G, Felix T, et al. (2020) Editing a γ-globin repressor binding site restores fetal hemoglobin synthesis and corrects the sickle cell disease phenotype. Science Advances 12 Feb 2020 ( https://advances.sciencemag.org/content/6/7/eaay9392?utm_source=TrendMD&utm_medium=cpc&utm_campaign=TrendMD_1 ) Demirci S, Leonard A, Tisdale JF. (2020) Genome editing strategies for fetal hemoglobin induction in beta-hemoglobinopathies. Human Molecular Genetics 14 May 2020 . https://doi.org/10.1093/hmg/ddaa088 Contributors and Attributions - Dr. Todd Nickle and Isabelle Barrette-Ng (Mount Royal University) The content on this page is licensed under CC SA 3.0 licensing guidelines.	libretexts	2025-03-17T22:27:31.368456	2019-10-01T21:21:14	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/", "url": "https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/06%3A_Regulation_of_Gene_Expression/6.02%3A_Eukaryotic_Gene_Regulation/6.2.01%3A_Gene_Expression_in_Evolution", "book_url": "https://commons.libretexts.org/book/bio-25718", "title": "6.2.1: Gene Expression in Evolution", "author": "Stefanie West Leacock" }
https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/06%3A_Regulation_of_Gene_Expression/6.02%3A_Eukaryotic_Gene_Regulation/6.2.02%3A_Regulating_Transcription_at_the_Chromatin_Level	6.2.2: Regulating Transcription at the Chromatin Level ↵ Learning Objectives - Predict regions of the genome that are more likely to have “open” or “closed” chromatin and understand how acetylation affects chromatin. - Identify the molecular nature of DNA methylation, the types of bases that are methylated, and the effect of DNA methylation on gene expression. Eukaryotes regulate transcription via promoter sequences close to the transcription unit (as in prokaryotes) and also use more distant enhancer sequences to provide more variation in the timing, level, and location of transcription, however, there are still additional levels of genetic control. This consists of two major mechanism: (1) large-scale changes in chromatin structure, and (2) modification of bases in the DNA sequence. These two are often inter-connected. Chromatin remodeling Despite the simplified way in which we often represent DNA, DNA is almost always associated with various chromatin proteins. For example, histones remain associated with the DNA even during transcription. Thus the rate of transcription is also controlled by the accessibility of DNA to RNA polymerase and regulatory proteins. So, in regions with highly compact chromatin transcription is unlikely, even if all the necessary cis - and trans - factors are present in the nucleus. The extent of chromatin compaction in various regions is regulated through the action of chromatin remodeling proteins. These protein complexes include enzymes that add or remove chemical tags, such as methyl or acetyl groups, to various DNA bound proteins, often the histone proteins in nucleosomes. These post-translational modifications alter the local chromatin density and thus the availability for transcription. Acetylated histones, for example, tend to be associated with actively transcribed genes, whereas deacetylated histone are associated with genes that are silenced. Figure $\PageIndex{1}$: Acetylation of histone proteins is associated with more a more open chromatin configuration. Acetylation is a reversible process. (Origianl-Deyholos-CC:AN) Likewise, methylation of DNA itself is also associated with transcription regulation. Cytosine bases, particularly when followed by a guanine are important targets for DNA methylation. Using a p to represent the phosphodiester bond between the nucleotides, these are often called CpG sites , and regions in which many CpG sites are nearby are known as CpG islands. Methylated cytosine within clusters of CpG sites is often associated with transcriptionally inactive DNA. Thinking about CpG Why is C followed by G an important site for DNA methylation? Think about the sequence of both strands of DNA. What is the complementary sequence of 5' ...CG... 3' ? Does this allow both strands to be methylated? Figure $\PageIndex{2}$: A methylation reaction produces 5-methylcytosine (5mC). Methyl groups may also be removed by DNA demethylases. (flickr-Beardy Git-CC:AND) Common post-translational modifications to histones These post-translational modifications are the result of enzymes that covalently link functional groups to amino acids at specific positions in histones. - Acetylation - Methylation - Phosphorylation - Ubiquitination Combinations of these modifications was first postulated to be a "code" by which the regulation of genes is interpreted by David Allis and colleagues in 2001. Heritability of Chromatin The modification of DNA and its associated proteins is enzymatically reversible (acetylases/deacetylases; methyltranferases/demethylases) and thus a cyclical activity. However, when DNA is replicated, existing modifications on the parental strand will be added to the newly synthesized strand as well, maintaining the pattern in daughter cells until the modification is enzymatically removed. Regulation of these chemical modifications provides another layer through which eukaryotic cells control the transcription of specific genes. Code readers In addition to influencing an overall level of chromatin accessibility, modified histones may interact specifically with proteins that recognize their modifications to promote recruitment of additional proteins for functions in repair, replication, or transcription. Contributors and Attributions - Dr. Todd Nickle and Isabelle Barrette-Ng (Mount Royal University) The content on this page is licensed under CC SA 3.0 licensing guidelines.	libretexts	2025-03-17T22:27:31.434103	2019-10-01T21:21:15	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/", "url": "https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/06%3A_Regulation_of_Gene_Expression/6.02%3A_Eukaryotic_Gene_Regulation/6.2.02%3A_Regulating_Transcription_at_the_Chromatin_Level", "book_url": "https://commons.libretexts.org/book/bio-25718", "title": "6.2.2: Regulating Transcription at the Chromatin Level", "author": "Stefanie West Leacock" }
https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/06%3A_Regulation_of_Gene_Expression/6.03%3A_Epigenetics	6.3: Epigenetics Learning Objectives - Describe examples of gene regulation through DNA methylation and mechanisms of genomic imprinting. Explain how methyltransferases allow methylation to be heritable. - Explain how DNA methylation can be associated with monoallelic expression. The term epigenetics describes any heritable change in phenotype that is not associated with a change in the chromosomal DNA sequence. Originally it meant the processes through which the genes were expressed to give the phenotype; that is, the changes in gene expression that occur during normal development of multicellular organisms. This includes the change in transcriptional state of a DNA sequence (gene) via DNA or chromatin protein reversible modifications. Thus, DNA methylation and histone protein methylation, phosphorylation, and acetylation have been targeted as mechanisms for heritable changes in cells as they grow from a single cell (zygote) and differentiate to a multicellular organism. Here, dividing cells commit to differentiate into different tissues such as muscle, neuron, and fibroblast due to the genes that they express or silence. Some genes are irreversibly silenced, through epigenetic mechanisms, in some cell types, but not in others. This doesn’t involve any change in DNA sequence. Remember, these epigenetic effects are not permanent changes and thus cannot be selectable in an evolutionary context. However, mutations in the genes that regulate the epigenetic effect can be selected. Definition: Epigenetics Epigenetics describes any heritable change in phenotype that is not associated with a change in the chromosomal DNA sequence. Heritability of epigenetic modifications During DNA replication, parental strands are used as a template to create complementary strands. However, modified bases or histones are not copied directly. Instead, cells have enzymes that recognize partially modified DNA and add modifications to the newly synthesized DNA. One example of an enzyme that plays this role is DNMT1 (DNA methyltransferase 1). When chromosome with methylated DNA is replicated, the parental strand retains the methylation, but the new strand does not, resulting in hemi-methylated DNA. Recall that DNA methylation usually occurs at CpG sequences, such that a CpG also exists on the complementary strand at the same position. DNMT1 recognizes hemi-methylated DNA and adds the corresponding methyl group to the new strand. Research Examples Researchers have found many cases of environmentally induced changes in gene expression that can be passed on to subsequent generations – a multi-generational effect. These altered expression patterns represent the diversity of expression for a genome. This extended phenotype, the ability to influence traits in the next generation, is a topic of current research and only some examples will be discussed here. Epigenetics and maternal care Watch this introductory video about experiments that tested the relationship between maternal care and offspring in rats. In experiments, Meaney and colleagues examined the influence of maternal licking on epigenetic modifications of genes involved in stress. Previous studies had shown that maternal care was associated with reduced fear and stress responses in adult rats. To determine whether this effect was due to changes in gene expression, epigenetic modifications near the glucocorticoid receptor (GR) gene were examined. In rats raised by mothers exhibiting high licking-grooming behaviors, methylation of a CpG in the GR was absent and the region was associated with histone acetylation (Weaver et al, 2008). Both of these modifications are typically associated with active chromatin, or gene expression. This GR acts in a feedback loop to reduce some stress responses. Therefore, these findings are consistent with the rat behaviors with high levels of maternal care associated with higher expression of GR, reducing the stress response of adult rats. This example illustrates how the DNA nucleotide sequence of the offspring does not change, but their experiences and environments alters their gene expression profile and therefore their phenotype. Imprinting For some genes, the allele inherited from the oocyte is expressed differently than the allele that is inherited from the sperm. This pattern is distinct from sex-linkage and is true even if both alleles are wild-type and autosomal. During gamete development (gametogenesis), each parent imprints epigenetic information on some genes that will affect the activity of the gene in the offspring. Imprinting does not change the DNA sequence, but does involve methylation of DNA or histones, and generally silences the expression of one of the parent’s alleles. In humans, some genes are expressed only from the paternal allele, and other genes are expressed only from the maternal allele. The imprinting marks are reprogrammed before the next generation of gametes are formed. For example, a sperm-producing individual inherits epigenetic information from both and oocyte and sperm, but the epigenetic marks will be erased before sperm development and only one the sperm pattern of imprinting will be passed to his offspring, either male or female. Because imprinting often produces monoallelic expression of a gene, if one copy is mutated or lost, phenotypic changes may be observed. This phenomenon produces an exception to Mendelian rules about recessive alleles not producing a phenotype when heterozygous. Imprinted genes, of which there are currently about 100 genes in humans, appear to explain many parent-of-origin effects. For example, Prader-Willi Syndrome (PWS) and Angelman Syndrome (AS) are two phenotypically different conditions in humans that result from deletion of a specific region of chromosome 15, which contains several genes. Whether the deletion results in PWS or in AS depends on the parent-of-origin. If the deletion is inherited from the father, PWS results. Conversely, if the deletion is inherited from the mother, AS is the result. The gene(s) involved in PWS is maternally silenced by imprinting, therefore the deletion of its paternally-inherited allele results in a complete deficiency of a required protein. On the other hand, the paternal allele of the gene involved in AS is silenced by imprinting, so deletion of the maternal allele results in deficiency of the protein encoded by that gene. Transgenerational inheritance of nutritional influences Nutrition is one aspect of the environment that has been particularly well-studied from an epigenetic perspective in both mice and humans. Adults who were conceived during the Dutch famine of 1944-1945 have IGF2 genes that are less methylated than their siblings born before or after the famine, even decades after birth (Heijmans et al, 2008). The IGF2 protein is a signaling molecule that promotes growth and cell division. Further research is ongoing to determine how much impact this change in methylation has on phenotypes. A study of an isolated Swedish village called Överkalix provides an example of transgenerational inheritance of nutritional factors. Detailed historical records allowed researchers to infer the nutritional status of villagers going back to 1890. The researchers then studied the health of two generations of these villagers’ offspring, using medical records. A significant correlation was found between the mortality risk of grandsons and the food availability of their paternal grandfathers. This effect was not seen in the granddaughters. Furthermore, the nutrition of paternal grandmothers, or either of the maternal grandparents did not affect the health of the grandsons. It was therefore proposed that epigenetic information affecting health (specifically diabetes and heart disease) was passed from the grandfathers, to the grandsons, through the male line (Pembrey et al, 2006). At least one study has recently replicated this finding in an independent population from Uppsala, Sweden (Vågerö et al, 2021) In mouse models, the agouti gene produces a signaling molecule that regulates pigment-producing cells and brain cells that affect feeding and body weight. Normally, agouti is silenced by methylation, and these mice are brown and have a normal weight. When agouti is demethylated by feeding certain chemicals (for example) or by mutating a gene that controls methylation, some mice become yellow and overweight. Although the nucleotide sequence of the gene remains unchanged, the methylation at the locus decreases, causing overexpression of the protein (review 4.4: Exceptions to simple dominance ). Methylation of agouti and normal weight and pigmentation of offspring can be restored if their mothers are fed folic acid and other vitamins during pregnancy. Vernalization as an example of epigenetics Many plant species in temperate regions are winter annuals, meaning that their seeds germinate in the late summer, and grow vegetatively through early fall before entering a dormant phase during the winter, often under a cover of snow. In the spring, the plant resumes growth and is able to produce seeds before other species that germinated in the spring. In order for this life strategy to work, the winter annual must not resume growth or start flower production until winter has ended. R he requirement to experience a long period of cold temperatures prior to flowering is called vernalization. How does a plant sense that winter has passed? The signal for resuming growth cannot simply be warm air temperature, since occasional warm days, followed by long periods of freezing, are common in temperate climates. Researchers have discovered that winter annuals use epigenetic mechanisms to sense and “remember” that winter has occurred. Fortunately for the researchers who were interested in vernalization, some varieties of Arabidopsis are winter annuals. Through mutational analysis of Arabidopsis , researchers found that a gene called FLC ( FLOWERING LOCUS C ) encodes a transcription repressor acting on several of the genes involved in early stages of flowering (Michaels and Amasino, 1999). In the fall and under other warm conditions, the histones associated with FLC are acetylated and so FLC is transcribed at high levels; expression of flowering genes is therefore entirely repressed. However, in response to cold temperatures, enzymes remove methyl and acetyl groups from the histones associated with the FLC locus. The longer the cold temperatures persist, the more acetyl groups are removed, until finally the FLC locus is no longer transcribed and the flowering genes are free to respond to other environmental and hormonal signals that induce flowering later in the spring. Because the deacetylated state of FLC is inherited as cells divide and the plant grows in the early spring, this is an example of a type of cellular memory mediated by an epigenetic mechanism. Query $\PageIndex{1}$ References Bygren LO, Tinghög P, Carstensen J, Edvinsson S, Kaati G, Pembrey ME, Sjöström M. Change in paternal grandmothers' early food supply influenced cardiovascular mortality of the female grandchildren. BMC Genet. 2014 Feb 20;15:12. doi: 10.1186/1471-2156-15-12. PMID: 24552514; PMCID: PMC3929550. He Y, Michaels SD, Amasino RM. Regulation of flowering time by histone acetylation in Arabidopsis. Science. 2003 Dec 5;302(5651):1751-4. doi: 10.1126/science.1091109. Epub 2003 Oct 30. PMID: 14593187. Heijmans BT, Tobi EW, Stein AD, et al. Persistent epigenetic differences associated with prenatal exposure to famine in humans. Proc Natl Acad Sci U S A . 2008;105(44):17046-17049. doi:10.1073/pnas.0806560105 ( https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2579375/ ) Michaels SD, Amasino RM, FLOWERING LOCUS C Encodes a Novel MADS Domain Protein That Acts as a Repressor of Flowering, The Plant Cell , Volume 11, Issue 5, May 1999, Pages 949–956, https://doi.org/10.1105/tpc.11.5.949 . Pembrey ME, Bygren LO, Kaati G, Edvinsson S, Northstone K, Sjöström M, Golding J; ALSPAC Study Team. Sex-specific, male-line transgenerational responses in humans. Eur J Hum Genet. 2006 Feb;14(2):159-66. doi: 10.1038/sj.ejhg.5201538. PMID: 16391557. Vågerö D, Pinger PR, Aronsson V, van den Berg GJ. Paternal grandfather's access to food predicts all-cause and cancer mortality in grandsons. Nat Commun. 2018 Dec 11;9(1):5124. doi: 10.1038/s41467-018-07617-9. Erratum in: Nat Commun. 2021 Mar 23;12(1):1954. PMID: 30538239; PMCID: PMC6290014. Weaver, I., Cervoni, N., Champagne, F. et al. Epigenetic programming by maternal behavior. Nat Neurosci 7, 847–854 (2004). https://doi.org/10.1038/nn1276 Contributors and Attributions - Dr. Todd Nickle and Isabelle Barrette-Ng (Mount Royal University) The content on this page is licensed under CC SA 3.0 licensing guidelines.	libretexts	2025-03-17T22:27:31.543934	2019-10-01T21:21:15	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/", "url": "https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/06%3A_Regulation_of_Gene_Expression/6.03%3A_Epigenetics", "book_url": "https://commons.libretexts.org/book/bio-25718", "title": "6.3: Epigenetics", "author": "Stefanie West Leacock" }
https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/06%3A_Regulation_of_Gene_Expression/6.04%3A_Regulation_of_Gene_Expression_(Exercises)	6.4: Regulation of Gene Expression (Exercises) - - Last updated - Save as PDF These are homework exercises to accompany Nickle and Barrette-Ng's " Online Open Genetics " TextMap. Genetics is the scientific study of heredity and the variation of inherited characteristics. It includes the study of genes, themselves, how they function, interact, and produce the visible and measurable characteristics we see in individuals and populations of species as they change from one generation to the next, over time, and in different environments. Study Questions: 12.1 List all the mechanisms that can be used to regulate gene expression in eukaryotes. 12.2 With respect to the expression of β-galactosidase, what would be the phenotype of each of the following strains of E. coli ? a) I + , O + , Z + , Y + (no glucose, no lactose) b) I + , O + , Z + , Y + (no glucose, high lactose) c) I + , O + , Z + , Y + (high glucose, no lactose) d) I + , O + , Z + , Y + (high glucose, high lactose) e) I + , O + , Z - , Y + (no glucose, no lactose) f) I + , O + , Z - , Y + (high glucose, high lactose) g) I + , O + , Z + , Y - (high glucose, high lactose) h) I + , Oc, Z + , Y + (no glucose, no lactose) i) I + , Oc,Z + , Y + (no glucose, high lactose) j) I + , Oc, Z + , Y + (high glucose, no lactose) k) I + , Oc, Z + , Y + (high glucose, high lactose) l) I - , O + , Z + , Y + (no glucose, no lactose) m) I - , O + , Z + , Y + (no glucose, high lactose) n) I - , O + , Z + , Y + (high glucose, no lactose) o) I - , O + , Z + , Y + (high glucose, high lactose) p) I s , O + , Z + , Y + (no glucose, no lactose) q) I s , O + , Z + , Y + (no glucose, high lactose) r) I s , O + , Z + , Y + (high glucose, no lactose) s) I s , O + , Z + , Y + (high glucose, high lactose) 12.3 In the E. coli strains listed below, some genes are present on both the chromosome, and the extrachromosomal F - factor episome. The genotypes of the chromosome and episome are separated by a slash. What will be the β - galactosidase phenotype of these strains? All of the strains are grown in media that lacks glucose.a) I + , O + , Z + , Y + / O - , Z - , Y - (high lactose) b) I + , O + , Z + , Y + / O - , Z - , Y - (no lactose) c) I + , O + , Z - , Y + / O - , Z + , Y + (high lactose) d) I + , O + , Z - , Y + / O - , Z + , Y + (no lactose) e) I + , O + , Z - , Y + / I - , O + , Z + , Y + (high lactose) f) I + , O + , Z - , Y + / I - , O + , Z + , Y + (no lactose) g) I - , O + , Z + , Y + / I + , O + , Z - , Y + (high lactose) h) I - , O + , Z + , Y + / I + , O + , Z - , Y + (no lactose) i) I + , Oc, Z + , Y + / I + , O + , Z - , Y + (high lactose) j) I + , Oc, Z + , Y + / I + , O + , Z - , Y + (no lactose) k) I + , O + , Z - , Y + / I + , Oc, Z + , Y + (high lactose) l) I + , O + , Z - , Y + / I + , Oc, Z + , Y + (no lactose) m) I + , O + , Z - , Y + / I s , O + , Z + , Y + (high lactose) n) I + , O + , Z - , Y + / I s , O + , Z + , Y + (no lactose) o) I s , O + , Z + , Y + / I + , O + , Z - , Y + (high lactose) p) I s , O + , Z + , Y + / I + , O + , Z - , Y + (no lactose) 12.1 Transcriptional: initiation, processing & splicing, degradation Translational: initiation, processing, degradation Post-translational: modifications (e.g. phosphorylation), localization Others: histone modification, other chromatin remodeling, DNA methylation 12.2 Legend: +++ Lots of β-galactosidase activity + Moderate β-galactosidase activity -- No β-galactosidase activity -- a) I + , O + , Z + , Y + (no glucose, no lactose) +++ b) I + , O + , Z + , Y + (no glucose, high lactose) -- c) I + , O + , Z + , Y + (high glucose, no lactose) + d) I + , O + , Z + , Y + (high glucose, high lactose) -- e) I + , O + , Z - , Y + (no glucose, no lactose) -- f) I + , O + , Z - , Y + (high glucose, high lactose) + g) I + , O + , Z + , Y - (high glucose, high lactose) +++ h) I + , Oc, Z + , Y + (no glucose, no lactose) +++ i) I + , Oc,Z + , Y + (no glucose, high lactose) + j) I + , Oc, Z + , Y + (high glucose, no lactose) + k) I + , Oc, Z + , Y + (high glucose, high lactose) +++ l) I - , O + , Z + , Y + (no glucose, no lactose) +++ m) I - , O + , Z + , Y + (no glucose, high lactose) + n) I - , O + , Z + , Y + (high glucose, no lactose) + o) I - , O + , Z + , Y + (high glucose, high lactose) -- p) I s , O + , Z + , Y + (no glucose, no lactose) -- q) I s , O + , Z + , Y + (no glucose, high lactose) -- r) I s , O + , Z + , Y + (high glucose, no lactose) -- s) I s , O + , Z + , Y + (high glucose, high lactose) 12.3 Legend: +++ Lots of β-galactosidase activity + Moderate β-galactosidase activity -- No β-galactosidase activity +++ a) I + , O + , Z + , Y + / O - , Z - , Y - (high lactose) -- b) I + , O + , Z + , Y + / O - , Z - , Y - (no lactose) +++ c) I + , O + , Z - , Y + / O - , Z + , Y + (high lactose) + d) I + , O + , Z - , Y + / O - , Z + , Y + (no lactose) +++ e) I + , O + , Z - , Y + / I - , O + , Z + , Y + (high lactose) -- f) I + , O + , Z - , Y + / I - , O + , Z + , Y + (no lactose) +++ g) I - , O + , Z + , Y + / I + , O + , Z - , Y + (high lactose) -- h) I - , O + , Z + , Y + / I + , O + , Z - , Y + (no lactose) +++ i) I + , Oc, Z + , Y + / I + , O + , Z - , Y + (high lactose) +++ j) I + , Oc, Z + , Y + / I + , O + , Z - , Y + (no lactose) +++ k) I + , O + , Z - , Y + / I + , Oc, Z + , Y + (high lactose) +++ l) I + , O + , Z - , Y + / I + , Oc, Z + , Y + (no lactose) -- m) I + , O + , Z - , Y + / I s , O + , Z + , Y + (high lactose) -- n) I + , O + , Z - , Y + / I s , O + , Z + , Y + (no lactose) -- o) I s , O + , Z + , Y + / I + , O + , Z - , Y + (high lactose) -- p) I s , O + , Z + , Y + / I + , O + , Z - , Y + (no lactose) 12.4 You could demonstrate this with just I + O c Z - / I + O + Z + . The fact that this does not have constitutive lactose expression shows that the operator only acts on the same piece of DNA on which it is located. There are also other possible answers. 12.5 You could also demonstrate this with just I + O + Z - / I - O + Z + . The fact that this has the same lactose-inducible phenotype as wild-type hows that a functional lacI gene can act on operators on both the same piece of DNA from which it is transcribed, or on a different piece of DNA. There are also other possible answers. 12.6 For all of these, the answer is the same: The l ac operon would be inducible by lactose, but only moderate expression of the lac operon would be possible, even in the absence of glucose a) loss-of-function of adenylate cyclase b) loss of DNA binding ability of CAP c) loss of cAMP binding ability of CAP d) mutation of CAP binding site (CBS) cis -element so that CAP could not bind 12.7 Both involve trans -factors binding to corresponding cis -elements to regulate the initiation of transcription by recruiting or stabilizing the binding of RNApol and related transcriptional proteins at the promoter. In prokaryotes, genes may be regulated as a single operon. In eukaryotes, enhancers may be located much further from the promoter than in prokaryotes. 12.8 These fish would all have spiny tales like the deep-water population. 12.9 These could have arisen from loss-of-function mutation in FLC , or in the cis-element to which FLC normally binds. 12.10 If there was no deacetylation of FLC by HDAC, transcription of FLC might continue constantly, leading to constant suppression of flowering, even after winter.	libretexts	2025-03-17T22:27:31.670058	2019-10-01T21:21:16	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/", "url": "https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/06%3A_Regulation_of_Gene_Expression/6.04%3A_Regulation_of_Gene_Expression_(Exercises)", "book_url": "https://commons.libretexts.org/book/bio-25718", "title": "6.4: Regulation of Gene Expression (Exercises)", "author": "Stefanie West Leacock" }
https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/07%3A_Manipulating_and_Analyzing_Genes_and_Genomes/7.02%3A_Anthropological_Genetics	7.2: Anthropological Genetics - Define Anthropology and explain how anthropology and genetics are connected. - Identify breakthroughs in genetics that contributed to anthropology. - Apply anthropological knowledge and genetic techniques in real world situations. - Determine when scientific work is or is not appropriate using anthropological ethics. Section 1: What is Anthropology? Anthropology ( Anthropo means human) is the scientific study of all human species (including extinct ones) and nonhuman primates, and includes the impact of humans on the world throughout all time. This includes humans’ impact on the environment, other living organisms, and other human populations. Anthropology often examines cultures and how they develop, how languages develop and evolve over time, how humans have impacted animals and the environment, how humans interact with one another, belief systems like religion and mythology, and more. Anthropology is inherently interdisciplinary since humans have interacted with and impacted most fields of science. Anything humans are involved in is anthropological in nature. That said, anthropologists often specialize in four categories: Biological Anthropology, Archaeology, Cultural Anthropology, or Linguistic Anthropology. Biological Anthropology is what will be the focus of this section. - Biological Anthropology- the study of human (and non-human primate) evolution and adaptation. This field includes such specialties as forensic anthropology, primatology, and human osteology, among many others. Every pre-health major who intends to work with humans will be conducting anthropological work, even if it is never called that. Every teacher sharing knowledge is contributing to anthropology, and every lesson learned came from anthropological work and is inherently anthropological in nature. Section 2: Humans, and How Genetics Contributed to Anthropological Understanding Anthropological techniques enabled science to begin reconstructing the evolution of hominid species. Hominids diverged from other primates millions of years ago and the groups began evolving independently of one another. Before we could sequence genes, these techniques were used to seriate these hominins. To do this, anthropologists look at data regarding the body in order to determine certain characteristics. For example, we know that bipedal hominids evolved when they did based on the fossil record. Creatures that walk on four legs have a foramen magnum, the opening through which the spinal cord passes, that is situated toward the back of the skull. In contrast, bipedal hominids have a foramen magnum that is situated at the base of the skull. Further features are factored in, such as the size of the brain case , dentition, orientation of certain joints, and body size, among others. Certain features of the body, such as whether or not the epiphyseal plates are fused and the size of the pelvis if it belongs to an adult skeleton, can help us further identify remains. These techniques are also minimally invasive and do not result in destruction of any part of the remains, while genetic techniques require a sample. For some of these ancient species, very little remains still exist and are known to us, so genetic techniques are used sparingly. Whenever possible, dating techniques are used on suitable materials found in situ with these remains to determine its age range. However, DNA sequencing has been used to do such archaeological work as reconstructing the influx of humans into North America . As technology continues to advance, it has become feasible for scientists to sequence genetic material that is both very old and from a very small sample . We just have to find viable DNA to sequence, and in some of these ancient ancestors there is not a lot of DNA left to recover. The only currently extant species of hominids is Homo sapiens . However, proof of the overlap of certain species can be found in the DNA of some people. Neanderthal DNA is found in approximately 1-4% of living humans and Denisovan DNA (not represented in the figure) is found in 4-6% of living human populations. Modern humans, Homo sapiens , have existed for approximately 300,000 years. The first modern humans emerged in Africa and spread out over time from there. As these populations got further from the equator, the UV levels dropped, allowing offspring with lighter skin tones to live to adulthood and reproduce . Certain human populations experienced periods of isolation from outside gene flow. When these periods are extended over many generations, this can eventually produce phenotypic variations between the isolated population and other populations. This accounts for the differences in ethnicity seen in different regions of the globe. Before modern dating techniques were discovered, human evolution was determined using seriation techniques to provide order to morphological and evolutionary changes seen in the fossil record for hominids. This was followed by the advent of various absolute dating techniques, which allowed science to determine where fossils fall within the true timeline. Now, genetic analysis has allowed us to create an even more accurate reconstruction of how modern humans evolved. The Human Genome Project One of the most well known, if not the best known, works regarding human genetic analysis is the Human Genome Project , or HGP. The HGP was a multibillion dollar project featuring international collaboration to sequence the entirety of the human genome. The project was launched in 1990 and completed in 2003. This international collaboration also saw legislators get involved in order to ensure the knowledge could not be patented and had to be shared freely with everyone. These legislators included the U.S. President Bill Clinton and then Prime Minister Tony Blair. Further legislation was introduced in the wake of the HGP to protect people from being discriminated against based on their genetic information, including the Health Insurance Portability and Accountability Act ( HIPAA ) in 1996 and the Genetic Information Nondiscrimination Act ( GINA ) in 2008. This project helped science determine that all currently living humans share 99.9% the same DNA no matter their phenotypic differences. This work proved that humans are definitively a single species and race. Most of the variations between humans are SNPs. There is still much more to learn about the human genome, despite knowing its sequence. As with many scientific breakthroughs, answering one question (sequencing the human genome) created many more unanswered questions. Section 3: The Ethics of Working with Humans Anthropologists are passionate about humans. Otherwise, you would have chosen to work with another species! Humans are complicated subjects when compared to other living organisms. Humans have independent thought, their own feelings and drives, and a variety of beliefs that can impact how people view the world. In order to conduct science pertaining to humans, a different set of ethics comes into play versus when we work with animals. Professional societies are a source of information regarding standards and ethics in their fields. The American Anthropological Association provides guidance on ethics when working with humans or humans remains. - Do no harm. - Be open and honest regarding your work. This also means you should publish your findings regardless of outcome and as quickly as possible. - Obtain informed consent and necessary permissions. - Weigh competing ethical obligations due collaborators and affected parties. - Make your results accessible. - Protect and preserve your records. - Maintain respectful and ethical professional relationships. This link AAA Statement on Ethics - The American Anthropological Association includes further explanations of these guidelines. The Society for American Archaeology provides additional guidance to consider when dealing with human remains: - Stewardship- You are managing these remains for all parties. Take responsible care of this person, including being respectful of that person’s beliefs and the beliefs of their descendants. - Commercialization- Never use human remains for profit. Never sell any goods belonging to the deceased, including data about them. - Ensure Safe Educational and Workplace Environments- Always be mindful of respecting others, including the deceased. Be respectful of others working around you as well. Treat everyone equally. Free speech and respectful open debate are encouraged when interpreting the data. Differing perspectives are welcome! Working with humans requires a lot of respect and empathy, and this extends to being considerate of the deceased. A person’s rights and autonomy do not end at their death, so we must respect their wishes for how to treat their remains. Even when we are granted permission to use a person’s remains for scientific research, we must aim to conduct this work with respect to the deceased and their descendants. When working with remains, handle them with care. Bones should be handled over a cushioned surface. The skull should always be picked up with care. Do not use the eye orbits, foramen magnum, or zygomatic arches to pick up a skull. Carry it with both hands. Remember that even a human skeleton was once a person. Certain groups of people have specific regulations for handling remains belonging to their populations. In the United States, this often relates to the handling of Native American remains and funerary objects. The Native American Grave Protection and Repatriation Act, or NAGPRA, was enacted in 1990 and provides protections to these groups. Prior to this legislation, Native American remains were often excavated without regard for the remains or the descendant communities, who often have strong ties to their ancestors. Science has a long and questionable history with misusing subjects in experiments. Examples include the history of animal treatment , human rights violations such as the Tuskegee experiment , and even the history behind HeLa cells (referenced in 1.3.1 ). Throughout history, certain groups have faced challenges receiving adequate medical care. Henrietta Lacks was an African-American woman who was diagnosed with cervical cancer in 1951. She received treatment for her cancer at Johns Hopkins Hospital in Baltimore, MD, one of the few hospitals at the time who treated African-American patients for cancer. While undergoing treatment, a sample was taken from Mrs. Lacks’ tumor. Dr. George Gey, a physician employed at Johns Hopkins, was looking for “immortal” cells that could be used in cancer research. The cells within this sample fit the bill. The cells survived and multiplied at high rates; they are still used today. Mrs. Lacks passed away in late 1951. While the cells got their name from He nrietta La cks, HeLa cells, Mrs. Lacks was never told this sample was taken or used. Informed consent was never obtained, and Mrs. Lacks and her family were not credited in any way until decades afterward. Her cells have contributed to cancer research and the development of pharmaceuticals for polio, Parkinsons, and leukemia. Her cells have saved countless lives. Some of the scientists who used the cells went on to win the Nobel Prize. Mrs. Lacks played a pivotal role in all of these breakthroughs, and continues to contribute to scientific understanding. In order for science and medicine to heal the misgivings people can have about participating in things like clinical studies, we must ensure we are always honest, objective, and obtain informed consent. References and Resources: AAA Statement on Ethics. 15 March 2024. < https://americananthro.org/about/pol...ent-on-ethics/ >. Animal Welfare Act Timeline. 15 March 2024. < https://www.nal.usda.gov/collections...re-prohibition >. BBC Global. Henrietta Lacks: The 'immortal' cells that changed the world. 30 October 2020. < https://www.youtube.com/watch?v=pgB1IqGp8BE >. Dehay, Colette and Henry Kennedy. Evolution of the Human Brain. 31 July 2020. < https://www.science.org/doi/full/10....cience.abd1840 >. Etheredge, Laura. Henrietta Lacks. 7 March 2024. < https://www.britannica.com/biography/Henrietta-Lacks >. Ethics in Archaeology. 7 March 2024. < https://www.saa.org/career-practice/...in-archaeology >. Genetic Information. 16 June 2017. < https://www.hhs.gov/hipaa/for-profes...%20or%20Titles >. Genetics vs. Genomics Fact Sheet. 7 September 2018. < https://www.genome.gov/about-genomic...netic%20makeup >. Gibbons, Ann. Ancient Skulls May Belong to Elusive Humans Called Denisovans. 2 March 2017. < https://www.science.org/content/arti...led-denisovans >. Health Insurance Portability and Accountability Act of 1996 (HIPAA). 27 June 2022. < https://www.cdc.gov/phlp/publication...pic/hipaa.html >. Hernandez, Joe. Henrietta Lacks' descendants reach a settlement over the use of her 'stolen' cells. 1 August 2023. < https://www.npr.org/2023/08/01/11912...t-stolen-cells >. How Genetics Interacts with Biological Anthropology. 29 January 2024. < https://www.youtube.com/watch?v=RpUv...Uwimvg&index=3 >. Human Genome Project. 24 August 2022. < https://www.genome.gov/about-genomic...genome-project >. Introduction to Anthropology. 2 January 2024. https://www.youtube.com/watch?v=LYUz...SXp8vqxAUwimvg . Leen, Sarah. Human Skin Color Variation. 3 January 2024. < https://humanorigins.si.edu/evidence...olor-variation >. Professor Dave Explains. History of Biological Anthropology (Up to Genetics). 15 January 2024. < https://www.youtube.com/watch?v=FZUY...Uwimvg&index=3 >. The U.S. Public Health Service Untreated Syphilis Study at Tuskegee. 5 December 2022. < https://www.cdc.gov/tuskegee/timeline.htm >. Waters, Michael R. Late Pleistocene Exploration and Settlement of the Americas by Modern Humans. 12 July 2019. < https://www.science.org/doi/full/10....cience.aat5447 >. This page was written and curated by Tosha Aleck at the University of Arkansas - Little Rock.	libretexts	2025-03-17T22:27:31.868768	2024-04-11T18:55:03	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/07%3A_Manipulating_and_Analyzing_Genes_and_Genomes/7.02%3A_Anthropological_Genetics", "book_url": "https://commons.libretexts.org/book/bio-25718", "title": "7.2: Anthropological Genetics", "author": "Stefanie West Leacock" }
https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/08%3A_Cancer_Genetics	8: Cancer Genetics Cancer is a group of diseases that exhibit uncontrolled growth, invasion of adjacent tissues, and sometimes metastasis (the movement of cancer cells through the blood or lymph). In cancer cells, the regulatory mechanisms that control cell division and limit abnormal growth have been disrupted, usually by the accumulation of several mutations. Cancer is therefore essentially a genetic disease. Although some cancer-‐related mutations may be heritable, most cancers are sporadic, meaning they arise from new mutations that occur in the individual who has the disease. In this chapter we will examine the connection between cancer and genes. Contributors and Attributions - Dr. Todd Nickle and Isabelle Barrette-Ng (Mount Royal University) The content on this page is licensed under CC SA 3.0 licensing guidelines.	libretexts	2025-03-17T22:27:32.081488	2019-10-01T21:21:16	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/", "url": "https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/08%3A_Cancer_Genetics", "book_url": "https://commons.libretexts.org/book/bio-25718", "title": "8: Cancer Genetics", "author": "Stefanie West Leacock" }
https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/08%3A_Cancer_Genetics/8.01%3A__Classification_of_Cancers	8.1: Classification of Cancers selected template will load here This action is not available. Cancers can be classified based on the tissues in which they originate. Sarcomas are cancers that originate in mesoderm tissues, such as bone or muscle, and cancers arising in glandular tissues (e.g. breast, prostate) are classified as adenocarcinomas . Carcinomas originate in epithelial cells (both inside the body and on its surface) and are the most common types of cancer (~85%). Each of these classifications may be further sub-‐divided. For example, squamous cell carcinoma (SCC) , basal cell carcinoma (BCC) , and melanoma are all types of skin cancers originating respectively in the squamous cells, basal cells, or melanocytes of the skin.	libretexts	2025-03-17T22:27:32.143780	2019-10-01T21:21:17	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/", "url": "https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/08%3A_Cancer_Genetics/8.01%3A__Classification_of_Cancers", "book_url": "https://commons.libretexts.org/book/bio-25718", "title": "8.1: Classification of Cancers", "author": "Stefanie West Leacock" }
https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/08%3A_Cancer_Genetics/8.02%3A_Hallmarks_of_Cancer	8.2: Hallmarks of Cancer Researchers have identified molecular and cellular traits that characterize most cancers. An original six hallmarks of cancer were identified and later four additional hallmarks were added (Hanahan and Weinberg, 2000; Hanahan and Weinberg, 2011 https://www.cell.com/fulltext/S0092-8674(11)00127-9 ). Hallmarks of Cancer - Growth signal autonomy: Cancer cells can divide without the external signals normally required to stimulate division. - Insensitivity to growth inhibitory signals: Cancer cells are unaffected by external signals that inhibit division of normal cells. - Evasion of apoptosis: When excessive DNA damage and other abnormalities are detected, apoptosis (a type of programmed cell death) is induced in normal cells, but not in cancer cells. - Reproductive potential not limited by telomeres: Each division of a normal cell reduces the length of its telomeres. Normal cells arrest further division once telomeres reach a certain length. Cancer cells avoid this arrest and/or maintain the length of their telomeres. - Sustained angiogenesis: Most cancers require the growth of new blood vessels into the tumor. Normal angiogenesis is regulated by both inhibitory and stimulatory signals not required in cancer cells. - Tissue invasion and metastasis: Normal cells generally do not migrate (except in embryo development). Cancer cells invade other tissues including vital organs. - Deregulated metabolic pathways: Cancer cells use an abnormal metabolism to satisfy a high demand for energy and nutrients. - Evasion of the immune system: Cancer cells are able to evade the immune system. - Chromosomal instability: Severe chromosomal abnormalities are found in most cancers. - Inflammation: Local chronic inflammation is associated with many types of cancer. Cancer hallmarks reflect genetic changes Previous chapters have covered many topics that are related to these hallmarks. For each hallmark, try to think of a gene or process that could be mutated to produce the effect. Contributors and Attributions - Dr. Todd Nickle and Isabelle Barrette-Ng (Mount Royal University) The content on this page is licensed under CC SA 3.0 licensing guidelines.	libretexts	2025-03-17T22:27:32.201837	2019-10-01T21:21:18	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/", "url": "https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/08%3A_Cancer_Genetics/8.02%3A_Hallmarks_of_Cancer", "book_url": "https://commons.libretexts.org/book/bio-25718", "title": "8.2: Hallmarks of Cancer", "author": "Stefanie West Leacock" }
https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/08%3A_Cancer_Genetics/8.03%3A_Mutagens_and_Carcinogens	8.3: Mutagens and Carcinogens A carcinogen is any agent that directly increases the incidence of cancer. Most, but not all, carcinogens are mutagens. Carcinogens that do not directly damage DNA include substances that accelerate cell division, thereby leaving less opportunity for cell to repair induced mutations, or errors in replication. Carcinogens that act as mutagens may be biological, physical, or chemical in nature, although the term is most often used in relation to chemical substances. Human Papilloma Virus ( HPV , Figure $\PageIndex{4}$) is an example of a biological carcinogen. Almost all cervical cancers begin with infection by HPV, which contains genes that disrupt the normal pattern of cell division within the host cell. Any gene that leads to an uncontrolled increase in cell division is called an oncogene . The HPV E6 and E7 genes are considered oncogenes because they inhibit the host cell’s natural tumor suppressing proteins (include p53, described below). The product of the E5 gene mimics the host’s own signals for cell division, and these and other viral gene products may contribute to dysplasia, which is detected during a Pap smear (Figure $\PageIndex{5}$). Detection of abnormal cell morphology in a Pap smear is not necessarily evidence of cancer. It must be emphasized again that cells have many regulatory mechanisms to limit division and growth, and for cancer to occur, each of these mechanisms must be disrupted. This is one reason why only a minority of individuals with HPV infections ultimately develop cancer. Although most HPV-related cancers are cervical, HPV infection can also lead to cancer in other tissues, in both women and men. Figure $\PageIndex{4}$: Electron micrograph of HPV. (Wikipedia-Unknown-PD) Figure $\PageIndex{5}$: Dysplastic (left) and normal (right) cells from a Pap smear. (Flickr-Ed Uthman-CC:AS) Radiation is a well-known physical carcinogen, because of its potential to induce DNA damage within the body. The most damaging type of radiation is ionizing , meaning waves or particles with sufficient energy to strip electrons from the molecules they encounter, including DNA or molecules that can subsequently react with DNA. Ionizing radiation, which includes x-rays, gamma rays, and some wavelengths of ultraviolet rays, is distinct from the non-ionizing radiation of microwave ovens, cell phones, and radios. As with other carcinogens, mutation of multiple, independent genes that normally regulate cell division is required before cancer develops. Chemical carcinogens (Table $\PageIndex{2}$) can be either natural or synthetic compounds that, based on animal feeding trials or epidemiological (i.e. human population) studies, increase the incidence of cancer. The definition of a chemical as a carcinogen is problematic for several reasons. Some chemicals become carcinogenic only after they are metabolized into another compound in the body; not all species or individuals may metabolize chemicals in the same way. Also, the carcinogenic properties of a compound are usually dependent on its dose. It can be difficult to define a relevant dose for both lab animals and humans. Nevertheless, when a correlation between cancer incidence and chemical exposure is observed, it is usually possible to find ways to reduce exposure to that chemical. \| Table $\PageIndex{2}$: Some classes of chemical carcinogens (Pecorino 2008) \| \| 1. PAHs (polycyclic aromatic hydrocarbons) : e.g. benzo[a]pyrene and several other components of the smoke of cigarettes, wood, and fossil fuels 2. Aromatic amines : e.g. formed in food when meat (including fish, poultry) are cooked at high temperature 3. Nitrosamines and nitrosamides : e.g. found in tobacco and in some smoked meat and fish 4. Azo dyes : e.g. various dyes and pigments used in textiles, leather, paints. 5. Carbamates : e.g. ethyl carbamate (urethane) found in some distilled beverages and fermented foods 6. Halogenated compounds : e.g. pentachlorophenol used in some wood preservatives and pesticides. 7. Inorganic compounds : e.g. asbestos; may induce chronic inflammation and reactive oxygen species 8. Miscellaneous compounds : e.g. alkylating agents, phenolics \| Testing for carcinogens The Ames Test ( 4.1.3 Genetic screens ) can be used as one screen for carcinogenic (mutagenic) potential. Because all living things share the same genetic material (DNA), a chemical that can damage DNA in one cell type may also damage DNA in another cell type. Such tests do not perfectly predict carcinogenic activity; for example, a chemical that is not carcinogenic in one organism might be metabolized to a carcinogenic form in another organism. Contributors and Attributions - Dr. Todd Nickle and Isabelle Barrette-Ng (Mount Royal University) The content on this page is licensed under CC SA 3.0 licensing guidelines.	libretexts	2025-03-17T22:27:32.262537	2019-10-01T21:21:19	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/", "url": "https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/08%3A_Cancer_Genetics/8.03%3A_Mutagens_and_Carcinogens", "book_url": "https://commons.libretexts.org/book/bio-25718", "title": "8.3: Mutagens and Carcinogens", "author": "Stefanie West Leacock" }
https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/08%3A_Cancer_Genetics/8.04%3A_Oncogenes	8.4: Oncogenes The control of cell division involves many different genes. Some of these genes act as signaling molecules to activate normal progression through the cell cycle. One of the pre-requisites for cancer occurs when one or more of these activators of cell division become mutated. The mutation may involve a change in the coding sequence of the protein, so that it is more active than normal, or a change in the regulation of its expression, so that it is produced at higher levels than normal, or persists in the cell longer than normal. Genes that are a part of the normal regulation of cell division, but which after mutation contribute to cancer, are called proto-oncogenes . Once a proto-oncogene has been abnormally activated by mutation, it is called an oncogene. More than 100 genes have been defined as proto-oncogenes. These include genes at almost every step of the signaling pathways that normally induce cell to divide, including growth factors, receptors , signal transducers , and transcription factors. The ras oncogene was originally identified from cancer-causing viruses. ras is an example of a proto-oncogene; this protein acts as a transducer in signal transduction pathways, including the regulation of cell division. When a receptor protein receives a signal for cell division, the receptor activates ras , which in turn activates other signaling components, ultimately leading to activation of genes involved in cell division. Certain mutations of the ras sequence causes it to be in a permanently active form, which can lead to constitutive activation of the cell cycle. This mutation is dominant as are most oncogenes. An example of the role of ras in relaying a signal for cell division in the EGF pathway is shown in Figure $\PageIndex{2}$. Ras mutations in cancer There are three ras homologs in the human genome (HRas, NRas, and KRas). As many cancer genomes are sequenced, databases such as COSMIC ( http://www.sanger.ac.uk/cosmic ) can be used to search mutations across cancer types. Visit the site and search for HRAS, NRAS, or KRAS. Can you find answers to these questions? - What amino acid positions are frequently mutated? - In what cancer types is the gene most often mutated? - Are the mutations most often missense, nonsense, frameshift, or silent? Is this what you would have predicted -- why or why not? Exercise $\PageIndex{1}$ All human cells have proto-oncogenes. True or False? - Answer - True. Exercise $\PageIndex{2}$ Are the mutations that convert proto-oncogenes to oncogenes like to be loss-of-function or gain-of-function mutations? Explain your reasoning. - Answer - Oncogenic mutations are typically gain-of-function, resulting in increased protein activity and therefore increased cell proliferation. A loss-of-function mutation in a proto-oncogene, would result in reduced cell proliferation, not cancer. Contributors and Attributions - Dr. Todd Nickle and Isabelle Barrette-Ng (Mount Royal University) The content on this page is licensed under CC SA 3.0 licensing guidelines.	libretexts	2025-03-17T22:27:32.322651	2019-10-01T21:21:20	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/", "url": "https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/08%3A_Cancer_Genetics/8.04%3A_Oncogenes", "book_url": "https://commons.libretexts.org/book/bio-25718", "title": "8.4: Oncogenes", "author": "Stefanie West Leacock" }
https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/08%3A_Cancer_Genetics/8.05%3A_Tumor_Suppressor_Genes	8.5: Tumor Suppressor Genes More than 30 genes are classified as tumor suppressors . The normal functions of these genes include repair of DNA, induction of programmed cell death ( apoptosis ) and prevention of abnormal cell division. In contrast to proto-oncogenes, in tumor suppressors it is loss-of-function mutations that contribute to the progression of cancer. This means that tumor suppressor mutations tend to be recessive, and thus both alleles must be mutated in order to allow abnormal growth to proceed. It is perhaps not surprising that mutations in tumor suppressor genes, are more likely than oncogenes to be inherited. An example is the tumor suppressor gene, BRCA1 , which is involved in DNA-repair. Inherited mutations in BRCA1 increase a woman’s lifetime risk of breast cancer by up to seven times, although these heritable mutations account for only about 10% of breast cancer. Thus, sporadic rather than inherited mutations are the most common sources of both oncogenes and disabled tumor suppressor genes. An important tumor suppressor gene is a transcription factor named p53 . Other proteins in the cell sense DNA damage, or abnormalities in the cell cycle and activate p53 through several mechanisms including phosphorylation (attachment of phosphate to specific site on the protein) and transport into the nucleus. In its active form, p53 induces the transcription of genes with several different types of tumor suppressing functions, including DNA repair, cell cycle arrest, and apoptosis. Over 50% of human tumors contain mutations in p53. People who inherit only one function copy of p53 have a greatly increased incidence of early onset cancer. However, as with the other cancer related genes we have discussed, most mutations in p53 are sporadic, rather than inherited. Mutation of p53, through formation of pyrimidine dimers in the genes following exposure to UV light, has been causally linked to squamous cell and basal cell carcinomas (but not melanomas, highlighting the variety and complexities of mechanisms that can cause cancer). Inherited Cancer Predisposition While cancer itself is not inherited, mutations that can drive cancer can be passed from parent to offspring. Many of the commonly inherited cancer-associated mutations are in tumor suppressor genes. In most cases, individuals inherit only one mutant allele of the tumor suppressor; they are heterozygous for the recessive mutation. However, if the second copy (due to random chance) becomes mutated or deleted in a somatic cell, that cell (and all its descendants) now lack the tumor suppressor entirely. This process is known as loss-of-heterozygosity. Contributors and Attributions - Dr. Todd Nickle and Isabelle Barrette-Ng (Mount Royal University) The content on this page is licensed under CC SA 3.0 licensing guidelines.	libretexts	2025-03-17T22:27:32.379447	2019-10-01T21:21:20	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/", "url": "https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/08%3A_Cancer_Genetics/8.05%3A_Tumor_Suppressor_Genes", "book_url": "https://commons.libretexts.org/book/bio-25718", "title": "8.5: Tumor Suppressor Genes", "author": "Stefanie West Leacock" }
https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/08%3A_Cancer_Genetics/8.06%3A__Genetics_and_Targeted_Cancer_Treatment__The_Story_of_Gleevec_(Imatinib)	8.6: Genetics and Targeted Cancer Treatment – The Story of Gleevec™ (Imatinib) Chronic myelogenous leukemia (CML) Chronic myelogenous leukemia ( CML ) is a type of cancer of white blood cells, myeloid cells, that are mutated and proliferate uncontrollably through three stages (chronic, accelerated, and blast crisis) and lead eventually to death. Cytogenetics showed the myeloid cells of CML patients usually also have a consistent chromosome translocation (the mutant event) between the long arms of chromosomes 9 and 22, t(9:22)(q34;q11), known as the Philadelphia chromosome (Ph + ). This translocation involves breaks in two genes, c-abl and bcr , on chromosomes 9 and 22, respectively. The fusion of the translocation breaks result in a chimeric gene, called bcr-abl , that contains exons 1 and/or 2 from bcr (this varies from patient to patient) and 2-11 from abl and it produces a chimeric protein (BCR-ABL or p185 bcr-abl ) that is transcribed like bcr and contains abl enzyme sequences. This chimeric protein has a tyrosine-kinase from the abl gene sequences that is unique to the CML mutant cell. The consistent, unregulated expression of this gene and its kinase product causes activation of a variety of intracellular signaling pathways, promoting the uncontrolled proliferative and survival properties of CML cells (the cancer). Thus the BCR-ABL tyrosine kinase enzyme exists only in cancer cells (and not in healthy cells) and a drug that inhibits this activity could be used to target and prevent the uncontrolled growth of the cancerous CML cells. Inhibiting the Bcr-Abl tyrosine kinase activity Knowing that the kinase activity was the key to treatment, pharmaceutical companies screened chemical libraries of potential kinase inhibitory compounds. After initially finding low potency inhibitors, a relationship between structure and activity suggested other compounds that were optimized to inhibit the BCR-ABL tyrosine kinase activity. The lead compound was STI571, now called Gleevec ™ or imatinib (Figure $\PageIndex{9}$). This drug was shown to inhibit the BCR-ABL tyrosine kinase activity and to inhibit CML cell proliferation in vitro and in vivo . Gleevec™ works via targeted therapy—only the kinase activity in cancer cells was targeted and thereby killed through the drug's action. In this regard, Gleevec™ was one of the first cancer therapies to show the potential for this type of targeted action. It was dependent upon the genetic identification of the cause and protein target and is often cited as a paradigm for genetic research in cancer therapeutics. Figure $\PageIndex{1}$: Biochemical structure of Gleevec™ or Imatinib. . (Wikipedia-Fuse809-CC:AN) Caution This is a simplified presentation of the CML/cancer targeting by the drug Gleevec™. There are many more details than could be presented here. It is represents as a model of finding a drug for each type of cancer, rather than the one, single “magic bullet” that kills all cancers. Remember, there are always complexities in this type of research to treatment process, such as patient genetic and environmental variation that leads to differences in drug metabolism, uptake, and binding. Also, changes in drug dose, mutation of the bcr-abl gene, and other events can affect the effectiveness of the treatment and the relapse rate. Biological systems are extremely complex and difficult to modulate in the specific, targeted manner necessary to treat cancer ideally. Remember, the drug, Gleevec™, is not a cure, but only a treatment. It prevents the uncontrolled proliferation of the CML cells, but doesn’t kill them directly. The arrested cells will die eventually, but there is always a small pool of CML cells that will proliferate if the drug is discontinued. While sustained use of this expensive drug is beneficial to the pharmaceutical companies, it is certainly not the ideal situation for the patient. Contributors and Attributions - Dr. Todd Nickle and Isabelle Barrette-Ng (Mount Royal University) The content on this page is licensed under CC SA 3.0 licensing guidelines.	libretexts	2025-03-17T22:27:32.439684	2019-10-01T21:21:21	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/", "url": "https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/08%3A_Cancer_Genetics/8.06%3A__Genetics_and_Targeted_Cancer_Treatment__The_Story_of_Gleevec_(Imatinib)", "book_url": "https://commons.libretexts.org/book/bio-25718", "title": "8.6: Genetics and Targeted Cancer Treatment – The Story of Gleevec™ (Imatinib)", "author": "Stefanie West Leacock" }
https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/08%3A_Cancer_Genetics/8.07%3A_Cancer_Genetics_(Exercises)	8.7: Cancer Genetics (Exercises) selected template will load here This action is not available. These are homework exercises to accompany Nickle and Barrette-Ng's " Online Open Genetics " TextMap. Genetics is the scientific study of heredity and the variation of inherited characteristics. It includes the study of genes, themselves, how they function, interact, and produce the visible and measurable characteristics we see in individuals and populations of species as they change from one generation to the next, over time, and in different environments. 13.1 Why do oncogenes tend to be dominant, but mutations in tumor suppressors tend to be recessive? 13.2 What tumor suppressing functions are controlled by p53? How can a single gene affect so many different biological pathways? 13.3 Are all carcinogens mutagens? Are all mutagens carcinogens? Explain why or why not. 13.4 Imagine that a laboratory reports that feeding a chocolate to laboratory rats increases the incidence of cancer. What other details would you want to know before you stopped eating chocolate? 13.5 Do all women with HPV get cancer? Why or why not? Do all women with mutations in BRCA1 get cancer? Why or why not?	libretexts	2025-03-17T22:27:32.503059	2019-10-01T21:21:21	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/", "url": "https://bio.libretexts.org/Courses/University_of_Arkansas_Little_Rock/Genetics_BIOL3300_(Leacock)/Genetics_Textbook/08%3A_Cancer_Genetics/8.07%3A_Cancer_Genetics_(Exercises)", "book_url": "https://commons.libretexts.org/book/bio-25718", "title": "8.7: Cancer Genetics (Exercises)", "author": "Stefanie West Leacock" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/01%3A_The_Cell_Cycle/1.01%3A_The_Cell_Cycle_and_Changes_in_DNA_Content	1.1: The Cell Cycle and Changes in DNA Content - - Last updated - Save as PDF Four stages of a typical cell cycle The life cycle of eukaryotic cells can generally be divided into four stages and a typical cell cycle is shown in Figure $\PageIndex{13}$. When a cell is produced through fertilization or cell division, there is usually a lag before it undergoes DNA synthesis (replication). This lag period is called Gap 1 ( G 1 ), and ends with the onset of the DNA synthesis ( S ) phase, during which each chromosome is replicated. Following replication, there may be another lag, called Gap 2 ( G 2 ), before mitosis ( M ). Cells undergoing meiosis do not usually have a G 2 phase. Interphase is as term used to include those phases of the cell cycle excluding mitosis and meiosis. Many variants of this generalized cell cycle also exist. Some cells never leave G 1 phase, and are said to enter a permanent, non-dividing stage called G 0 . On the other hand, some cells undergo many rounds of DNA synthesis (S) without any mitosis or cell division, leading to endoreduplication. Understanding the control of the cell cycle is an active area of research, particularly because of the relationship between cell division and cancer. Measures of DNA content and chromosome content The amount of DNA within a cell changes following each of the following events: fertilization, DNA synthesis, mitosis, and meiosis (Fig 2.14). We use “ c ” to represent the DNA c ontent in a cell, and “ n ” to represent the n umber of complete sets of chromosomes. In a gamete (i.e. sperm or egg), the amount of DNA is 1c, and the number of chromosomes is 1n. Upon fertilization, both the DNA content and the number of chromosomes doubles to 2c and 2n, respectively. Following DNA replication, the DNA content doubles again to 4c, but each pair of sister chromatids is still counted as a single chromosome (a replicated chromosome ), so the number of chromosomes remains unchanged at 2n. If the cell undergoes mitosis, each daughter cell will return to 2c and 2n, because it will receive half of the DNA, and one of each pair of sister chromatids. In contrast, the 4 cells that come from meiosis of a 2n, 4c cell are each 1c and 1n, since each pair of sister chromatids, and each pair of homologous chromosomes, divides during meiosis.	libretexts	2025-03-17T22:27:32.614251	2021-01-03T20:11:36	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/01%3A_The_Cell_Cycle/1.01%3A_The_Cell_Cycle_and_Changes_in_DNA_Content", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "1.1: The Cell Cycle and Changes in DNA Content", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/01%3A_The_Cell_Cycle/1.02%3A_Bacterial_Cell_Division_and_the_Eukaryotic_Cell_Cycle	1.2: Bacterial Cell Division and the Eukaryotic Cell Cycle - - Last updated - Save as PDF The life of actively growing bacteria is not separated into a time for duplicating genes (i.e., DNA synthesis) and one for binary fission (dividing and partitioning the duplicated DNA into new cells). Instead, the single circular chromosmome of a typical bacterium is replicating even before fission is complete, so that the new daughter cells already contained partially duplicated chromosomes. Cell growth, replication and fission are illustrated below The roughly 30-60 minute life cycle of an actively growing bacterium is not divided into discrete phases. On the other hand, typical eukaryotic cells have a roughly 16-24 hour cell cycle (depending on cell type) that is divided into four separate phases. In the late 1800s, light microscopy revealed that some cells lost their nuclei while forming chromosomes (from chroma , colored; soma , bodies). In mitosis , paired, attached chromosomes ( chromatids ) were seen to separate and to be drawn along spindle fibers to opposite poles of dividing cells. Thus homologous chromosomes were equally partioned to the daughter cells at the end of cell division. Because of the same chromosomal behavior was observed in mitosis in diverse organisms, chromosomes were soon recognized as the stuff of inheritance, the carrier of genes! The short period of intense mitotic activity was in stark contrast to the much longer ‘ quiet ’ time in the life of the cell, called interphase . The events of mitosis itself were described as occurring in 4 phases occupyiing a short time as shown below Depending on whom you ask, cytokinesis (the cell movements of actually dividing a cell in two) is not part of mitosis. In that sense, we can think of three stages in the life of a cell: interphase, mitosis and cytokinesis. Of course, it turned out that interphase is not cellular ‘quiet time’ at all! A. Defining the Phases of the Cell Cycle Correlation of the inheritance of specific traits with that of chromosomes was demonstrated early in the early 20th century, most elegantly in genetic studies of the fruit fly, Drosophila melanogaster . At that time, chromosomes were assumed to contain the genetic material and that both were duplicated during mitosis. The first clue that this was not so came only after the discovery that DNA was in fact the chemical stuff of genes . The experiment distinguishing the time of chromosome formation from the time of DNA duplication is summarized below. - Cultured cells were incubated with 3 H-thymine , the radioactive base that cells will incorporate into thymidine triphosphate (dTTP), and then into DNA. - Cultured cells were incubated with 3 H-thymine, the radioactive base that cells will incorporate into thymidine triphosphate (dTTP), and then into DNA. - Slides were dipped in a light-sensitive emulsion containing the same light sensitive chemicals as found in the emulsion-side of film. - After some time to allow the radioactivity on the slide to ‘ expose ’ the emulsion, the slides were developed (in much the same way as developing film). - The resulting autoradiographs in the microscope revealed images in the form of dark spots created by exposure to hot (i.e., radioactive DNA. If DNA replicates in chromosomes undergoing mitosis, then when the developed film is placed back over the slide, any dark spots should lie over the cells in mitosis, and not over cells that are not actively dividing. The experimental is illustrated below Observation of the autoradiographs show that none of the cells in mitosis is radioactively labeled. But some of the cells in interphase were! Therefore, DNA synthesis must take place sometime in interphase, before mitosis and cytokinesis (illustrated below). 340 Experiments that Reveal Replication in Interphase of the Cell Cycle Next a series of pulse-chase experiments were done to determine when in the cell cycle DNA synthesis actually takes place. Cultured cells given a short pulse (exposure) to 3 H-thymine and then allowed to grow in non-radioactive medium for different times (the chase ). At the end of each chase time, cells were spread on a glass slide and again prepared for autoradiography. Analysis of the autoradiographs identified distinct periods of activity within interphase: Gap1 ( G 1 ), a time of DNA synthesis ( S ) and Gap 2 ( G 2 ). Here are the details of these very creative experiments, performed before it became possible to synchronize cells in culture so that they would all be growing and dividing at the same time. - Cells were exposed to 3H-thymine for just 5 minutes (the pulse ) and then centrifuged. The radioactive supernatant was then discarded. - The cells were rinsed and centrifuged again to remove as much labeled precursor as possible. - The cells were re-suspended in fresh medium containing unlabeled (i.e., nonradioactive) thymine and further incubated for different times (the chase periods).After dipping the slides in light-sensitive emulsion, exposing and developing the film, the autoradiographs were examined, with the following results: - After a 3-hour (or less) chase period, the slides looked just like they would immediately after the pulse. That is, none of the 7% of the cells that were in mitosis is radioactively labeled, but many interphase cells showed labeled nuclei, as shown below. - After 4 hours of chase, a few of the 7% of the cells that were in mitosis were labeled, along with others in interphase (below). - After a 5 hour chase, most cells in mitosis (still about 7% of cells on the slide) were labeled; many fewer cells in interphase were labeled (below). - After a 20 hour chase, none of the 7% of cells that were in mitosis is labeled. Instead, all of the labeled cells are in interphase (below). - After a 3-hour (or less) chase period, the slides looked just like they would immediately after the pulse. That is, none of the 7% of the cells that were in mitosis is radioactively labeled, but many interphase cells showed labeled nuclei, as shown below. - The graph below plots a count of radiolabeled mitotic cells against chase times . The plot defines the duration of events, or phases of the cell cycle as follows: - The first phase (interval #1 on the graph) must be the time between the end of DNA synthesis and the start of mitosis, defined as Gap 2 ( G 2 ). - Cell doubling times are easily measured. Assume that the cells in this experiment doubled every 20 hours. This would be consistent with the time interval of 20 hours between successive peaks in the number of radiolabeled mitotic cells after the pulse (interval #2 ). - Interval #3 is easy enough to define. It is the time when DNA is synthesized, from start to finish; this is the synthesis , or S phase. - One period of the cell cycle remains to be defined, but it is not on the graph! That would be the time between the end cell division (i.e., mitosis and cytokinesis) and the beginning of DNA synthesis ( replication ). That interval can be calculated from the graph as the time of the cell cycle (~20 hours) minus the sum of the other defined periods of the cycle. This phase is defined as the Gap 1 ( G 1 ) phase of the cycle. So at last, here is our cell cycle with a summary of events occurring in each phase. During all of interphase ( G 1 , S and G 2 ) , the cell grows in size, preparing for the next cell division. Growth in G 1 includes the synthesis of enzymes and other proteins that will be needed for replication. DNA is replicated during the S phase, along with the synthesis of new histone and other proteins that will be needed to assemble new chromatin . G 2 is the shortest time of interphase and is largely devoted to preparing the cell for the next round of mitosis and cytokinesis. Among the proteins whose synthesis increases in this time are the tubulins and proteins responsible for condensing chromatin into the paired chromatids representing the duplicated chromosomes. Cohesin is a more recently discovered protein made in the run-up to mitosis. It holds centromeres of chromatids together until they are ready to separate. 341 Events in the Phases of the Cell Cycle In a final note, typical dividing cells have generation times ranging from 16 to 24 hours. Atypical cells, like newly fertilized eggs, might divide every hour or so! In these cells, events that normally take many hours must be completed in just fractions of an hour. B. When Cells Stop Dividing Terminally differentiated cells are those that spend the rest of their lives performing a specific function. These cells no longer cycle. Instead, shortly after entering G 1 they are diverted into a phase called G 0 , as shown below. Referred to as terminally differentiated , these cells normally never divide again. With a few exceptions (e.g., many neurons), most terminally differentiated cells have a finite lifespan, and must be replaced by stem cells. Examples include red blood cells. With a half-life of about 60 days, they are regularly replaced by reticulocytes produced in bone marrow	libretexts	2025-03-17T22:27:32.684610	2021-01-03T20:11:37	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/01%3A_The_Cell_Cycle/1.02%3A_Bacterial_Cell_Division_and_the_Eukaryotic_Cell_Cycle", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "1.2: Bacterial Cell Division and the Eukaryotic Cell Cycle", "author": "Gerald Bergtrom" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/01%3A_The_Cell_Cycle/1.03%3A_Regulation_of_the_Cell_Cycle	1.3: Regulation of the Cell Cycle - - Last updated - Save as PDF Progress through the cell cycle is regulated. The cycle can be controlled or put on ‘pause’ at any one of several phase transitions. Such checkpoints monitor whether the cell is on track to complete a successful cell division event. Superimposed on these controls are signals that promote cell differentiation. Embryonic cells differentiate as the embryo develops. Even after terminal differentiation of cells that form all adult tissues and organs, adult stem cells will divide and differentiate to replace worn out cells. Once differentiated, cells are typically signaled in G 1 to enter G 0 and stop cycling. In some circumstances cells in G 0 are recruited to resume cycling. However, if this occurs to by mistake, the cells may be transformed to cancer cells. Here we consider how the normal transition between phases of the cell cycle is controlled. A. Discovery and Characterization of Maturation Promoting Factor (MPF) Growing, dividing cells monitor their progress through the phases. Cells produce internal chemical signals that tell them when it’s time to begin replication or mitosis, or even when to enter into G 0 when they reach their terminally differentiated state. The experiment that first demonstrated a chemical regulator of the cell cycle involved fusing very large frog’s eggs! The experiment is described below. The hypothesis tested here was that frog oocyte cytoplasm from germinal vesicle stage oocytes (i.e., in mid-meiosis) contains a chemical that caused the cell to lose its nuclear membrane, condense its chromatin into chromosomes and enter meiosis. Cytoplasm was withdrawn from one of these mid-meiotic oocytes with a fine hypodermic needle, and then injected into a pre-meiotic oocyte. The mid-meiotic oocyte cytoplasm induced premature meiosis in the immature oocyte. A maturation promoting factor ( MPF ) could be isolated from the mid-meiotic cells and injected into pre-meiotic cells; it caused them to enter meiosis. MPF turns out to be a protein kinase made up of two polypeptide subunits as shown below. MPF was then also shown to stimulate somatic cells in G 2 to enter premature mitosis. So conveniently, MPF can also be Mitosis Promoting Factor ! Hereafter we will discuss the effects of MPF as being equivalent in mitosis and meiosis. When active, MPF targets many cellular proteins. 342 Discovery of MPF Kinase and Its Role in Meiosis and Mitosis Assays of MPF activity as well as the actual levels of the two subunits over time during the cell cycle are graphed below. One subunit of MPF is cyclin , a regulatory polypeptide. The other subunit, cyclin-dependent kinase ( cdk ), contains the kinase enzyme active site . Both subunits must be bound to make an active kinase. Cyclin was so-named because its levels rise gradually after cytokinesis, peak at the next mitosis, and then fall. Levels of the cdk subunit do not change significantly during the life of the cell. Because the kinase activity of MPF requires cyclin , it tracks the rise in cyclin near the end of the G 2 , and its fall after mitosis. Cyclin begins to accumulate in G 1 , rising gradually and binding to more and more cdk subunits. MPF reaches a threshold concentration in G 2 that triggers entry into mitosis. For their discovery of these central molecules Leland H. Hartwell, R. Timothy Hunt, and Paul M. Nurse won the 2001 Nobel Prize in Physiology or Medicine. B. Other Cyclins, CDKs and Cell Cycle Checkpoints Other chemical signals accumulate at different points in the cell cycle. For example, when cells in S are fused with cells in G 1 , the G 1 cells begin synthesizing DNA (visualized as 3 H-thymine incorporation). An experiment showing control of progress to different phases of the cell cycle is illustrated below. An S phase factor could be isolated from the S phase cells. This factor also turns out to be a two-subunit protein kinase, albeit a different one from MPF. Just as MPF signals cells in G 2 to begin mitosis, the S phase kinase signals cells in G 1 to enter the S phase of the cell cycle. MPF and the S phase kinase govern activities at two of several cell cycle checkpoints . In each case, the activity of the kinases is governed by prior progress through the cell cycle. In other words, if the cell is not ready to begin mitosis, active MPF production is delayed until it is. Likewise, the S phase kinase will not be activated until the cell is ready to begin DNA synthesis. 343 Cell Cycle Control at Check Points and the Go "Phase" The sequence of signals that control progress through the cell cycle is probably more intricate and extensive than we currently know, but the best-described checkpoints are in G 1 , G 2 and M (below). We generally envision checkpoints as monitoring and blocking progress until essential events of a current phase of the cell cycle phase are completed. These kinases are part of molecular sensing mechanisms that act by phosphorylating cytoplasmic and/or nuclear proteins required by upcoming phases of the cycle. Let’s take a closer look at some events that are monitored at these checkpoints in more detail. 1. The G 1 Checkpoint The G 1 checkpoint controls the transition from the G 1 to the S phase of the cell cycle. If actively dividing cells (e.g., stem cells) in G 1 fail to complete their preparation for replication, the S-phase kinase won’t be produced and the cells won’t proceed the S phase until the preparatory biochemistry catches up with the rest of the cycle. To enter S, a cell must be ready to make proteins of replication, like DNA polymerases, helicases, and primases among others. Only when these molecules have accumulated to (or become active at) appropriate levels, is it “safe” to enter S and begin replicating DNA. This description of G 1 checkpoint activity is consistent with the idea that all checkpoints delay cycling until a prior phase is complete. What about cells that are fully differentiated? Such terminally differentiated cells stop producing the active G 1 checkpoint kinase and stop dividing. These cells are arrested in G 0 (see below). As an interesting side-note, recall that somatic cells are diploid and germ cells (sperm, egg) are haploid . So, are cells in G 2 that have already doubled their DNA ‘tetraploid’, however briefly? Whether or not we can call G 2 cells tetraploid (officially, probably not), it is clear that G 1 cells and G 0 cells are diploid! 2. The G 2 Checkpoint Passage through the G 2 checkpoint is only possible if DNA made in the prior S phase is not damaged. Or if it was, that the damage has been (or can be) repaired (review the proofreading functions of DNA polymerase and the various DNA repair pathways). Cells that do successfully complete replication and pass the G 2 checkpoint must prepare to make the proteins necessary for the upcoming mitotic phase. These include nuclear proteins necessary to condense chromatin into chromosomes, tubulins for making microtubules , etc. Only when levels of these and other required proteins reach a threshold can the cell begin mitosis. Consider the following two tasks required of the G 2 checkpoint (in fact, any checkpoint): - sensing whether prior phase activities have been successfully completed. - delaying transition to the next phase if those activities are unfinished. But what if sensing is imperfect and a checkpoint is leaky? A recent study suggests that either the G 2 checkpoint is leaky, or at least, that incomplete activities in the S phase are tolerated, and that some DNA repair is not resolved until mitosis is underway in M! Check it out at DNA repair and replication during mitosis. 3. M Checkpoint The M checkpoint is monitored by the original MPF phosphorylation of proteins that: (a) bind to chromatin causing it to condense and form chromatids, (b) lead to the breakdown of the nuclear envelope, and (c) enable spindle fiber formation,. In addition, tension in the spindle apparatus at metaphase tugs at the kinetochores holding the duplicated chromatids together. When this tension reaches a threshold, MPF peaks and an activated separase enzyme causes the chromatids to separate at their centromeres. Beginning in anaphase , tension in the spindle apparatus draws the new chromosomes to opposite poles of the cell. Near the end of mitosis and cytokinesis, proteins phosphorylated by MPF initiate the breakdown of cyclin in the cell. Passing the M checkpoint means that the cell will complete mitosis and cytokinesis, and that each daughter cell will enter a new G 1 phase. Dividing yeast cells only seem to have the three checkpoints discussed here. More complex eukaryotes use more cyclins and cdks to control the cell cycle at additional checkpoints. Different cyclins show cyclic patterns of synthesis, while cdks remain at constant levels throughout the cell cycle (as in MPF). Different gene families encode evolutionarily conserved cdks or cyclins . But each cyclin / cdk pair has been coopted in evolution to monitor different cell cycle events and to catalyze phosphorylation of phase-specific proteins. To learn more, see Elledge SJ (1996) Cell Cycle Checkpoints: Preventing an Identity Crisis . Science 274:1664-1672. C. The G 0 State This is not really a phase of the cell cycle, since cells in G 0 have reached a terminally differentiated state and have stopped dividing. In development, terminally differentiated cells in tissues and organs no longer divide. Nevertheless, most cells have finite half-lives (recall our red blood cells that must be replaced every 60 days or so). Because cells in many tissues are in G 0 and can’t divide, they must be replaced by stem cells, which can divide and differentiate. Some cells live so long in G 0 that they are nearly never replaced (muscle cells, neurons). Other cells live short lives in G 0 (e.g., stem cells, some embryonic cells). For example, a lymphocyte is a differentiated immune system white blood cell type. However, exposure of lymphocytes to foreign chemicals or pathogens activates mitogens that cause them to re-enter the cell cycle from G 0 . The newly divided cells then make the antibodies that neutralize the chemicals and fight off the pathogens. The retinoblastoma ( Rb ) protein is an example of a mitogen. Like other mitogens, the Rb protein is a transcription factor that turns on genes that lead to cell proliferation. What if cells continue cycling when they aren’t supposed to? Or, what if they are inappropriately signaled to exit G o ? Such cells are in trouble! Having escaped normal controls on cell division, they can become a focal point of cancer cell growth. You can guess from its name that the retinoblastoma gene was discovered as a mutation that causes retinal cancer. For more about the normal function of the Rb protein and its interaction with a G 1 cdk, check out the link below. What if cells continue cycling when they aren’t supposed to? Or, what if they are inappropriately signaled to exit G 0 ? Such cells are in trouble! Having escaped normal controls on cell division, they can become a focal point of cancer cell growth. You can guess from its name that the retinoblastoma gene was discovered as a mutation that causes retinal cancer. For more about the normal function of the Rb protein and its interaction with a G 1 cdk , check out the link below.	libretexts	2025-03-17T22:27:32.760086	2021-01-03T20:11:38	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/01%3A_The_Cell_Cycle/1.03%3A_Regulation_of_the_Cell_Cycle", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "1.3: Regulation of the Cell Cycle", "author": "Gerald Bergtrom" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/02%3A_Mitosis_and_Meiosis/2.01%3A_Mitosis	2.1: Mitosis selected template will load here This action is not available. Cell growth and division is essential to asexual reproduction and the development of multicellular organisms. Accordingly, the primary function of mitosis is to ensure that at division each daughter cell inherits identical genetic material, i.e. exactly one copy of each chromosome. To make this happen, replicated chromosomes condense ( prophase ), and are positioned near the middle of the dividing cell ( metaphase ), and then each of the sister chromatids from each chromosome migrates towards opposite poles of the dividing cell ( anaphase ), until the identical sets of unreplicated chromosomes are completely separated from each other within the two newly formed daughter cells ( telophase ) (Figures 2.10 and 2.11). This is followed by the division of the cytoplasm ( cytokinesis ) to complete the process. The movement of chromosomes occurs through microtubules that attach to the chromosomes at the centromeres.	libretexts	2025-03-17T22:27:32.851906	2021-01-03T20:11:40	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/02%3A_Mitosis_and_Meiosis/2.01%3A_Mitosis", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "2.1: Mitosis", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/02%3A_Mitosis_and_Meiosis/2.02%3A_Meiosis	2.2: Meiosis - - Last updated - Save as PDF Most eukaryotes replicate sexually - a cell from one individual joins with a cell from another to create the next generation. For this to be successful, the cells that fuse must contain half the number of chromosomes as in the adult organism. Otherwise, the number of chromosomes would double with each generation! The reduction in chromosome number is achieved by the process of meiosis . In meiosis, there are usually two steps, Meiosis I and II. In Meiosis I homologous chromosomes segregate, while in Meiosis II sister chromatids segregate. Most multicellular organisms use meiosis to produce gametes , the cells that fuse to make offspring. Some single celled eukaryotes such as yeast also use meiosis. Meiosis I and II Meiosis begins similarly to mitosis (a cell has replicated its chromosomes and grown large enough to divide), but requires two rounds of division (Figure $\PageIndex{8}$). In the first, known as meiosis I, the homologous chromosomes separate and segregate. During meiosis II the sister chromatids separate and segregate. Note how meosis I and II are both divided into prophase, metaphase, anaphase, and telophase. After two rounds of cytokinesis, four cells will be produced, each with a single copy of each chromosome. Cells that will undergo the process of meiosis are called meiocytes and are diploid (2N). Meiosis is divided into two stages designated by the roman numerals I and II. Meiosis I is called a reductional division, because it reduces the number of chromosomes inherited by each of the daughter cells. Meiosis I is further divided into Prophase I, Metaphase I, Anaphase I, and Telophase I, which are roughly similar to the corresponding stages of mitosis, except that in Prophase I and Metaphase I, homologous chromosomes pair with each other, or synapse , and are called bivalents (Figs. 2.12). This is an important difference between mitosis and meiosis, because it affects the segregation of alleles, and also allows for recombination to occur through crossing-over, as described later. During Anaphase I, one member of each pair of homologous chromosomes migrates to each daughter cell (1N). Meiosis II resembles mitosis, with one sister chromatid from each chromosome separating to produce two daughter cells. Because Meiosis II, like mitosis, results in the segregation of sister chromatids, Meiosis II is called an equational division. In meiosis I replicated, homologous chromosomes pair up , or synapse , during prophase I, lining up in the middle of the cell during metaphase I, and separating during anaphase I. For this to happen the homologous chromosomes need to be brought together while they condense during prophase I. These attachments are formed in two ways. Proteins bind to both homologous chromosomes along their entire length and form the synaptonemal complex (synapse means junction). These proteins hold the chromosomes in a transient structure called a bivalent . The proteins are released when the cell enters anaphase I. Within the synaptonemal complex a second event, crossingover , occurs. These are places where DNA repair enzymes break the DNA two non-sister chromatids in similar locations and then covalently reattach non-sister chromatids together to create a crossover between non-sister chromatids. This reorganization of chromatids will persist for the remainder of meiosis and result in recombination of alleles in the gametes. Crossovers function to hold homologous chromosomes together during meiosis I so they segregate successfully and they also cause the reshuffling of gene/allele combinations to create genetic diversity, which can have an important effect on evolution (see Chapter 7). Stages of Prophase I In meiosis, Prophase I is divided up into five visual stages, that are steps along a continuum of events. Leptotene, zygotene, pachytene, diplotene and diakinesis. From interphase, a cell enters leptotene as the nuclear material begins to condense into long visible threads (chromosomes). During Zygotene homologous chromosomes begin to pair up (synapse) and form an elaborate structure called the synaptonemal complex along their length. At pachytene homologous chromosomes are fully synapsed (two chromosomes and four chromatids) to form bivalents . Crossing over takes place in pachytene. After this, the pairing begins to loosen and individual chromatids become apparent in diplotene . This is when the consequences of each crossing over event can be seen as a chiasma (plural: chiasmata ). Diakinesis follows as the chromosomes continue to condense and individualize. This is followed by metaphase I were the paired chromosomes orient on the metaphase plate in preparation for segregation (reductional). Meiosis II and Gamete Maturation At the completion of meiosis I there are two cells, each with one, replicated copy of each chromosome (1N). Because the number of chromosomes per cell has decreased (2->1), meiosis I is called a reductional cell division . In the second part of meiosis the chromosomes will once again be brought to the middle of the cell, but this time it is the sister chromatids that will segregate during anaphase. After cytokinesis there will be four cells, each containing only one unreplicated chromosome of each type. Meiosis II resembles mitosis in that the number of chromosomes per cell is unchanged - both are equational cell divisions – but in meiosis II all cells won’t have the same genetic composition. There will be allelic differences among the gametes. In animals and plants the cells produced by meiosis need to mature before they become functional gametes. In male animals the four products of meiosis are called spermatids. They grow tails and become functional sperm cells. In female animals the gametes are eggs. In order that each egg contains the maximum amount of nutrients only one of the four products of meiosis becomes an egg. The other three cells end up as tiny disposable cells called polar bodies . In plants the products of meiosis reproduce a few times using mitosis as they develop into functional male or female gametes.	libretexts	2025-03-17T22:27:32.912593	2021-01-03T20:11:41	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/02%3A_Mitosis_and_Meiosis/2.02%3A_Meiosis", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "2.2: Meiosis", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/03%3A_Alleles_and_Probabilities/3.01%3A_Genes_are_the_Basic_Units_of_Inheritance	3.1: Genes are the Basic Units of Inheritance selected template will load here This action is not available. The once prevalent (but now discredited) concept of blending inheritance proposed that some undefined essence, in its entirety, contained all of the heritable information for an individual. It was thought that mating combined the essences from each parent, much like the mixing of two colors of paint. Once blended together, the individual characteristics of the parents could not be separated again. However, Gregor Mendel (Fig 1.10) was one of the first to take a quantitative, scientific approach to the study of heredity. He started with well-characterized strains, repeated his experiments many times, and kept careful records of his observations. Working with peas, Mendel showed that white-flowered plants could be produced by crossing two purple-flowered plants, but only if the purple-flowered plants themselves had at least one white-flowered parent (Fig 1.11). This was evidence that the genetic factor that produced white-flowers had not blended irreversibly with the factor for purple-flowers. Mendel’s observations disprove blending inheritance and favor an alternative concept, called particulate inheritance , in which heredity is the product of discrete factors that control independent traits.	libretexts	2025-03-17T22:27:33.120140	2021-01-03T20:11:45	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/03%3A_Alleles_and_Probabilities/3.01%3A_Genes_are_the_Basic_Units_of_Inheritance", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "3.1: Genes are the Basic Units of Inheritance", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/03%3A_Alleles_and_Probabilities/3.02%3A_Model_Organisms_Facilitate_Genetic_Advances	3.2: Model Organisms Facilitate Genetic Advances - - Last updated - Save as PDF Model organisms Many of the great advances in genetics were made using species that are not especially important from a medical, economic, or even ecological perspective. Geneticists, from Mendel onwards, have sought the best organisms for their experiments. Today, a small number of species are widely used as model organisms in genetics (Fig 1.17). All of these species have specific characteristics that make large number of them easy to grow and analyze in laboratories: (1) they are small, (2) fast growing with a short generation time, (3) produce lots of progeny from matings that can be easily controlled, (4) have small genomes (small C-value), and (5) are diploid (i.e. chromosomes are present in pairs). The most commonly used model organism are: - The prokaryote bacterium, Escherichia coli, is the simplest genetic model organism and is often used to clone DNA sequences from other model species. - Yeast ( Saccharomyces cerevisiae ) is a good general model for the basic functions of eukaryotic cells. - The roundworm, Caenorhabditis elegans is a useful model for the development of multicellular organisms, in part because it is transparent throughout its life cycle, and its cells undergo a well-characterized series of divisions to produce the adult body. - The fruit fly ( Drosophila melanogaster ) has been studied longer, and probably in more detail, than any of the other genetic model organisms still in use, and is a useful model for studying development as well as physiology and even behavior. - The mouse ( Mus musculus ) is the model organism most closely related to humans, however there are some practical difficulties working with mice, such as cost, slow reproductive time, and ethical considerations. - The zebrafish ( Danio rerio ) has more recently been developed by researchers as a genetic model for vertebrates.Unlike mice, zebrafish embryos develop quickly and externally to their mothers, and are transparent, making it easier to study the development of internal structures and organs. - Finally, a small weed, Arabidopsis thaliana , is the most widely studied plant genetic model organism. This provides knowledge that can be applied to other plant species, such as wheat, rice, and corn.	libretexts	2025-03-17T22:27:33.178541	2021-01-03T20:11:48	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/03%3A_Alleles_and_Probabilities/3.02%3A_Model_Organisms_Facilitate_Genetic_Advances", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "3.2: Model Organisms Facilitate Genetic Advances", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/03%3A_Alleles_and_Probabilities/3.03%3A__Mendels_First_Law	3.3: Mendel’s First Law - - Last updated - Save as PDF Character Traits Exist in Pairs that Segregate at Meiosis Through careful study of patterns of inheritance, Mendel recognized that a single trait could exist in different versions, or alleles , even within an individual plant or animal. For example, he found two allelic forms of a gene for seed color: one allele gave green seeds, and the other gave yellow seeds. Mendel also observed that although different alleles could influence a single trait, they remained indivisible and could be inherited separately. This is the basis of Mendel’s First Law , also called The Law of Equal Segregation , which states: during gamete formation, the two alleles at a gene locus segregate from each other; each gamete has an equal probability of containing either allele. Hetero-, Homo-, Hemizygosity Mendel’s First Law is especially remarkable because he made his observations and conclusions (1865) without knowing about the relationships between genes, chromosomes, and DNA. We now know the reason why more than one allele of a gene can be present in an individual: most eukaryotic organisms have at least two sets of homologous chromosomes. For organisms that are predominantly diploid, such as humans or Mendel’s peas, chromosomes exist as pairs, with one homolog inherited from each parent. Diploid cells therefore contain two different alleles of each gene, with one allele on each member of a pair of homologous chromosomes. If both alleles of a particular gene are identical, the individual is said to be homozygous for that gene. On the other hand, if the alleles are different from each other, the genotype is heterozygous . In cases where there is only one copy of a gene present, for example if there is a deletion on the homologous chromosome, we use the term hemizygous . Although a typical diploid individual can have at most two different alleles of a particular gene, many more than two different alleles can exist in a population of individuals. In a natural population the most common allelic form is usually called the wild-type allele. However, in many populations there can be multiple variants at the DNA sequence level that are visibly indistinguishable as all exhibit a normal, wild type appearance. There can also be various mutant alleles (in wild populations and in lab strains) that vary from wild type in their appearance, each with a different change at the DNA sequence level. Such collections of mutations are known as an allelic series .	libretexts	2025-03-17T22:27:33.237715	2021-01-03T20:11:50	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/03%3A_Alleles_and_Probabilities/3.03%3A__Mendels_First_Law", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "3.3: Mendel’s First Law", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/03%3A_Alleles_and_Probabilities/3.04%3A__Biochemical_Basis_of_Dominance	3.4: Biochemical Basis of Dominance selected template will load here This action is not available. Given that a heterozygote’s phenotype cannot simply be predicted from the phenotype of homozygotes, what does the type of dominance tell us about the biochemical nature of the gene product? How does dominance work at the biochemical level? There are several different biochemical mechanisms that may make one allele dominant to another. For the majority of genes studied, the normal (i.e. wild-type) alleles are haplosufficient . So in diploids, even with a mutation that causes a complete loss of function in one allele, the other allele, a wild-type allele, will provide sufficient normal biochemical activity to yield a wild type phenotype and thus be dominant and dictate the heterozygote phenotype. On the other hand, in some biochemical pathways, a single wild-type allele is not enough protein and may be haplo in sufficient to produce enough biochemical activity to result in a normal phenotype, when heterozygous with a non-functioning mutant allele. In this case, the non-functional mutant allele will be dominant (or semi-dominant) to a wild-type allele. Mutant alleles may also encode products that have new and/or different biochemical activities instead of, or in addition to, the normal ones. These novel activities could cause a new phenotype that would be dominantly expressed.	libretexts	2025-03-17T22:27:33.302686	2021-01-03T20:11:52	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/03%3A_Alleles_and_Probabilities/3.04%3A__Biochemical_Basis_of_Dominance", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "3.4: Biochemical Basis of Dominance", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/03%3A_Alleles_and_Probabilities/3.05%3A__Crossing_Techniques_Used_in_Classical_Genetics	3.5: Crossing Techniques Used in Classical Genetics - - Last updated - Save as PDF Classical Genetics Not only did Mendel solve the mystery of inheritance as units (genes), he also invented several testing and analysis techniques still used today. Classical genetics is the science of solving biological questions using controlled matings of model organisms. It began with Mendel in 1865 but did not take off until Thomas Morgan began working with fruit flies in 1908. Later, starting with Watson and Crick’s structure of DNA in 1953, classical genetics was joined by molecular genetics , the science of solving biological questions using DNA, RNA, and proteins isolated from organisms. The genetics of DNA cloning began in 1970 with the discovery of restriction enzymes. True Breeding Lines Geneticists make use of true breeding lines just as Mendel did (Figure $\PageIndex{6}$a). These are in-bred populations of plants or animals in which all parents and their offspring (over many generations) have the same phenotypes with respect to a particular trait. True breeding lines are useful, because they are typically assumed to be homozygous for the alleles that affect the trait of interest. When two individuals that are homozygous for the same alleles are crossed, all of their offspring will all also be homozygous. The continuation of such crosses constitutes a true breeding line or strain. A large variety of different strains, each with a different, true breeding character, can be collected and maintained for genetic research. Monohybrid Crosses A monohybrid cross is one in which both parents are heterozygous (or a hybrid) for a single (mono) trait. The trait might be petal colour in pea plants (Figure $\PageIndex{6}$b). Recall from chapter 1 that the generations in a cross are named P (parental), F 1 (first filial), F 2 (second filial), and so on. Punnett Squares Given the genotypes of any two parents, we can predict all of the possible genotypes of the offspring. Furthermore, if we also know the dominance relationships for all of the alleles, we can predict the phenotypes of the offspring. A convenient method for calculating the expected genotypic and phenotypic ratios from a cross was invented by Reginald Punnett. A Punnett square is a matrix in which all of the possible gametes produced by one parent are listed along one axis, and the gametes from the other parent are listed along the other axis. Each possible combination of gametes is listed at the intersection of each row and column. The F 1 cross from Figure $\PageIndex{6}$b would be drawn as in Figure $\PageIndex{7}$. Punnett squares can also be used to calculate the frequency of offspring. The frequency of each offspring is the frequency of the male gametes multiplied by the frequency of the female gamete. \| A \| a \| \| \| A \| AA \| Aa \| \| a \| Aa \| aa \| Figure $\PageIndex{7}$: A Punnett square showing a monohybrid cro ss. (Original-Deholos (Fireworks)-CC:AN) Test Crosses Knowing the genotypes of an individual is usually an important part of a genetic experiment. However, genotypes cannot be observed directly; they must be inferred based on phenotypes. Because of dominance, it is often not possible to distinguish between a heterozygote and a homozgyote based on phenotype alone (e.g. see the purple-flowered F 2 plants in Figure $\PageIndex{6}$b). To determine the genotype of a specific individual, a test cross can be performed, in which the individual with an uncertain genotype is crossed with an individual that is homozygous recessive for all of the loci being tested. For example, if you were given a pea plant with purple flowers it might be a homozygote ( AA ) or a heterozygote ( Aa ). You could cross this purple-flowered plant to a white-flowered plant as a tester , since you know the genotype of the tester is aa . Depending on the genotype of the purple-flowered parent (Figure $\PageIndex{8}$), you will observe different phenotypic ratios in the F 1 generation. If the purple-flowered parent was a homozgyote, all of the F 1 progeny will be purple. If the purple-flowered parent was a heterozygote, the F 1 progeny should segregate purple-flowered and white-flowered plants in a 1:1 ratio. \| A \| A \| \| \| a \| Aa \| Aa \| \| a \| Aa \| Aa \| \| A \| a \| \| \| a \| Aa \| aa \| \| a \| Aa \| aa \| Figure $\PageIndex{8}$: Punnett Squares showing the two possible outcomes of a test cros s. (Original-Deholos (Fireworks)-CC:AN)	libretexts	2025-03-17T22:27:33.374888	2021-01-03T20:11:53	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/03%3A_Alleles_and_Probabilities/3.05%3A__Crossing_Techniques_Used_in_Classical_Genetics", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "3.5: Crossing Techniques Used in Classical Genetics", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/03%3A_Alleles_and_Probabilities/3.06%3A_Dihybrid_Crosses	3.6: Dihybrid Crosses - - Last updated - Save as PDF Mendel’s Second Law Before Mendel, it had not yet been established that heritable traits were controlled by discrete factors. Therefor an important question was therefore whether distinct traits were controlled by discrete factors that were inherited independently of each other? To answer this, Mendel took two apparently unrelated traits, such as seed shape and seed color, and studied their inheritance together in one individual. He studied two variants of each trait: seed color was either green or yellow, and seed shape was either round or wrinkled. (He studied seven traits in all.) When either of these traits was studied individually, the phenotypes segregated in the classical 3:1 ratio among the progeny of a monohybrid cross (Figure $\PageIndex{2}$), with ¾ of the seeds green and ¼ yellow in one cross, and ¾ round and ¼ wrinkled in the other cross. Would this be true when both were in the same individual? To analyze the segregation of both traits at the same time in the same individual, he crossed a pure breeding line of green, wrinkled peas with a pure breeding line of yellow, round peas to produce F 1 progeny that were all green and round, and which were also dihybrids ; they carried two alleles at each of two loci (Figure $\PageIndex{3}$). If the inheritance of seed color was truly independent of seed shape, then when the F 1 dihybrids were crossed to each other, a 3:1 ratio of one trait should be observed within each phenotypic class of the other trait (Figure $\PageIndex{3}$). Using the product law, we would therefore predict that if ¾ of the progeny were green, and ¾ of the progeny were round, then ¾ × ¾ = 9/16 of the progeny would be both round and green (Table $\PageIndex{1}$). Likewise, ¾ × ¼ = 3/16 of the progeny would be both round and yellow, and so on. By applying the product rule to all of these combinations of phenotypes, we can predict a 9:3:3:1 phenotypic ratio among the progeny of a dihybrid cross, if certain conditions are met, including the independent segregation of the alleles at each locus. Indeed, 9:3:3:1 is very close to the ratio Mendel observed in his studies of dihybrid crosses, leading him to state his Second Law, the Law of Independent Assortment , which we now express as follows: two loci assort independently of each other during gamete formation. Definition: Mendel's Second Law Two loci assort independently of each other during gamete formation. \| Frequency of phenotypic crosses within separate monohybrid crosses: seed shape: ¾ round ¼ wrinkled seed color: ¾ yellow ¼ green Frequency of phenotypic crosses within a dihybrid cross: ¾ round × ¾ yellow = 9/16 round & yellow ¾ round × ¼ green = 3/16 round & green ¼ wrinkled × ¾ yellow = 3/16 wrinkled & yellow ¼ wrinkled × ¼ green = 1/16 wrinkled & green \| The 9:3:3:1 phenotypic ratio that we calculated using the product rule can also be obtained using Punnett Square (Figure $\PageIndex{4}$). First, we list the genotypes of the possible gametes along each axis of the Punnett Square. In a diploid with two heterozygous genes of interest, there are up to four combinations of alleles in the gametes of each parent. The gametes from the respective rows and column are then combined in the each cell of the array. When working with two loci, genotypes are written with the symbols for both alleles of one locus, followed by both alleles of the next locus (e.g. AaBb , not ABab ). Note that the order in which the loci are written does not imply anything about the actual position of the loci on the chromosomes. To calculate the expected phenotypic ratios, we assign a phenotype to each of the 16 genotypes in the Punnett Square, based on our knowledge of the alleles and their dominance relationships. In the case of Mendel’s seeds, any genotype with at least one R allele and one Y allele will be round and yellow; these genotypes are shown in the nine, green-shaded cells in Figure $\PageIndex{4}$. We can represent all of four of the different genotypes shown in these cells with the notation ( R_Y _), where the blank line (__), means “any allele”. The three offspring that have at least one R allele and are homozygous recessive for y (i.e. R_yy ) will have a round, green phenotype. Conversely the three progeny that are homozygous recessive r , but have at least one Y allele ( rrY_ ) will have wrinkled, yellow seeds. Finally, the rarest phenotypic class of wrinkled, yellow seeds is produced by the doubly homozygous recessive genotype, rryy , which is expected to occur in only one of the sixteen possible offspring represented in the square. Assumptions of the 9:3:3:1 ratio Both the product rule and the Punnett Square approaches showed that a 9:3:3:1 phenotypic ratio is expected among the progeny of a dihybrid cross such as Mendel’s RrYy × RrYy . In making these calculations, we assumed that: - both loci assort independently; - one allele at each locus is completely dominant; and - each of four possible phenotypes can be distinguished unambiguously, with no interactions between the two genes that would alter the phenotypes. Deviations from the 9:3:3:1 phenotypic ratio may indicate that one or more of the above conditions has not been met. Modified ratios in the progeny of a dihybrid cross can therefore reveal useful information about the genes involved. Linkage is one of the most important reasons for distortion of the ratios expected from independent assortment. Linked genes are located close together on the same chromosome. This close proximity alters the frequency of allele combinations in the gametes. We will return to the concept of linkage in Chapter 7. Deviations from 9:3:3:1 ratios can also be due to interactions between genes. These interactions will be discussed in the remainder of this chapter. For simplicity, we will focus on examples that involve easily scored phenotypes, such as pigmentation. Nevertheless, keep in mind that the analysis of segregation ratios of any markers can provide insight into a wide range of biological processes they represent.	libretexts	2025-03-17T22:27:33.441304	2021-01-03T20:11:56	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/03%3A_Alleles_and_Probabilities/3.06%3A_Dihybrid_Crosses", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "3.6: Dihybrid Crosses", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/03%3A_Alleles_and_Probabilities/3.07%3A_Why_Use_Nomenclature	3.7: Why Use Nomenclature? selected template will load here This action is not available. Word and practical problems in biology can get confusing in a hurry, particularly if you’re distracted by something like exam stress! When you are manipulating several ideas, it is good practice to be thoughtful and follow rules that keep you consistent in your interpretation. As an instructor, I have seen work in which the student clearly got flustered and forgot that the mutation he or she was working on was dominant. This often leads to an answer that is inconsistent with the data. There are a few simple rules we can use for nomenclature. This first appendix uses a simplified system to communicate allele characteristics. The next appendix will show a more complicated system that carries even more information in the gene symbols.	libretexts	2025-03-17T22:27:33.504254	2021-01-03T20:11:57	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/03%3A_Alleles_and_Probabilities/3.07%3A_Why_Use_Nomenclature", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "3.7: Why Use Nomenclature?", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/03%3A_Alleles_and_Probabilities/3.08%3A_Basic_Nomenclature	3.8: Basic Nomenclature - - Last updated - Save as PDF Single Letter System Sometimes what you want to do is a little rough work for investigating your genetic model. A genetic model is a diagram of the logic that you propose for a particular type of inheritance. For instance, if you cross a true‐breeding purple plant with a true‐breeding white plant (Figure A1.2), you will get a heterozygote (the middle plant). If we name the gene after the mutation ( a is the first letter in “albino”), we know that the heterozygote will have one capital letter “ A ” and a lower case “ a ”. The heterozygote is the F 1 generation (“first filial”, which means it’s the first child from parents that are crossed). The F 1 is purple, which means the “ a ” allele is recessive; only one copy of the “ A ” allele is needed for enough purple pigment to make it identical to one true-breeding parent. This is complete dominance. We cannot know from the information given which allele is wild type or mutant. One hypothesis, though, is that purple pigments are required to attract pollinators and therefore would help the plant in the wild. Albino plants could be a mutant and might not generate as many seeds for lack of pollinators. If the context of your genetics problem doesn’t indicate which allele is wild‐type, it’s good practice to name your allele based on the recessive trait. Often the recessive allele is the mutation. We might consider that the “ a ” allele is null and makes no pigment at all. One or two “ A ” alleles make enough protein to cause the plant to be purple. We can’t know from the information given which allele is wild type or mutant. One hypothesis, though, is that purple pigments are required to attract pollinators and therefore would help the plant in the wild. Albino plants could be a mutant and might not generate as many seeds for lack of pollinators. If the context of your genetics problem doesn’t indicate which allele is wild‐type, it’s good practice to name your allele based on the recessive trait. Often the recessive allele is the mutation. We might consider that the “a” allele is null and makes no pigment at all. One or two “A” alleles make enough protein to cause the plant to be purple. Figure A1.2 already assumes that the capital letter ( A ) stands for an allele that encodes a protein for purple pigment and the recessive allele ( a ) doesn’t make pigment. Thus the Aa heterozygote is sufficient evidence to adopt upper- and lower-case letter “ A ”s to communicate the characteristics of purple and white alleles. A note of caution . When you’re writing down gene symbols for homework or on an exam, be sure to make the characters distinct. A typewritten “ y ” is easy to distinguish from the upper case “ Y ” but not as easy when writing it down. Instructors who ask you to show your work need to be able to follow your logic train. More important than that is YOU have to be able to follow your own reasoning. Students often switch symbols and come up with an answer that is inconsistent with the data given because of this. Consider underlining your capitals or putting a line through one of them to make it distinct (e.g. Y for the dominant allele; y for the recessive). Name the gene after the mutant phenotype Some instructors would accept “ P ” for “ purple ” for the previous cross. However, the better answer is to follow an established system. During “exam fog”, it’s easy to get lost if you are inconsistent with how you develop your symbols. During your study period and when you’re practicing genetics problems, be thoughtful about the gene names you choose. Let’s always choose a letter based on the mutant phenotype for our gene symbol. If we are presented with a ladybug mutant that is small, we might choose “ d ” for “ dwarf ”. Geneticists sometimes set up a research program based on unusual phenotypes of the organism they are studying. The fact that a mutant phenotype that is heritable exists tells us that there is a genetic control for the trait and that it might be isolated in the lab. When you look at your classmates, you don’t necessarily note that none of them has an arm growing from the tops of their heads. If one student had this trait, however, you couldn’t help but notice it. If you found out it showed up in that student’s ancestry in a predictable fashion, you might reasonably suggest that there is a genetic basis for that. If it happened to be controlled by a single gene, you might call the gene “ extra arm ” or “ arm head ”. If it happened to be a dominant trait, you might use the letter “ X ” or “ A ” for the mutant allele. The wild type allele would be “ x ” or “ a ”, respectively. you don’t know which is mutant, use the recessive trait for the gene name What if you don’t know which allele is mutant? What if you’re presented with two true-‐ breeding frogs: one that is gold and one that is yellow. If you don’t know what the predominant colour in nature is you can’t know which one is mutant. If you crossed them and all the progeny are gold, then you know the dominant allele encodes a protein to make it gold . The recessive, therefore, is “ yellow ” and you should name the gene “ y ” after the recessive phenotype. This means the dominant allele would be “ Y ”. Your offspring would therefore be Yy and the gold parent would be YY . The yellow parent would be yy . Apply these ideas at the Online Open Genetics exercises. 1.3 One Letter System Superscripts Sometimes a letter is used as the name of a gene, and superscripts can modify it to indicate the different alleles. One common single letter code for an allelic series is “ I ". Red blood cells can have their cell membranes modified by sugar tags that give rise to our blood type. One allele of I gives rise to blood type A and is therefore called I A . An enzyme encoded by I B modifies sugars to create blood type B. A heterozygote I A I B demonstrates both sugar tags because those alleles are expressed – they are codominant. People with blood type O only possess alleles for the I gene that don’t work and are therefore recessive – they don’t modify the extracellular sugar tags. Because it is recessive, individuals are homozygous for i : they are ii . Chapter 13, Section 7 has more detail on this allelic series. Sometimes a superscript “plus sign” is used to denote the wild type allele. One might use the symbol W + to indicate a wild-‐type allele that promotes wing growth. Note that the generic “wing” gene name isn’t a best practice – name the gene after the mutant phenotype! A wingless mutant would be W ‐ . You should never use a “+” and shift the case of the letter unless you are dealing with a special case such as the codominance in the blood type example above. The capital “ I ” letter indicates it is dominant to “ i ". The superscript A and B for the codominant alleles indicate the dominant alleles are different from each other. Superscripts can be symbols, a single letter, or many letters. They modify the gene name only in the superscripted symbols: the regular-‐sized letters are identical between them (see Table 1). This means that Abc + and abc would be different genes ( i.e. not allelic); Abc + and Abc are alleles, as are abc + and abc . Note that a superscript is not mandatory for all alleles of that gene, depending on the convention. Alleles of bacterial genes are typically indicated with a superscript + or -. For example, a bacterial allele that creates an enzyme that makes methionine would be met + , and a defective allele of that gene is met - . \| white apr or white apricot \| An allele of the white gene which has an “apricot” phenotype \| \| Abc + Abc \| Two alleles for the Abc gene (wild-type and mutant, respectively). Note the mutant allele is dominant. \| \| w + ; w a ; w \| Three alleles in a series for the w gene. The first is wild type; the second two are different mutant alleles. \| \| bio + ; bio - \| A wild-type allele of a biotin gene and its recessive counterpart. This is likely a bacterial gene due to the convention. \|	libretexts	2025-03-17T22:27:33.574169	2021-01-03T20:11:57	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/03%3A_Alleles_and_Probabilities/3.08%3A_Basic_Nomenclature", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "3.8: Basic Nomenclature", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/03%3A_Alleles_and_Probabilities/3.09%3A_Linked_Genes	3.9: Linked Genes - - Last updated - Save as PDF Mendel was lucky. He studied a variety of traits in pea plants and his data were consistent with his idea of traits being encoded by pairs of discrete heritable units. He did not call these “genes” and had no idea about their chromosomal origins or chemical makeup. It turns out that the genes he studied were either on different chromosomes and so assorted independently (Chapter 9), or so far apart on the same chromosome that linkage could not be detected (Chapter 10). Symbols for a gene can be drawn on a page to communicate their position on a chromosome. To do this, we use a forward slash (/) to demonstrate what is on each chromosome. Figure A1.4 shows how we might conceptualize the position of a gene on two chromosomes by collapsing the chromosomes into a single line. There’s no question about where the gene is located when only one trait is under investigation: it will be at the same position on each homolog. Two genes, however, can be one of three possibilities. Each possibility has implications for gene mapping and predicting ratios from a dihybrid cross. Figure A1.5 shows the positions of genes for an unlinked situation as well as linked genes in coupling and repulsion configurations. If genes are unlinked, put the allele symbols for one gene on either side of one slash followed by a semicolon (indicating that it’s unlinked) and the other gene with the alleles separated by a second slash ( A/a ; B/b ). When genes are linked, only one slash is used: remember, the slash stands for a pair of homologous chromosomes. Genes in coupling would have the dominant genes together on one side of the slash and recessives on the other side ( AB/ab ). Repulsion would represent the other arrangement ( Ab/aB ). Practice your skills with identifying linked and unlinked genes online in module 1.5. Some examples of different forms of gene symbols are shown in Table 2. Keep in mind that sometimes you have flexibility in which system of nomenclature you use, but sometimes it is dictated to you, for example in publications or other formal submissions. You are discouraged from inventing your own system or mixing up different systems because it will confuse your readers (or graders!). \| Examples \| Interpretation \| \|---\|---\| \| A and a \| Uppercase letters represent dominant alleles and lowercase letters indicate recessive alleles. Mendel invented this system but it is not commonly used in publications because not all alleles show complete dominance and many genes have more than two alleles. It’s quick and easy for you to use when working out genetics problems when you are sure each gene involves only two alleles. \| \| a + and a \| Superscripts are used to indicate alleles. For wild type alleles the symbol is a superscript +. The mutant allele of gene a would be recessive. \| \| met + and met - \| This is typical of a prokaryote gene symbol. It could be referring to wild-type (functional) and mutant (nonfunctional) alleles of a gene that makes a protein in the methionine synthesis pathway. \| \| AA or A/A \| Sometimes a forward slash is used to indicate that the two symbols are alleles of the same gene, but on homologous chromosomes. Both representations in this row are identical: it represents a homozygous dominant. \| \| Aa/Aa or Aa/aa \| Note that this example shows two alleles of the gene Aa . We know that the gene symbol is two letters because the slash separates the allele found on each of the homologous chromosomes. We cannot tell if the mutant phenotype is recessive because there’s no indication which is wild type. \| \| Grn + shr/Grn shr \| The three-letter system is used here. “ Grn ” might mean that the phenotype is “green”, but we can’t be sure. What we do know is that the mutant allele codes for a protein leading to a dominant phenotype. The wild-type allele must be recessive to the mutant allele. Maybe “ shr ” means “shrunken” or “short”, but we know that the mutant phenotype can only be seen in the homozygous recessive configuration. The phenotype for this organism is mutant for both Grn and shr traits. Final note: the genes are on the same chromosome based on the position of the slash. \| \| bob + /bob ; mia/mia \| This also uses the three-letter system. The organism is heterozygous for bob but shows the wild-type trait in its phenotype. It is homozygous recessive for mia and therefore shows that mutant phenotype. The genes are unlinked. \| A more advanced system of nomenclature is outlined in Appendix 2. New rules are introduced to help you identify sex linked genes and predict phenotypes from gene symbols alone.	libretexts	2025-03-17T22:27:33.636205	2021-01-03T20:11:58	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/03%3A_Alleles_and_Probabilities/3.09%3A_Linked_Genes", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "3.9: Linked Genes", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/04%3A_Hypothesis_Testing/4.01%3A__Phenotypic_Ratios_May_Not_Be_As_Expected	4.1: Phenotypic Ratios May Not Be As Expected selected template will load here This action is not available. For a variety of reasons, the phenotypic ratios observed from real crosses rarely match the exact ratios expected based on a Punnett Square or other prediction techniques. There are many possible explanations for deviations from expected ratios. Sometimes these deviations are due to sampling effects , in other words, the random selection of a non-representative subset of individuals for observation. On the other hand, it may be because certain genotypes have a less than 100% survival rate. For example, Drosophila crosses sometimes give unexpected results because the more mutant alleles a zygote has the less likely it is to survive to become an adult. Genotypes that cause death for embryos or larvae are underrepresented when adult flies are counted. A statistical procedure called the chi-square ( χ 2 ) test can be used to help a geneticist decide whether the deviation between observed and expected ratios is due to sampling effects, or whether the difference is so large that some other explanation must be sought by re-examining the assumptions used to calculate the expected ratio. The procedure for performing a chi-square test is covered in the labs.	libretexts	2025-03-17T22:27:33.728747	2021-01-03T20:12:00	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/04%3A_Hypothesis_Testing/4.01%3A__Phenotypic_Ratios_May_Not_Be_As_Expected", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "4.1: Phenotypic Ratios May Not Be As Expected", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/05%3A_Sex_Determination_Chromosomes_and_Linkage/5.01%3A_Karyotypes_Describe_Chromosome_Number_and_Structure	5.1: Karyotypes Describe Chromosome Number and Structure - - Last updated - Save as PDF Karyograms are images of real chromosomes Each eukaryotic species has its nuclear genome divided among a number of chromosomes that is characteristic of that species. For example, a haploid human nucleus (i.e. sperm or egg) normally has 23 chromosomes (n=23), and a diploid human nucleus has 23 pairs of chromosomes (2n=46). A karyotype is the complete set of chromosomes of an individual. The cell was in metaphase so each of the 46 structures is a replicated chromosome even though it is hard to see the two sister chromatids for each chromosome at this resolution. As expected there are 46 chromosomes. Note that the chromosomes have different lengths. In fact, human chromosomes were named based upon this feature. Our largest chromosome is called 1, our next longest is 2, and so on. By convention the chromosomes are arranged into the pattern shown in Figure $\PageIndex{15}$ and the resulting image is called a karyogram . A karyogram allows a geneticist to determine a person's karyotype - a written description of their chromosomes including anything out of the ordinary. Figure $\PageIndex{15}$: Karyogram of a normal human male karytype.(Wikipedia-NHGRI-PD) Various stains and fluorescent dyes are used to produce characteristic banding patterns to distinguish all 23 chromosomes. The number of chromosomes varies between species, but there appears to be very little correlation between chromosome number and either the complexity of an organism or its total amount genomic DNA. Autosomes and Sex Chromosomes In the figure above note that most of the chromosomes are paired (same length, centromere location, and banding pattern). These chromosomes are called autosomes . However note that two of the chromosomes, the X and the Y do not look alike. These are sex chromosomes . In humans males have one of each while females have two X chromosomes. Autosomes are those chromosomes present in the same number in males and females while sex chromosomes are those that are not. When sex chromosomes were first discovered their function was unknown and the name X was used to indicate this mystery. The next ones were named Y, then Z, and then W. The combination of sex chromosomes within a species is associated with either male or female individuals. In mammals, fruit flies, and some flowering plants embryos, those with two X chromosomes develop into females while those with an X and a Y become males. In birds, moths, and butterflies males are ZZ and females are ZW. Because sex chromosomes have arisen multiple times during evolution the molecular mechanism(s) through which they determine sex differs among those organisms. For example, although humans and Drosophila both have X and Y sex chromosomes, they have different mechanisms for determining sex . In mammals, the sex chromosomes evolved just after the divergence of the monotreme lineage from the lineage that led to placental and marsupial mammals. Thus nearly every mammal species uses the same sex determination system. During embryogenesis the gonads will develop into either ovaries or testes. A gene present only on the Y chromosome called TDF encodes a protein that makes the gonads mature into testes. XX embryos do not have this gene and their gonads mature into ovaries instead (default). Once formed the testes produce sex hormones that direct the rest of the developing embryo to become male, while the ovaries make different sex hormones that promote female development. The testes and ovaries are also the organs where gametes (sperm or eggs) are produced. How do the sex chromosome behave during meiosis? Well, in those individuals with two of the same chromosome (i.e. homogametic sexes: XX females and ZZ males) the chromosomes pair and segregate during meiosis I the same as autosomes do. During meiosis in XY males or ZW females ( heterogametic sexes) the sex chromosomes pair with each other (Figure $\PageIndex{16}$). In mammals the consequence of this is that all egg cells will carry an X chromosome while the sperm cells will carry either an X or a Y chromosome. Half of the offspring will receive two X chromosomes and become female while half will receive an X and a Y and become male. Figure $\PageIndex{16}$: Meiosis in an XY mammal. The stages shown are anaphase I, anaphase II, and mature sperm. Note how half of the sperm contain Y chromosomes and half contain X chromosomes. (Original-Harrington-CC:AN) Aneuploidy - Changes in Chromosome Number Analysis of karyotypes can identify chromosomal abnormalities, including aneuploidy , which is the addition or subtraction of a chromosome from a pair of homologs. More specifically, the absence of one member of a pair of homologous chromosomes is called monosomy (only one remains). On the other hand, in a trisomy , there are three, rather than two (disomy), homologs of a particular chromosome. Different types of aneuploidy are sometimes represented symbolically; if 2n symbolizes the normal number of chromosomes in a cell, then 2n-1 indicates monosomy and 2n+1 represents trisomy. The addition or loss of a whole chromosome is a mutation, a change in the genotype of a cell or organism. The most familiar human aneuploidy is trisomy-21 (i.e. three copies of chromosome 21), which is one cause of Down syndrome . Most (but not all) other human aneuploidies are lethal at an early stage of embryonic development. Note that aneuploidy usually affects only one set of homologs within a karyotype, and is therefore distinct from polyploidy , in which the entire chromosome set is duplicated (see below). Aneuploidy is almost always deleterious, whereas polyploidy appears to be beneficial in some organisms, particularly many species of food plants. Aneuploidy can arise due to a non-disjunction event, which is the failure of at least one pair of chromosomes or chromatids to segregate during mitosis or meiosis. Non-disjunction will generate gametes with extra and missing chromosomes. Chromosomal abnormalities Structural defects in chromosomes are another type of abnormality that can be detected in karyotypes (Fig 2.17). These defects include deletions, duplications , and inversions , which all involve changes in a segment of a single chromosome. Insertions and translocations involve two non-homologous chromosomes. In an insertion, DNA from one chromosome is moved to a non-homologous chromosome in a unidirectional manner. In a translocation, the transfer of chromosomal segments is bidirectional and reciprocal – a reciprocal translocation. Figure $\PageIndex{17}$: Structural abberations in chromosomes.(Wikipedia-Zephyris-GFDL) Structural defects affect only part of a chromosome (a subset of genes), and so tend to be less harmful than aneuploidy. In fact, there are many examples of ancient chromosomal rearrangements in the genomes of species including our own. Duplications of some small chromosomal segments, in particular, may have some evolutionary advantage by providing extra copies of some genes, which can then evolve in new, potentially beneficial, ways. Chromosomal abnormalities arise in many different ways, some of which can be traced to rare errors in natural cellular processes such as DNA replication. Chromosome breakage also occurs infrequently as the result of physical damage (such as ionizing radiation), movement of some types of transposons, and other factors. During the repair of a broken chromosome, deletions, insertions, translocations and even inversions can be introduced.	libretexts	2025-03-17T22:27:33.884248	2021-01-03T20:12:05	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/05%3A_Sex_Determination_Chromosomes_and_Linkage/5.01%3A_Karyotypes_Describe_Chromosome_Number_and_Structure", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "5.1: Karyotypes Describe Chromosome Number and Structure", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/05%3A_Sex_Determination_Chromosomes_and_Linkage/5.02%3A__Sex-Linkage-_An_Exception_to_Mendels_First_Law	5.2: Sex-Linkage- An Exception to Mendel’s First Law - - Last updated - Save as PDF In the previous chapter we introduced sex chromosomes and autosomes. For loci on autosomes, the alleles follow the normal Mendelian pattern of inheritance. However, for loci on the sex chromosomes this is mostly not true, because most of the loci on the typical X-chromosome are absent from the Y-chromosome, even though they act as a homologous pair during meiosis. Instead, they will follow a sex-linked pattern of inheritance. X-Linked Genes: the white gene in Drosophila melanogaster A well-studied sex-linked gene is the white gene on the X chromosome of Drosophila melanogaster . Normally flies have red eyes but flies with a mutant allele of this gene called white - ( w - ) have white eyes because the red pigments are absent. Because this mutation is recessive to the wild type w + allele females that are heterozygous have normal red eyes. Female flies that are homozygous for the mutant allele have white eyes. Because there is no white gene on the Y chromosome, male flies can only be hemizygous for the wild type allele or the mutant allele. A researcher may not know beforehand whether a novel mutation is sex-linked. The definitive method to test for sex-linkage is reciprocal crosses (Figure $\PageIndex{10}$). This means to cross a male and a female that have different phenotypes, and then conduct a second set of crosses, in which the phenotypes are reversed relative to the sex of the parents in the first cross. For example, if you were to set up reciprocal crosses with flies from pure-breeding w + and w - strains the results would be as shown in Figure $\PageIndex{10}$. Whenever reciprocal crosses give different results in the F1 and F2 and whenever the male and female offspring have different phenotypes the usual explanation is sex-linkage. Remember, if the locus were autosomal the F1 and F2 progeny would be different from either of these crosses. A similar pattern of sex-linked inheritance is seen for X-chromosome loci in other species with an XX-XY sex chromosome system, including mammals and humans. The ZZ-ZW system is similar, but reversed (see below). Sex Determination in animals. There are various mechanisms for sex determination in animals. These include sex chromosomes, chromosome dosage, and environment. For example in humans and other mammals XY embryos develop as males while XX embryos become females. This difference in development is due to the presence of only a single gene , the TDF-Y gene, on the Y-chromosome. Its presence and expression dictates that the sex of the individual will be male. Its absence results in a female phenotype. Although Drosophila melanogaster also has an XX-XY sex chromosomes, its sex determination system uses a different method, that of X:Autosome (X:A) ratio . In this system it is the ratio of autosome chromosome sets (A) relative to the number of X-chromosomes (X) that determines the sex. Individuals with two autosome sets and two X-chromosomes (2A:2X) will develop as females, while those with only one X-chromosome (2A:1X) will develop as males. The presence/absence of the Y-chromosome and its genes are not significant. In other species of animals the number of chromosome sets can determine sex. For example the haploid-diploid system is used in bees, ants, and wasps. Typically haploids are male and diploids are female. In other species, the environment can determine an individuals sex. In alligators (and some other reptiles) the temperature of development dictates the sex, while in many reef fish, the population sex ratio can cause some individuals to change sex. Dosage Compensation for Loci on Sex Chromosomes. Mammals and Drosophila both have XX - XY sex determination systems. However, because these systems evolved independently they work differently with regard to compensating for the difference in gene dosage (and sex determination – see above). Remember, in most cases the sex chromosomes act as a homologous pair even though the Y-chromosome has lost most of the loci when compared to the X-chromosome. Typically, the X and the Y chromosomes were once similar but, for unclear reasons, the Y chromosomes have degenerated, slowly mutating and loosing its loci. In modern day mammals the Y chromosomes have very few genes left while the X chromosomes remain as they were. This is a general feature of all organisms that use chromosome based sex determination systems. Chromosomes found in both sexes (the X or the Z) have retained their genes while the chromosome found in only one sex (the Y or the W) have lost most of their genes. In either case there is a gene dosage difference between the sexes: e.g. XX females have two doses of X-chromosome genes while XY males only have one. This gene dosage needs to be compensated in a process called dosage compensation . There are two major mechanisms. In Drosophila and many other insects, to make up for the males only having a single X chromosome the genes on it are expressed at twice the normal rate. This mechanism of dosage compensation restores a balance between proteins encoded by X-linked genes and those made by autosomal genes . In mammals a different mechanism is used, called X-chromosome inactivation . X-chromosome Inactivation in Mammals In mammals the dosage compensation system operates in females, not males. In XX embryos one X in each cell is randomly chosen and marked for inactivation. From this point forward this chromosome will be inactive, hence its name X inactive (X i ). The other X chromosome, the X active (X a ), is unaffected. The X i is replicated during S phase and transmitted during mitosis the same as any other chromosome but most of its genes are never allowed to turn on. The chromosome appears as a condensed mass within interphase nuclei called the Barr body. With the inactivation of genes on one X-chromosome, females have the same number of functioning X-linked genes as males. This random inactivation of one X-chromosome leads to a commonly observe phenomenon in cats. A familiar X-linked gene is the Orange gene ( O ) in cats. The O O allele encodes an enzyme that results in orange pigment for the hair. The O B allele causes the hairs to be black. The phenotypes of various genotypes of cats are shown in Figure $\PageIndex{11}$. Note that the heterozygous females have an orange and black mottled phenotype known as tortoiseshell. This is due to patches of skin cells having different X-chromosomes inactivated. In each orange hair the X i chromosome carrying the O B allele is inactivated. The O O allele on the X a is functional and orange pigments are made. In black hairs the reverse is true, the X i chromosome with the O O allele is inactive and the X a chromosome with the O B allele is active. Because the inactivation decision happens early during embryogenesis, the cells continue to divide to make large patches on the adult cat skin where one or the other X is inactivated. The Orange gene in cats is a good demonstration of how the mammalian dosage compensation system affects gene expression. However, most X-linked genes do not produce such dramatic mosaic phenotypes in heterozygous females. A more typical example is the F8 gene in humans. It makes Factor VIII blood clotting proteins in liver cells. If a male is hemizygous for a mutant allele the result is hemophilia type A. Females homozygous for mutant alleles will also have hemophilia. Heterozygous females, those people who are F8 + / F8 - , do not have hemophilia because even though half of their liver cells do not make Factor VIII (because the X with the F8 + allele is inactive) the other 50% can (Figure $\PageIndex{12}$). Because some of their liver cells are exporting Factor VIII proteins into the blood stream they have the ability to form blood clots throughout their bodies. The genetic mosaicism in the cells of their bodies does not produce a visible mosaic phenotype. Other Sex-Linked Genes – Z-linked genes One last example is a Z-linked gene that influences feather colour in turkeys. Turkeys are birds, which use the ZZ-ZW sex chromosome system. The E allele makes the feathers bronze and the e allele makes the feathers brown (Figure $\PageIndex{13}$). Only male turkeys can be heterozygous for this locus, because they have two Z chromosomes. They are also uniformly bronze because the E allele is completely dominant to the e allele and birds use a dosage compensation system similar to Drosophila and not mammals. Reciprocal crosses between turkeys from pure-breeding bronze and brown breeds would reveal that this gene is in fact Z-linked. Mechanisms of Sex Determination Systems Sex is a phenotype. Typically, in most species, there are multiple characteristics, in addition to sex organs, that distinguish male from female individuals (although some species are normally hermaphrodites where both sex organs are present in the same individual). The morphology and physiology of male and females is a phenotype just like hair or eye colour or wing shape. The sex of an organism is part of its phenotype and can be genetically (or environmentally) determined. For each species, the genetic determination relies on one of several gene or chromosome based mechanisms. See Figure $\PageIndex{14}$ for a summary. There are, for other species, also a variety of environmental mechanisms, too (rearing temperature, social interactions, parthenogenesis ). Whatever the sex choice mechanism, however, there are two different means by which the cells of an organism carry out this decision: hormonal or cell-autonomous Hormonal mechanism: With this system, used by mammals for example, including humans, the zygote initially develops into a sexually undifferentiated embryo that can become either sex. Then, depending on the sex choice of the genital ridge cells, they will grow and differentiate into male (testis) or female (ovary) gonads, which will then produce the appropriate hormones (e.g. testosterone or estrogen). This hormone will circulate throughout the body and cause all the other tissues to develop and differentiate accordingly, into a male or female phenotype for that individual. Thus, the circulating hormone “tells” all the cells and tissues what sex to be and which sexual phenotype to be. A freemartin is a type of chimera found in cattle (and some other mammals). Externally it appears as a female but is infertile, and has masculinized behavior and non-functioning ovaries. The animal originates as a female (XX), but acquires male (XY) cells or tissues in utero by exchange of some cellular material from a male twin. The female reproductive development is altered by anti-Müllerian hormone from the male twin, acquired via vascular connections between placentas. Cell-autonomous mechanism: With this system, used by many animals, including birds and insects, the zygote cell initially has a sex phenotype set at the cell level. All cells intrinsically know, individually, which sex they are and develop accordingly, giving the appropriate sexual characteristics and phenotype. Each cell is autonomous with respect to its sex; there are no sex hormone cues to determine the sex expressed. This autonomy can lead to sexual gynandromorphs , which are mosaics that display both male and female characteristics in a mosaic fasion, typically split down the midline of the organism. These rare individuals are thought to be the result of an improper sex chromosome segregation that occurs in a cell very early in development so that one half of the individual has cells with a male chromosome set while the other half has cells with a female set. If the species is sexually dimorphic (external morphology easily distinguishs males from females) they are easily visible and are even sometimes seen in the wild. See Figure $\PageIndex{15}$ for a local example. A search on the internet will bring up many more examples. While gynandromorphs are seen in cell-autonomous species, such as insects and birds, they are not seen in hormonally determined species, such as mammals, because all the cells display the same sex phenotype caused by the circulating sex hormones. Sexual gynandromorphs appear to be absent in reptiles, amphibians, and fish indicating that they don’t use a cell-autonomous mechanism. Nevertheless, there are genetic mosaic individuals in these groups but they do not appear to involve sex determined traits, which is required for a true gynandromorph. They often involve mosaicism of alleles at a single gene locus that affect external morphology (e.g. color). - A gynandromorph is an organism that made up of mosaic tissues of male and female genotypes and displays both male and female characteristics. - A mosaic is an organism or a tissue that contains two or more types of genetically different cells derived from the same zygote. - A chimera is a single organism composed of genetically distinct cells derived from different zygotes.	libretexts	2025-03-17T22:27:33.960644	2021-01-03T20:12:07	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/05%3A_Sex_Determination_Chromosomes_and_Linkage/5.02%3A__Sex-Linkage-_An_Exception_to_Mendels_First_Law", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "5.2: Sex-Linkage- An Exception to Mendel’s First Law", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/06%3A_Pedigrees/6.01%3A_Pedigree_Analysis	6.1: Pedigree Analysis - - Last updated - Save as PDF Pedigree charts are diagrams that show the phenotypes and/or genotypes for a particular organism and its ancestors. While commonly used in human families to track genetic diseases, they can be used for any species and any inherited trait. Geneticists use a standardized set of symbols to represent an individual’s sex, family relationships and phenotype. These diagrams are used to determine the mode of inheritance of a particular disease or trait, and to predict the probability of its appearance among offspring. Pedigree analysis is therefore an important tool in both basic research and genetic counseling . Each pedigree chart represents all of the available information about the inheritance of a single trait (most often a disease) within a family. The pedigree chart is therefore drawn using factual information, but there is always some possibility of errors in this information, especially when relying on family members’ recollections or even clinical diagnoses. In real pedigrees, further complications can arise due to incomplete penetrance (including age of onset) and variable expressivity of disease alleles, but for the examples presented in this book, we will presume complete accuracy of the pedigrees. A pedigree may be drawn when trying to determine the nature of a newly discovered disease, or when an individual with a family history of a disease wants to know the probability of passing the disease on to their children. In either case, a tree is drawn, as shown in Figure $\PageIndex{2}$, with circles to represent females, and squares to represent males. Matings are drawn as a line joining a male and female, while a consanguineous mating (closely related is two lines. The affected individual that brings the family to the attention of a geneticist is called the proband (or propositus). If an individual is known to have symptoms of the disease ( affected ), the symbol is filled in. Sometimes a half-filled in symbol is used to indicate a known carrier of a disease; this is someone who does not have any symptoms of the disease, but who passed the disease on to subsequent generations because they are a heterozygote. Note that when a pedigree is constructed, it is often unknown whether a particular individual is a carrier or not, so not all carriers are always explicitly indicated in a pedigree. For simplicity, in this chapter we will assume that the pedigrees presented are accurate, and represent fully penetrant traits.	libretexts	2025-03-17T22:27:34.124798	2021-01-03T20:12:11	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/06%3A_Pedigrees/6.01%3A_Pedigree_Analysis", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "6.1: Pedigree Analysis", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/06%3A_Pedigrees/6.02%3A_Inferring_the_Mode_of_Inheritance	6.2: Inferring the Mode of Inheritance - - Last updated - Save as PDF Given a pedigree of an uncharacterized disease or trait, one of the first tasks is to determine which modes of inheritance are possible and then which mode of inheritance is most likely. This information is essential in calculating the probability that the trait will be inherited in any future offspring. We will mostly consider five major types of inheritance: autosomal dominant (AD), autosomal recessive (AR), X-linked dominant (XD), X-linked recessive (XR), and Y-linked (Y). Autosomal Dominant (AD) When a disease is caused by a dominant allele of a gene, every person with that allele will show symptoms of the disease (assuming complete penetrance), and only one disease allele needs to be inherited for an individual to be affected. Thus, every affected individual must have an affected parent. A pedigree with affected individuals in every generation is typical of AD diseases. However, beware that other modes of inheritance can also show the disease in every generation, as described below. It is also possible for an affected individual with an AD disease to have a family without any affected children, if the affected parent is a heterozygote. This is particularly true in small families, where the probability of every child inheriting the normal, rather than disease allele is not extremely small. Note that AD diseases are usually rare in populations, therefore affected individuals with AD diseases tend to be heterozygotes (otherwise, both parents would have had to been affected with the same rare disease). Achondroplastic dwarfism, and polydactyly are both examples of human conditions that may follow an AD mode of inheritance. X-linked dominant (XD) In X-linked dominant inheritance, the gene responsible for the disease is located on the X-chromosome, and the allele that causes the disease is dominant to the normal allele in females. Because females have twice as many X-chromosomes as males, females tend to be more frequently affected than males in the population. However, not all pedigrees provide sufficient information to distinguish XD and AD. One definitive indication that a trait is inherited as AD, and not XD, is that an affected father passes the disease to a son; this type of transmission is not possible with XD, since males inherit their X chromosome from their mothers. Autosomal recessive (AR) Diseases that are inherited in an autosomal recessive pattern require that both parents of an affected individual carry at least one copy of the disease allele. With AR traits, many individuals in a pedigree can be carriers, probably without knowing it. Compared to pedigrees of dominant traits, AR pedigrees tend to show fewer affected individuals and are more likely than AD or XD to “skip a generation”. Thus, the major feature that distinguishes AR from AD or XD is that unaffected individuals can have affected offspring. X-linked recessive (XR) Because males have only one X-chromosome, any male that inherits an X-linked recessive disease allele will be affected by it (assuming complete penetrance). Therefore, in XR modes of inheritance, males tend to be affected more frequently than females in a population. This is in contrast to AR and AD, where both sexes tend to be affected equally, and XD, in which females are affected more frequently. Note, however, in the small sample sizes typical of human families, it is usually not possible to accurately determine whether one sex is affected more frequently than others. On the other hand, one feature of a pedigree that can be used to definitively establish that an inheritance pattern is not XR is the presence of an affected daughter from unaffected parents; because she would have had to inherit one X-chromosome from her father, he would also have been affected in XR. Y-linked and Mitochondrial Inheritance. Two additional modes are Y-linked and Mitochondrial inheritance . Only males are affected in human Y-linked inheritance (and other species with the X/Y sex determining system). There is only father to son transmission. This is the easiest mode of inheritance to identify, but it is one of the rarest because there are so few genes located on the Y-chromosome. An example of Y-linked inheritance is the hairy-ear-rim phenotype seen in some Indian families. As expected this trait is passed on from father to all sons and no daughters. Y-chromosome DNA polymorphisms can be used to follow the male lineage in large families or through ancient ancestral lineages. For example, the Y-chromosome of Mongolian ruler Genghis Khan (1162-1227 CE), and his male relatives, accounts for ~8% of the Y-chromosome lineage of men in Asia (0.5% world wide). Mutations in Mitochondrial DNA are inherited through the maternal line (from your mother). There are some human diseases associated with mutations in mitochondria genes. These mutations can affect both males and females, but males cannot pass them on as the mitochondria are inherited via the egg, not the sperm. Mitochondrial DNA polymorphisms are also used to investigate evolutionary lineages, both ancient and recent. Because of the relative similarity of sequence mtDNA is also used in species identification in ecology studies.	libretexts	2025-03-17T22:27:34.189725	2021-01-03T20:12:11	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/06%3A_Pedigrees/6.02%3A_Inferring_the_Mode_of_Inheritance", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "6.2: Inferring the Mode of Inheritance", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/06%3A_Pedigrees/6.03%3A_Sporadic_and_Non-Heritable_Diseases	6.3: Sporadic and Non-Heritable Diseases selected template will load here This action is not available. Not all of the characterized human traits and diseases are attributed to mutant alleles at a single gene locus. Many diseases that have a heritable component, have more complex inheritance patterns due to (1) the involvement of multiple genes, and/or (2) environmental factors. On the other hand, some non-genetic diseases may appear to be heritable because they affect multiple members of the same family, but this is due to the family members being exposed to the same toxins or other environmental factors (e.g. in their homes). Finally, diseases with similar symptoms may have different causes, some of which may be genetic while others are not. One example of this is ALS (amyotrophic lateral sclerosis); approximately 5-10% of cases are inherited in an AD pattern, while the majority of the remaining cases appear to be sporadic , in other words, not caused by a mutation inherited from a parent. We now know that different genes or proteins are affected in the inherited and sporadic forms of ALS. The physicist Stephen Hawking (Figure $\PageIndex{10}$) and baseball player Lou Gehrig both suffered from sporadic ALS.	libretexts	2025-03-17T22:27:34.252079	2021-01-03T20:12:13	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/06%3A_Pedigrees/6.03%3A_Sporadic_and_Non-Heritable_Diseases", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "6.3: Sporadic and Non-Heritable Diseases", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/06%3A_Pedigrees/6.04%3A_Calculating_Probabilities	6.4: Calculating Probabilities - - Last updated - Save as PDF Once the mode of inheritance of a disease or trait is identified, some inferences about the genotype of individuals in a pedigree can be made, based on their phenotypes and where they appear in the family tree. Given these genotypes, it is possible to calculate the probability of a particular genotype being inherited in subsequent generations. This can be useful in genetic counseling, for example when prospective parents wish to know the likelihood of their offspring inheriting a disease for which they have a family history. Probabilities in pedigrees are calculated using knowledge of Mendelian inheritance and the same basic methods as are used in other fields. The first formula is the product rule : the joint probability of two independent events is the product of their individual probabilities; this is the probability of one event AND another event occurring. For example, the probability of a rolling a “five” with a single throw of a single six-sided die is 1/6, and the probability of rolling “five” in each of three successive rolls is 1/6 x 1/6 x 1/6 = 1/216. The second useful formula is the sum rule , which states that the combined probability of two independent events is the sum of their individual probabilities. This is the probability of one event OR another event occurring. For example, the probability of rolling a five or six in a single throw of a dice is 1/6 + 1/6 = 1/3. With these rules in mind, we can calculate the probability that two carriers (i.e. heterozygotes) of an AR disease will have a child affected with the disease as ½ x ½ = ¼, since for each parent, the probability of any gametes carrying the disease allele is ½. This is consistent with what we already know from calculating probabilities using a Punnett Square (e.g. in a monohybrid cross Aa x Aa , ¼ of the offspring are aa ). We can likewise calculate probabilities in the more complex pedigree shown in Figure $\PageIndex{11}$. Assuming the disease has an AR pattern of inheritance, what is the probability that individual 14 will be affected? We can assume that individuals #1, #2, #3 and #4 are heterozygotes ( Aa ), because they each had at least one affected ( aa ) child, but they are not affected themselves. This means that there is a 2/3 chance that individual #6 is also Aa . This is because according to Mendelian inheritance, when two heterozygotes mate, there is a 1:2:1 distribution of genotypes AA : Aa : aa . However, because #6 is unaffected, he can’t be aa , so he is either Aa or AA , but the probability of him being Aa is twice as likely as AA . By the same reasoning, there is likewise a 2/3 chance that #9 is a heterozygous carrier of the disease allele. If individual 6 is a heterozygous for the disease allele, then there is a ½ chance that #12 will also be a heterozygote (i.e. if the mating of #6 and #7 is Aa × AA , half of the progeny will be Aa ; we are also assuming that #7, who is unrelated, does not carry any disease alleles). Therefore, the combined probability that #12 is also a heterozygote is 2/3 x 1/2 = 1/3. This reasoning also applies to individual #13, i.e. there is a 1/3 probability that he is a heterozygote for the disease. Thus, the overall probability that both individual #12 and #13 are heterozygous, and that a particular offspring of theirs will be homozygous for the disease alleles is 1/3 x 1/3 x 1/4 = 1/36.	libretexts	2025-03-17T22:27:34.309871	2021-01-03T20:12:14	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/06%3A_Pedigrees/6.04%3A_Calculating_Probabilities", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "6.4: Calculating Probabilities", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/07%3A_Multiple_Gene_Alleles/7.01%3A_Relationships_Between_Genes_Genotypes_and_Phenotypes	7.1: Relationships Between Genes, Genotypes and Phenotypes - - Last updated - Save as PDF Terminology A specific position along a chromosome is called a locus . Each gene occupies a specific locus (so the terms locus and gene are often used interchangeably). Each locus will have an allelic form (allele). The complete set of alleles (at all loci of interest) in an individual is its genotype . Typically, when writing out a genotype, only the alleles at the locus (loci) of interest are considered – all the others are present and assumed to be wild type. The visible or detectable effect of these alleles on the structure or function of that individual is called its phenotype – what it looks like. The phenotype studied in any particular genetic experiment may range from simple, visible traits such as hair color, to more complex phenotypes including disease susceptibility or behavior. If two alleles are present in an individual, then various interactions between them may influence their expression in the phenotype. Complete Dominance Let us return to an example of a simple phenotype: flower color in Mendel’s peas. We have already said that one allele as a homozygote produces purple flowers, while the other allele as a homozygote produces white flowers (see Figures 1.8 and 3.3). But what about an individual that has one purple allele and one white allele; what is the phenotype of an individual whose genotype is heterozygous? This can only be determined by experimental observation. We know from observation that individuals heterozygous for the purple and white alleles of the flower color gene have purple flowers. Thus, the allele associated with purple color is therefore said to be dominant to the allele that produces the white color. The white allele, whose phenotype is masked by the purple allele in a heterozygote, is recessive to the purple allele. To represent this relationship, often, a dominant allele will be represented by a capital letter (e.g. A ) while a recessive allele will be represented in lower case (e.g. a ). However, many different systems of genetic symbols are in use. The most common are shown in Table $\PageIndex{1}$. Also note that genes and alleles are usually written in italics and chromosomes and proteins are not. For example, the white gene in Drosophila melanogaster on the X chromosome encodes a protein called WHITE. \| Examples \| Interpretation \| \|---\|---\| \| A and a \| Uppercase letters represent dominant alleles and lowercase letters indicate recessive alleles. Mendel invented this system but it is not commonly used because not all alleles show complete dominance and many genes have more than two alleles. \| \| a + and a 1 \| Superscripts or subscripts are used to indicate alleles. For wild type alleles the symbol is a superscript +. \| \| AA or A/A \| Sometimes a forward slash is used to indicate that the two symbols are alleles of the same gene, but on homologous chromosomes. \| Incomplete Dominance Besides dominance and recessivity, other relationships can exist between alleles. In incomplete dominance (also called semi-dominance, Figure $\PageIndex{4}$), both alleles affect the trait additively, and the phenotype of the heterozygote is intermediate between either of the homozygotes. For example, alleles for color in carnation flowers (and many other species) exhibit incomplete dominance. Plants with an allele for red petals ( A 1 ) and an allele for white petals ( A 2 ) have pink petals. We say that the A 1 and the A 2 alleles show incomplete dominance because neither allele is completely dominant over the other. Co-Dominance Co-dominance is another type of allelic relationship, in which a heterozygous individual expresses the phenotype of both alleles simultaneously. An example of co-dominance is found within the ABO blood group of humans. The ABO gene has three common alleles which were named (for historical reasons) I A , I B , and i . People homozygous for I A or I B display only A or B type antigens, respectively, on the surface of their blood cells, and therefore have either type A or type B blood (Figure $\PageIndex{5}$). Heterozygous I A I B individuals have both A and B antigens on their cells, and so have type AB blood. Notice that the heterozygote expresses both alleles simultaneously, and is not some kind of novel intermediate between A and B. Co-dominance is therefore distinct from incomplete dominance, although they are sometimes confused. Figure $\PageIndex{5}$: Relationship between genotype and phenotype for three alleles of the human ABO gene. The I A and I B alleles show co-dominance. The I A allele is completely dominant to the i allele. The I B allele is completely dominant to the i allele. (Original-Deholos -CC:AN)It is also important to note that the third allele, i , does not make either antigen and is recessive to the other alleles. I A /i or I B /i individuals display A or B antigens respectively. People homozygous for the i allele have type O blood. This is a useful reminder that different types of dominance relationships can exist, even for alleles of the same gene. Many types of molecular markers, which we will discuss in a later chapter, display a co-dominant relationship among alleles.	libretexts	2025-03-17T22:27:34.404385	2021-01-03T20:12:16	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/07%3A_Multiple_Gene_Alleles/7.01%3A_Relationships_Between_Genes_Genotypes_and_Phenotypes", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "7.1: Relationships Between Genes, Genotypes and Phenotypes", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/07%3A_Multiple_Gene_Alleles/7.02%3A_Complementation_tests_and_Allelism	7.2: Complementation tests and Allelism - - Last updated - Save as PDF – One, or more than one gene? As explained earlier in this chapter, mutant screening is one of the beginning steps geneticists use to investigate biological processes. When geneticists obtain two independently derived mutants (either from natural populations or during a mutant screen) with similar phenotypes, an immediate question is whether or not the mutant phenotype is due to a loss of function in the same gene, or are they mutant in different genes that both affect the same phenotype (e.g., in the same pathway). That is, are they allelic mutations , or non-allelic mutations , respectively? This question can be resolved using complementation tests , which bring together, or combine, the two mutations under consideration into the same organism to assess the combined phenotype. hypothetical example of purple flowers The easiest way to understand a complementation test is by example (Fig.4.9). The pigment in a purple flower could depend on a biochemical pathway much like the biochemical pathways leading to the production of arginine in Neurospora (review in Chapter 1). A plant that lacks the function of gene A (genotype aa ) would produce mutant, white flowers that looked just like the flowers of a plant that lacked the function of gene B (genotype bb ). (The genetics of two loci are discussed more in the following chapters.) Both A and B are enzymes in the same pathway that leads from a colorless compound#1, thorough colorless compound#2, to the purple pigment. Blocks at either step will result in a mutant white, not wild type purple, flower. Strains with mutations in gene A can be represented as the genotype aa , while strains with mutations in gene B can be represented as bb . Given that there are two genes here, A and B, then each of these mutant strains can be more completely represented as aaBB and AAbb . (LEARNING NOTE: Student often forget that genotypes usually only show mutant loci, however, one must remember all the other genes are assumed to be wild type.) If these two strains are crossed together the resulting progeny will all be AaBb . They will have both a wild type, functional A gene and B gene and will thus have a pigmented, purple flower, a wild type phenotype. This is an example of complementation . Together, each strain provides what the other is lacking ( AaBb ). The mutations are in different genes and are thus called non-allelic mutations. Now, if we are presented with a third pure-breeding, independently derived white-flower mutant strain, we won't initially know if it is mutant in gene A or gene B (or possibly some other gene altogether). We can use complementation testing to determine which gene is mutated. To perform a complementation test, two homozygous individuals with similar mutant phenotypes are crossed (Figure $\PageIndex{10}$). If the F1 progeny all have the same mutant phenotype (Case 1 - Figure $\PageIndex{10}$A), then we infer that the same gene is mutated in each parent. These mutations would then be called allelic mutations - in the same gene locus. These mutations FAIL to COMPLEMENT one another (still mutant). These could be either the exact same mutant alleles, or different mutations in the same gene (allelic). Conversely, if the F1 progeny all appear to be wild-type (Case 2 - Figure $\PageIndex{10}$B), then each of the parents most likely carries a mutation in a different gene. These mutations would then be called non-allelic mutations - in a different gene locus. These mutations do COMPLEMENT one another. Note: For mutations to be used in complementation tests they are (1) usually true-breeding (homozygous at the mutant locus), and (2) must be recessive mutations. Dominant mutation CANNOT be used in complementation tests. Also, remember, some mutant strains may have more than one gene locus mutated and thus would fail to complement mutants from more than one other locus (or group). \| A. \| B. \| \|\| \| Figure $\PageIndex{10}$A – Observation: In a typical complementation test, the genotypes of two parents are unknown (although they must be pure breeding, homozygous mutants). If the F1 progeny all have a mutant phenotype (Case 1), there is no complementation. If the F1 progeny are all wild-type, the mutations have successfully complemented each other. \| Figure $\PageIndex{10}$B – Interpretation: The pure breeding, homozygous mutant parents had unknown genotypes before the complementation test, but it could be assumed that they had either mutations in the same genes (Case 1) or in different genes (Case 2). In Case 1, all of the progeny would have the mutant phenotype, because they would all have the same, homozygous genotype as the parents. In Case 2, each parent has a mutation in a different gene, therefore none of the F 1 progeny would be homozygous mutant at either locus. Note that the genotype in Case 1 could be written as either aa or aaBB. (Original-Deyholos-CC:AN) \|	libretexts	2025-03-17T22:27:34.466389	2021-01-03T20:12:17	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/07%3A_Multiple_Gene_Alleles/7.02%3A_Complementation_tests_and_Allelism", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "7.2: Complementation tests and Allelism", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/08%3A_Gene_Interactions_and_Epistasis/8.01%3A_Epistasis_and_Other_Gene_Interactions	8.1: Epistasis and Other Gene Interactions - - Last updated - Save as PDF Some dihybrid crosses produce a phenotypic ratio that differs from 9:3:3:1, such as 9:3:4, 12:3:1, 9:7, or 15:1. Note that each of these modified ratios can be obtained by summing one or more of the 9:3:3:1 classes expected from our original dihybrid cross. In the following sections, we will look at some modified phenotypic ratios obtained from dihybrid crosses and what they might tell us about interactions between genes. Recessive epistasis Epistasis (which means “standing upon”) occurs when the phenotype of one locus masks, or prevents, the phenotype of another locus. Thus, following a dihybrid cross fewer than the typical four phenotypic classes will be observed with epistasis. As we have already discussed, in the absence of epistasis, there are four phenotypic classes among the progeny of a dihybrid cross. The four phenotypic classes correspond to the genotypes: A_B_, A_bb, aaB_, and aabb . If either of the singly homozygous recessive genotypes (i.e. A_bb or aaB_ ) has the same phenotype as the double homozygous recessive ( aabb ), then a 9:3:4 phenotypic ratio will be obtained. For example, in the Labrador Retriever breed of dogs (Figure $\PageIndex{5}$), the B locus encodes a gene for an important step in the production of melanin. The dominant allele, B is more efficient at pigment production than the recessive b allele, thus B _ hair appears black, and bb hair appears brown. A second locus, which we will call E , controls the deposition of melanin in the hairs. At least one functional E allele is required to deposit any pigment, whether it is black or brown. Thus, all retrievers that are ee fail to deposit any melanin (and so appear pale yellow), regardless of the genotype at the B locus (Figure $\PageIndex{6}$). The ee genotype is therefore said to be epistatic to both the B and b alleles, since the homozygous ee phenotype masks the phenotype of the B locus. The B/b locus is said to be hypostatic to the ee genotype. Because the masking allele is in this case is recessive, this is called recessive epistasis . Dominant epistasis In some cases, a dominant allele at one locus may mask the phenotype of a second locus. This is called dominant epistasis , which produces a segregation ratio such as 12:3:1 , which can be viewed as a modification of the 9:3:3:1 ratio in which the A_B_ class is combined with one of the other genotypic classes that contains a dominant allele. One of the best known examples of a 12:3:1 segregation ratio is fruit color in some types of squash (Figure $\PageIndex{7}$). Alleles of a locus that we will call B produce either yellow ( B _) or green ( bb ) fruit. However, in the presence of a dominant allele at a second locus that we call A , no pigment is produced at all, and fruit are white. The dominant A allele is therefore epistatic to both B and bb combinations (Figure $\PageIndex{8}$). One possible biological interpretation of this segregation pattern is that the function of the A allele somehow blocks an early stage of pigment synthesis, before neither yellow or green pigments are produced. Figure $\PageIndex{8}$: Genotypes and phenotypes among the progeny of a dihybrid cross of squash plants heterozygous for two loci affecting fruit color. (Original-Deyholos-CC:AN)	libretexts	2025-03-17T22:27:34.552802	2021-01-03T20:12:19	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/08%3A_Gene_Interactions_and_Epistasis/8.01%3A_Epistasis_and_Other_Gene_Interactions", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "8.1: Epistasis and Other Gene Interactions", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/08%3A_Gene_Interactions_and_Epistasis/8.02%3A_Example_of_Multiple_Genes_Affecting_One_Character	8.2: Example of Multiple Genes Affecting One Character - - Last updated - Save as PDF Cat Fur Genetics Most aspects of the fur phenotypes of common cats can be explained by the action of just a few genes (Table 6-2). Other genes, not described here, may further modify these traits and account for the phenotypes seen in tabby cats and in more exotic breeds, such as Siamese. For example, the X-linked Orange gene has two allelic forms. The O O allele produces orange fur, while the O B alleles produce non-orange (often black) fur. Note however, that because of X-chromosome inactivation the result is mosaicism in expression. In O O / O B female heterozygotes patches of black and orange are seen, which produces the tortoise shell pattern (Figure 6-13 A,B). This is a rare example of co-dominance since the phenotype of both alleles can be seen. Note that the cat in part A has short fur compared to the cat in part B; recessive alleles at an independent locus (L/l) produce long ( ll ) rather than short ( L_ ) fur. Alleles of the dilute gene affect the intensity of pigmentation, regardless of whether that pigmentation is due to black or orange pigment. Part C shows a black cat with at least one dominant allele of dilute ( D_ ), in contrast to the cat in D, which is grey rather than black, because it has the dd genotype. Epistasis is demonstrated by an allele of only one of the genes in Table $\PageIndex{2}$. One dominant allele of white masking ( W ) prevents normal development of melanocytes (pigment producing cells). Therefore, cats with genotype ( W_ ) will have entirely white fur regardless of the genotype at the Orange or dilute loci (part E). Although this locus produces a white colour, W_ is not the same as albinism, which is a much rarer phenotype caused by mutations in other genes. Albino cats can be distinguished by having red eyes, while W_ cats have eyes that are not red. Piebald spotting is the occurrence of patches of white fur. These patches vary in size due to many reasons, including genotype. Homozygous cats with genotype ss do not have any patches of white, while cats of genotype Ss and SS do have patches of white, and the homozygotes tend to have a larger proportion of white fur than heterozygotes (part F). The combination of piebald spotting and tortoise shell patterning produce a calico cat , which has separate patches of orange, black, and white fur. \| Trait \| Phenotype \| Genotype \| Comments \| \|---\|---\|---\|---\| \| fur length \| short \| LL or Ll \| L is completely dominant \| \| long \| ll \| \|\| \| all white fur (non-albino) \| 100% white fur \| WW or Ww \| If the cat has red eyes it is albino, not W_ . W is epistatic to all other fur color genes; if cat is W_ , can’t infer genotypes for any other fur color genes. \| \| ww \| \|\|\| \| piebald spotting \| > 50% white patches (but not 100%) \| SS \| S is incompletely dominant and shows variable expressivity \| \| < 50% white patches \| Ss \| \|\| \| no white patches \| ss \| \|\| \| orange fur \| all orange fur \| X O X O or X O Y \| O is X-linked \| \| tortoise shell variegation \| X O X B \| \|\| \| no orange fur (often black) \| X B X B or X B Y \| \|\| \| dilute pigmentation \| pigmentation is intense \| Dd or dd \| D is completely dominant \| \| pigmentation is dilute (e.g. gray rather than black; cream rather than orange; light brown rather than brown) \| dd \| \|\| \| tabby \| tabby pattern \| AA or Aa \| This is a simplification of the tabby phenotype, which involves multiple genes \| \| solid coloration \| aa \| \|\| \| sex \| female \| XX \| \| \| male \| XY \| References - Adapted f rom Christensen (2000) Genetics 155:999-1004 )	libretexts	2025-03-17T22:27:34.623871	2021-01-03T20:12:19	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/08%3A_Gene_Interactions_and_Epistasis/8.02%3A_Example_of_Multiple_Genes_Affecting_One_Character", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "8.2: Example of Multiple Genes Affecting One Character", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/10%3A_Nucleic_Acids/10.01%3A_DNA_is_the_Genetic_Material	10.1: DNA is the Genetic Material - - Last updated - Save as PDF By the early 1900’s, biochemists had isolated hundreds of different chemicals from living cells. Which of these was the genetic material? Proteins seemed like promising candidates, since they were abundant, diverse, and complex molecules. However, a few key experiments demonstrated that DNA, rather than protein, is the genetic material. Griffith’s Transformation Experiment (1928) Microbiologists identified two strains of the bacterium Streptococcus pneumoniae . The R-strain produced rough colonies on a bacterial plate, while the other S-strain was smooth (Figure $\PageIndex{2}$). More importantly, the S-strain bacteria caused fatal infections when injected into mice, while the R-strain did not (top, Figure $\PageIndex{3}$). Neither did “heat-treated” S-strain cells. Griffith in 1929 noticed that upon mixing “heat-treated” S-strain cells together with some R-type bacteria (neither should kill the mice), the mice died and there were S-strain, pathogenic cells recoverable. Thus, some non-living component from the S-type strains contained genetic information that could be transferred to and transform the living R-type strain cells into S-type cells. Avery, MacLeod and McCarty’s Experiment (1944) What kind of molecule from within the S-type cells was responsible for the transformation? To answer this, researchers named Avery, MacLeod and McCarty separated the S-type cells into various components, such as proteins, polysaccharides, lipids, and nucleic acids. Only the nucleic acids from S-type cells were able to make the R-strains smooth and fatal. Furthermore, when cellular extracts of S-type cells were treated with DNase (an enzyme that digests DNA), the transformation ability was lost. The researchers therefore concluded that DNA was the genetic material, which in this case controlled the appearance (smooth or rough) and pathogenicity of the bacteria. Hershey and Chase’s Experiment (1952) Further evidence that DNA is the genetic material came from experiments conducted by Hershey and Chase . These researchers studied the transmission of genetic information in a virus called the T2 bacteriophage, which used Escherichia coli as its host bacterium (Figure $\PageIndex{4}$). Like all viruses, T2 hijacks the cellular machinery of its host to manufacture more viruses. The T2 phage itself only contains both protein and DNA, but no other class of potential genetic material. To determine which of these two types of molecules contained the genetic blueprint for the virus, Hershey and Chase grew viral cultures in the presence of radioactive isotopes of either phosphorus ( 32 P) or sulphur ( 35 S). The phage incorporated these isotopes into their DNA and proteins, respectively (Fig 1.5). The researchers then infected E. coli with the radiolabeled viruses, and looked to see whether 32 P or 35 S entered the bacteria. After ensuring that all viruses had been removed from the surface of the cells, the researchers observed that infection with 32 P labeled viruses (but not the 35 S labeled viruses) resulted in radioactive bacteria. This demonstrated that DNA was the material that contained genetic instructions. Meselson and Stahl experiment (1958) From the complementary strands model of DNA, proposed by Watson and Crick in 1953, there were three straightforward possible mechanisms for DNA replication: (1) semi-conservative, (2) conservative, and (3) dispersive (Fig 1.6). - The semi-conservative model proposes the two strands of a DNA molecule separate during replication and then strand acts as a template for synthesis of a new, complementary strand. - The conservative model proposes that the entire DNA duplex acts as a single template for the synthesis of an entirely new duplex. - The dispersive model has the two strands of the double helix breaking into units that which are then replicated and reassembled, with the new duplexes containing alternating segments from one strand to the other. Each of these three models makes a different prediction about the how DNA strands should be distributed following two rounds of replication. These predictions can be tested in the following experiment by following the nitrogen component in DNA in E. coli as it goes through several rounds of replication. Meselson and Stahl used different isotopes of Nitrogen, which is a major component in DNA. Nitrogen-14 ( 14 N ) is the most abundant natural isotope, while Nitrogen-15 ( 15 N ) is rare, but also denser. Neither is radioactive; each can be followed by a difference in density – “ light ” 14 vs “ heavy ”15 atomic weight in a CsCl density gradient ultra-centrifugation of DNA. The experiment starts with E. coli grown for several generations on medium containing only 15 N. It will have denser DNA. When extracted and separated in a CsCl density gradient tube, this “heavy” DNA will move to a position nearer the bottom of the tube in the more dense solution of CsCl (left side in Figure $\PageIndex{7}$). DNA extracted from E. coli grown on normal, 14 N containing medium will migrate more towards the less dense top of the tube. If these E. coli cells are transferred to a medium containing only 14 N, the “light” isotope, and grown for one generation, then their DNA will be composed of one-half 15 N and one-half 14 N. If the this DNA is extracted and applied to a CsCl gradient , the observed result is that one band appears at the point midway between the locations predicted for wholly 15 N DNA and wholly 14 N DNA (Figure $\PageIndex{7}$). This “single-band” observation is inconsistent with the predicted outcome from the conservative model of DNA replication (disproves this model), but is consistent with both that expected for the semi-conservative and dispersive models. If the E. coli is permitted to go through another round of replication in the 14 N medium, and the DNA extracted and separated on a CsCl gradient tube, then two bands were seen by Meselson and Shahl: one at the 14 N- 15 N intermediate position and one at the wholly 14 N position (Figure $\PageIndex{7}$). This result is inconsistent with the dispersive model (a single band between the 14 N- 15 N position and the wholly 14 N position) and thus disproves this model. The two band observation is consistent with the semi-conservative model which predicts one wholly 14 N duplex and one 14 N- 15 N duplex. Additional rounds of replication also support the semi-conservative model/hypothesis of DNA replication. Thus, the semi-conservative model is the currently accepted mechanism for DNA replication. Note however, that we now also know from more recent experiments that whole chromosomes, which can be millions of bases in length, are also semi-conservatively replicated. These experiments, published in 1958, are a wonderful example of how science works. Researchers start with three clearly defined models (hypotheses). These models were tested, and two (conservative and dispersive) were found to be inconsistent with the observations and thus disproven. The third hypothesis, semi-conservative, was consistent with the observations and thereby supported and accepted as mechanism of DNA replication. Note, however, this is not “proof” of the model, just strong evidence for it; hypotheses are not “proven”, only disproven or supported. RNA and protein While DNA is the genetic material for the vast majority of organisms, there are some viruses that use RNA as their genetic material . These viruses can be either single or double stranded and include SARS, influenza, hepatitis C and polio, as well as the retroviruses like HIV-AIDS. Typically there is DNA used at some stage in their life cycle to replicate their RNA genome. Also, the case of Prion infections agents transmit characteristics via only a protein (no nucleic acid present). Prions infect by transmitting a misfolded protein state from one aberrant protein molecule to a normally folded molecule. These agents are responsible for bovine spongiform encephalopathy (BSE, also known as "mad cow disease") in cattle and deer and Creutzfeldt–Jakob disease (CJD) in humans. All known prion diseases act by altering the structure of the brain or other neural tissue and all are currently untreatable and ultimately fatal.	libretexts	2025-03-17T22:27:34.789977	2021-01-03T20:12:21	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/10%3A_Nucleic_Acids/10.01%3A_DNA_is_the_Genetic_Material", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "10.1: DNA is the Genetic Material", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/10%3A_Nucleic_Acids/10.02%3A__The_Structure_of_DNA	10.2: The Structure of DNA - - Last updated - Save as PDF The experiments outlined in the previous sections proved that DNA was the genetic material, but very little was known about its structure at the time. Chargaff’s Rules When Watson and Crick set out in the 1940’s to determine the structure of DNA, it was already known that DNA is made up of a series four different types of molecules, called bases or nucleotides: adenine (A), cytosine (C), thymine (T), guanine (G). Watson and Crick also knew of Chargaff’s Rules , which were a set of observations about the relative amount of each nucleotide that was present in almost any extract of DNA. Chargaff had observed that for any given species, the abundance of A was the same as T, and G was the same as C. This was essential to Watson & Crick’s model. The Double Helix Using proportional metal models of the individual nucleotides, Watson and Crick deduced a structure for DNA that was consistent with Chargaff’s Rules and with x-ray crystallography data that was obtained (with some controversy) from another researcher named Rosalind Franklin. In Watson and Crick’s famous double helix , each of the two strands contains DNA bases connected through covalent bonds to a sugar-phosphate backbone (Fig 1.8, 1.9). Because one side of each sugar molecule is always connected to the opposite side of the next sugar molecule, each strand of DNA has polarity: these are called the 5’ (5-prime) end and the 3’ (3-prime) end, in accordance with the nomenclature of the carbons in the sugars. The two strands of the double helix run in anti-parallel (i.e. opposite) directions, with the 5’ end of one strand adjacent to the 3’ end of the other strand. The double helix has a right-handed twist, (rather than the left-handed twist that is often represented incorrectly in popular media). The DNA bases extend from the backbone towards the center of the helix, with a pair of bases from each strand forming hydrogen bonds that help to hold the two strands together. Under most conditions, the two strands are slightly offset, which creates a major groove on one face of the double helix, and a minor groove on the other. Because of the structure of the bases, A can only form hydrogen bonds with T, and G can only form hydrogen bonds with C (remember Chargaff’s Rules). Each strand is therefore said to be complementary to the other, and so each strand also contains enough information to act as a template for the synthesis of the other. This complementary redundancy is important in DNA replication and repair. How can this molecule, DNA, contain the genetic material?	libretexts	2025-03-17T22:27:34.849547	2021-01-03T20:12:23	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/10%3A_Nucleic_Acids/10.02%3A__The_Structure_of_DNA", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "10.2: The Structure of DNA", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/11%3A_Amplifying_and_Manipulating_DNA_Fragments/11.01%3A_Prelude_to_Molecular_Genetics	11.1: Prelude to Molecular Genetics selected template will load here This action is not available. Genetics is the study of the inheritance and variation of biological traits. We have previously noted that it is possible to conduct genetic research without directly studying DNA. Indeed some of the greatest geneticists had no special knowledge of DNA at all, but relied instead on analysis of phenotypes, inheritance patterns, and their ratios in carefully designed crosses. Today, classical genetics is often complemented by molecular biology , to give molecular genetics , which involves the study of DNA and other macromolecules that have been isolated from an organism. Usually, molecular genetics experiments involve some combination of techniques to isolate and analyze the DNA or RNA transcribed from a particular gene. In some cases, the DNA may be subsequently manipulated by mutation or by recombination with other DNA fragments. Techniques of molecular genetics have wide application in many fields of biology, as well as forensics, biotechnology, and medicine.	libretexts	2025-03-17T22:27:35.060635	2021-01-03T20:12:27	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/11%3A_Amplifying_and_Manipulating_DNA_Fragments/11.01%3A_Prelude_to_Molecular_Genetics", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "11.1: Prelude to Molecular Genetics", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/11%3A_Amplifying_and_Manipulating_DNA_Fragments/11.02%3A_Isolating_Genomic_DNA	11.2: Isolating Genomic DNA selected template will load here This action is not available. DNA purification strategies rely on the chemical properties of DNA that distinguish it from other molecules in the cell, namely that it is a very long, negatively charged molecule. To extract purified DNA from a tissue sample, cells are broken open by grinding or lysing in a solution that contains chemicals that protect the DNA while disrupting other components of the cell (Figure $\PageIndex{2}$). These chemicals may include detergents , which dissolve lipid membranes and denature proteins. A cation such as Na + helps to stabilize the negatively charged DNA and separate it from proteins such as histones. A chelating agent , such as EDTA , is added to protect DNA by sequestering Mg 2+ ions, which can otherwise serve as a necessary co-factor for nucleases (enzymes that digest DNA). As a result, free, double-stranded DNA molecules are released from the chromatin into the extraction buffer, which also contains proteins and all other cellular components. (The basics of this procedure can be done with household chemicals and are presented on YouTube.) The free DNA molecules are subsequently isolated by one of several methods. Commonly, proteins are removed by adjusting the salt concentration so they precipitate. The supernatant , which contains DNA and other, smaller metabolites, is then mixed with ethanol, which causes the DNA to precipitate. A small pellet of DNA can be collected by centrifugation, and after removal of the ethanol, the DNA pellet can be dissolved in water (usually with a small amount of EDTA and a pH buffer) for the use in other reactions. Note that this process has purified all of the DNA from a tissue sample; if we want to further isolate a specific gene or DNA fragment, we must use additional techniques, such as PCR.	libretexts	2025-03-17T22:27:35.124120	2021-01-03T20:12:28	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/11%3A_Amplifying_and_Manipulating_DNA_Fragments/11.02%3A_Isolating_Genomic_DNA", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "11.2: Isolating Genomic DNA", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/11%3A_Amplifying_and_Manipulating_DNA_Fragments/11.03%3A__Isolating_or_Detecting_a_Specific_Sequence_by_PCR	11.3: Isolating or Detecting a Specific Sequence by PCR - - Last updated - Save as PDF Components of the PCR Reaction The Polymerase Chain Reaction (PCR) is a method of DNA replication that is performed in a test tube (i.e. in vitro ). Here “polymerase” refers to a DNA polymerase enzyme extracted and purified from bacteria, and “chain reaction” refers to the ability of this technique produce millions of copies of a DNA molecule, by using each newly replicated double helix as a template to synthesize two new DNA double helices. PCR is therefore a very efficient method of amplifying DNA. Besides its ability to make large amounts of DNA, there is a second characteristic of PCR that makes it extremely useful. Recall that most DNA polymerases can only add nucleotides to the end of an existing strand of DNA, and therefore require a primer to initiate the process of replication. For PCR, chemically synthesized primers of about 20 nucleotides are used. In an ideal PCR, primers only hybridize to their exact complementary sequence on the template strand (Figure $\PageIndex{3}$). The experimenter can therefore control exactly what region of a DNA template is amplified by controlling the sequence of the primers used in the reaction. To conduct a PCR amplification, an experimenter combines in a small, thin-walled tube (Figure $\PageIndex{4}$), all of the necessary components for DNA replication, including DNA polymerase and solutions containing nucleotides (dATP, dCTP, dGTP, dTTP), a DNA template, DNA primers, a pH buffer, and ions (e.g. Mg 2+ ) required by the polymerase. Successful PCR reactions have been conducted using only a single DNA molecule as a template, but in practice, most PCR reactions contain many thousands of template molecules. The template DNA (e.g. total genomic DNA) has usually already been purified from cells or tissues using the techniques described above. However, in some situations it is possible to put whole cells directly in a PCR reaction for use as a template. An essential aspect of PCR is thermal-cycling , meaning the exposure of the reaction to a series of precisely defined temperatures (Figure $\PageIndex{5}$). The reaction mixture is first heated to 95°C. This causes the hydrogen bonds between the strands of the template DNA molecules to melt, or denature . This produces two single-stranded DNA molecules from each double helix (Figure $\PageIndex{6}$). In the next step ( annealing ), the mixture is cooled to 45-65°C. The exact temperature depends on the primer sequence used and the objectives of the experiment. This allows the formation of double stranded helices between complementary DNA molecules, including the annealing of primers to the template. In the final step ( extension ) the mixture is heated to 72°C. This is the temperature at which the particular DNA polymerase used in PCR is most active. During extension, the new DNA strand is synthesized, starting from the 3' end of the primer, along the length of the template strand. The entire PCR process is very quick, with each temperature phase usually lasting 30 seconds or less. Each cycle of three temperatures (denaturation, annealing, extension) is usually repeated about 30 times, amplifying the target region approximately 2 30 -fold. Notice from the figure that most of the newly synthesized strands in PCR begin and end with sequences either identical to or complementary to the primer sequences; although a few strands are longer than this, they are in such a small minority that they can almost always be ignored. Figure $\PageIndex{6}$: PCR with the three phases of the thermalcycle numbered. The template strand (blue) is replicated from primers (red), with newly synthesized strands in green. The green strands flanked by two primer binding sites will increase in abundance exponentially through successive PCR cycles. (Wikipedia-madprime-GFDL) The earliest PCR reactions used a polymerase from E. coli . Because the high temperature of the denaturation step destroyed the enzyme, new polymerase had to be added after each cycle. To overcome this, researchers identified thermostable DNA polymerases such as Taq DNA pol , from Thermus acquaticus , a thermophilic bacterium that lives in hot springs. Taq, and similar thermostable polymerases from other hot environments, are able to remain functional in the repeated cycles of amplification. Taq polymerase cannot usually amplify fragments longer than about 3kbp, but under some specialized conditions, PCR can amplify fragments up to approximately 10kbp. Other polymerases, either by themselves or in combination with Taq, are used to increase the length of amplified fragments or to increase the fidelity of the replication. After completion of the thermalcycling (amplification), an aliquot from the PCR reaction is usually loaded onto an electrophoretic gel (described below) to determine whether a DNA fragment of the expected length was successfully amplified or not. Usually, the original template DNA will be so dilute that it will not be visible on the gel, only the amplified PCR product. The presence of a sharp band of the expected length indicates that PCR was able to amplify its target. If the purpose of the PCR was to test for the presence of a particular template sequence, this is the end of the experiment. Otherwise, the remaining PCR product can be used as starting material for a variety of other techniques such as sequencing or cloning. An Application of PCR: the StarLink Affair PCR is very sensitive (meaning it can amplify very small starting amounts of DNA), and specific (meaning it can amplify only the target sequence from a mixture of many DNA sequences). This made PCR the perfect tool to test whether genetically modified corn was present in consumer products on supermarket shelves. Although currently (2013) 85% of corn in the United States is genetically modified, and contains genes that government regulators have approved for human consumption, back in 2000, environmental groups showed that a strain of genetically modified corn, which had only been approved for use as animal feed, had been mixed in with corn used in producing human food, like taco shells. To do this, the groups purchased taco shells from stores in the Washington DC area, extracted DNA from the taco shells and used it as a template in a PCR reaction with primers specific to the unauthorized gene ( Cry9C ). Their suspicions were confirmed when they ran this PCR product on an agarose gel and saw a band of expected size. The PCR test was able to detect one transgenic kernel in a whole bushel of corn (1 per 100,000). The company (Aventis) that sold the transgenic seed to farmers had to pay for the destruction of large amounts of corn, and was the target of a class action law-suit by angry consumers who claimed they had been made sick by the taco shells. While no legitimate cases of harm were ever proven, and the plaintiffs were awarded $9 million, of which $3 million went to the legal fees, and the remainder of the judgment went to the consumers in the form of coupons for taco shells. The affair damaged the company, and exposed a weakness in the way the genetically modified crops were handled in the United States at the time.	libretexts	2025-03-17T22:27:35.186718	2021-01-03T20:12:28	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/11%3A_Amplifying_and_Manipulating_DNA_Fragments/11.03%3A__Isolating_or_Detecting_a_Specific_Sequence_by_PCR", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "11.3: Isolating or Detecting a Specific Sequence by PCR", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/11%3A_Amplifying_and_Manipulating_DNA_Fragments/11.05%3A_Origins_of_Molecular_Polymorphisms	11.5: Origins of Molecular Polymorphisms selected template will load here This action is not available. Mutations of DNA sequences can arise in many ways (Chapter 4). Some of these changes occur during DNA replication processes, resulting in an insertion, deletion, or substitution of one or a few nucleotides. Larger mutations can be caused by mobile genetic elements such as transposons, which are inserted more or less randomly into chromosomal DNA, sometimes occurring in clusters. In these and other types of repetitive DNA sequences, the number of repeated units is highly prone to change through unequal crossovers and other replication events.	libretexts	2025-03-17T22:27:35.289047	2021-01-03T20:12:29	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/11%3A_Amplifying_and_Manipulating_DNA_Fragments/11.05%3A_Origins_of_Molecular_Polymorphisms", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "11.5: Origins of Molecular Polymorphisms", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/11%3A_Amplifying_and_Manipulating_DNA_Fragments/11.06%3A_Classification_and_Detection_of_Molecular_Markers	11.6: Classification and Detection of Molecular Markers selected template will load here This action is not available. Regardless of their origins, molecular markers can be classified as polymorphisms that either vary in the length of a DNA sequence, or vary only in the identity of nucleotides at a particular position on a chromosome (Figure $\PageIndex{1}$). In both cases, because two or more alternative versions of the DNA sequence exist, we can treat each variant as a different allele of a single locus. Each allele gives a different molecular phenotype . For example, polymorphisms of SSRs (short sequence repeats) can be distinguished based on the length of PCR products: one allele of a particular SSR locus might produce a 100bp band, while the same primers used with a different allele as a template might produce a 120bp band (Figure $\PageIndex{2}$). A different type of marker, called a SNP (single nucleotide polymorphism), is an example of polymorphism that varies in nucleotide identity, but not length. SNPs are the most common of any molecular markers, and the genotypes of thousands of SNP loci can be determined in parallel, using new, hybridization based instruments. Note that the alleles of most molecular markers are co-dominant, since it is possible to distinguish the molecular phenotype of a heterozygote from either homozygote. Mutations that do not affect the function of protein sequences or gene expression are likely to persist in a population as polymorphisms, since there will be no selection either in favor or against them (i.e. they are neutral ). Note that the although the rate of spontaneous mutation in natural populations is sufficiently high so as to generate millions of polymorphisms that accumulate over thousands of generations, the rate of mutation is on the other hand sufficiently low that existing polymorphisms are stable throughout the few generations we study in a typical genetic experiment.	libretexts	2025-03-17T22:27:35.352279	2021-01-03T20:12:29	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/11%3A_Amplifying_and_Manipulating_DNA_Fragments/11.06%3A_Classification_and_Detection_of_Molecular_Markers", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "11.6: Classification and Detection of Molecular Markers", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/11%3A_Amplifying_and_Manipulating_DNA_Fragments/11.07%3A_Cutting_and_Pasting_DNA-_Restriction_Digests_and_DNA_Ligation	11.7: Cutting and Pasting DNA- Restriction Digests and DNA Ligation - - Last updated - Save as PDF Restriction Enzymes Many bacteria have enzymes that recognize specific DNA sequences (usually 4 or 6 nucleotides) and then cut the double stranded DNA helix at this sequence (Figure $\PageIndex{7}$). These enzymes are called site-specific restriction endonucleases , or more simply “ restriction enzymes ”, and they naturally function as part of bacterial defenses against viruses and other sources of foreign DNA. To cut DNA at known locations, researchers use restriction enzymes that have been purified from various bacterial species, and which can be purchased from various commercial sources. These enzymes are usually named after the bacterium from which they were first isolated. For example, EcoRI and EcoRV are both enzymes from E. coli . EcoRI cuts double stranded DNA at the sequence GAATTC, but note that this enzyme, like many others, does not cut in exactly the middle of the restriction sequence (Figure $\PageIndex{8}$). The ends of a molecule cut by EcoRI have an overhanging region of single stranded DNA, and so are sometimes called sticky-ends . On the other hand, EcoRV is an example of an enzyme that cuts both strands in exactly the middle of its recognition sequence, producing what are called blunt-ends , which lack overhangs. DNA Ligation The process of DNA ligation occurs when DNA strands are covalently joined, end-to-end through the action of an enzyme called DNA ligase . Sticky-ended molecules with complementary overhanging sequences are said to have compatible ends , which facilitate their joining to form recombinant DNA. Likewise, two blunt-ended sequences are also considered compatible to join together, although they do not ligate together as efficiently as sticky-ends. Note: sticky-ended molecules with non-complementary sequences will not ligate together with DNA ligase. Ligation is therefore central to the production of recombinant DNA, including the insertion of a double stranded DNA fragment into a plasmid vector.	libretexts	2025-03-17T22:27:35.410631	2021-01-03T20:12:30	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/11%3A_Amplifying_and_Manipulating_DNA_Fragments/11.07%3A_Cutting_and_Pasting_DNA-_Restriction_Digests_and_DNA_Ligation", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "11.7: Cutting and Pasting DNA- Restriction Digests and DNA Ligation", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/11%3A_Amplifying_and_Manipulating_DNA_Fragments/11.08%3A_Make_and_Screen_a_cDNA_Library	11.8: Make and Screen a cDNA Library - - Last updated - Save as PDF The first step in making a cDNA library is to isolate cellular mRNA. This mRNA extract should represent all of the transcripts in the cells at the time of isolation, or the cell’s transcriptome . This term is used by analogy to genome. However, a genome is all of the genetic information of an organism. In contrast, a transcriptome (usually eukaryotic) reflects all of the genes expressed in a given cell type at a moment in time. Reversetranscribed cDNAs from an mRNA extract are also referred to as a transcriptome…, and likewise, a cDNA library. A cDNA library is a tube full of bacterial cells that have taken up (i.e., been transformed with) plasmids recombined with cDNAs. cDNA libraries made from mRNAs taken from different cell types or the same cells grown under different conditions are in effect, different transcriptomes. Each reflects mRNAs transcribed in cells at the moment of their extraction. When cells in a cDNA library are spread out on a nutrient agar petri dish, each cell grows into a colony of cells; each cell in the colony is a clone of a starting cell. cDNA libraries can be used isolate and sequence the DNA encoding a polypeptide that you are studying. Recall that the mature mRNA in eukaryotic cells has been spliced. This means that cDNAs from eukaryotic cells do not include introns. Introns, as well as sequences of enhancers and other regulatory elements in and surrounding a gene must be studied in genomic libraries, to be discussed later. Here we look at how to make a cDNA library. A. cDNA Construction mRNA is only a few percent of a eukaryotic cell; most is rRNA. But that small amount of mRNA can be separated from other cellular RNAs by virtue of their 3’ poly(A) tails. Simply pass a total RNA extract over an oligo-d(T) column (illustrated below). The strings of thymidine (T) can H-bond with the poly(A) tails of mRNAs, tethering them to the column. All RNAs without a 3’ poly(A) tail will flow through the column as waste. A second buffer is passed over the column to destabilize the A-T H-bonds to allow elution of an mRNA fraction. When free’ oligo d(T) is added to the eluted mRNA, it forms H-bonds with the poly(A) tails of the mRNAs, serving as a primer for the synthesis of cDNA copies of the poly(A) mRNAs originally in the cells. Finally, four deoxynucleotide DNA precursors and reverse transcriptase (originally isolated from chicken retrovirus-infected cells) are added to start reverse transcription. The synthesis of a cDNA strand complementary to an mRNA is shown below. After heating to separate the cDNAs from the mRNAs, the cDNA is replicated to produce double-stranded, or (ds)cDNA, as illustrated below. Synthesis of the second cDNA strand is also catalyzed by reverse transcriptase! The enzyme recognizes DNA as well as RNA templates, and has the same 5’-to-3’ DNA polymerizing activity as DNA polymerases. After 2nd cDNA strand synthesis, S1 nuclease (a single-stranded endonuclease originally isolated from an East Asian fungus!) is added to open the loop of the (ds) cDNA structure and trim the rest of the single-stranded DNA. What remains is the (ds) cDNA. B. Cloning cDNAs into Plasmid Vectors To understand cDNA cloning and other aspects of making recombinant DNA, we need to talk a bit more about the recombinant DNA tool kit. In addition to reverse transcriptase and S1 nuclease, other necessary enzymes in the ‘kit’ include restriction endonucleases ( restriction enzymes ) and DNA ligase . The natural function of restriction enzymes in bacteria is to recognize specific restriction site sequences in phage DNA (most often palindromic DNA sequences), hydrolyze it and thus avoid infection. Restriction enzymes that make a scissors cut through the two strands of the double helix leaves blunt ends . Restriction enzymes that make a staggered cut on each strand at their restriction site leave behind complementary (‘sticky’) ends (below). If you mix two of double-stranded DNA fragments with the same sticky ends from different sources (e.g., different species), they will form H-bonds at their complementary ends, making it easy to recombine plasmid DNA with (ds)cDNA, that have the same complementary ‘sticky ends’. Using the language of recombinant DNA technologies, let’s look at how plasmid vectors and cDNAs can be made to recombine. 1. Preparing Recombinant Plasmid Vectors Containing cDNA Inserts Vectors are carrier DNAs engineered to recombine with foreign DNAs of interest. When a recombinant vector with its foreign DNA insert gets into a host cell, it can replicate many copies of itself, enough in fact for easy isolation and study. cDNAs are typically inserted into plasmid vectors that are usually purchased “off-the-shelf”. They can be cut with a restriction enzyme at a suitable location, leaving those sticky ends. On the other hand, it would not do to digest (ds)cDNA with restriction endonucleases since the goal is not to clone cDNA fragments, but entire cDNA molecules. Therefore, it will be necessary to attach linkers to either end of the (ds)cDNAs. Plasmid DNAs and cDNA-linker constructs can then be digested with the same restriction enzyme to produce compatible ‘sticky ends’. Steps in the preparation of vector and (ds)cDNA for recombination are shown below. To prepare for recombination, a plasmid vector is digested with a restriction enzyme to open the DNA circle. To have compatible sticky ends , double-stranded cDNAs to be inserted are mixed with linkers and DNA ligase to put a linker DNA at both ends of the (ds) cDNA. DNA ligase is another tool in the recombinant DNA toolkit. Linkers are short, synthetic double-stranded DNA oligomers containing restriction sites recognized and cut by the same restriction enzyme as the plasmid. Once the linkers are attached to the ends of the plasmid DNAs, they are digested with the appropriate restriction enzyme. This leaves both the (ds)cDNAs and the plasmid vectors with complementary sticky ends. 2. Recombining Plasmids and cDNA Inserts and Transforming Host Cells The next step is to mix the cut plasmids with the digested linker-cDNAs in just the right proportions so that the most of the cDNA (linker) ends will anneal (form Hbonds) with the most of the sticky plasmid ends. Adding DNA ligase to the plasmid/linker-cDNA mixture forms phosphodiester bonds between plasmid and cDNA insert, completing the recombinant circle of DNA, as shown below. In early cloning experiments, an important consideration was to generate plasmids with only one copy of a given cDNA insert, rather than lots of re-ligated plasmids with no inserts or lots of plasmids with multiple inserts. Using betterengineered vector and linker combinations, this issue became less important. 3. Transforming Host Cells with Recombinant Plasmids The recombinant DNA molecules are now ready for ‘cloning’. They are added to E. coli (sometimes other host cells) made permeable so that they can be easily transformed . Recall that transformation as defined by Griffith is bacterial uptake of foreign DNA leading to a genetic change. The transforming principle in cloning is the recombinant plasmid! The transformation step is shown below. The tube full of transformed cells is the cDNA Library. After all these treatments, not all plasmid molecules in the mix are recombinant; some cells in the mix haven’t even taken up a plasmid. So when the recombinant cells are plated on agar, how do you tell which of the colonies that grow came from cells that took up a recombinant plasmid? Both the host strain of E. coli and plasmid vectors used these days were further engineered to solve this problem. One such plasmid vector carries an antibiotic resistance gene . In this case, ampicillin-sensitive cells would be transformed with recombinant plasmids containing the resistance gene . When these cells are plated on media containing ampicillin (a form of penicillin), they grow, as illustrated below. Untransformed cells (cells that failed to take up a plasmid) lack the ampicillin resistance gene and thus, do not grow on ampicillin-medium. But, there is still a question. How can you tell whether the cells that grew were transformed by a recombinant plasmid containing a cDNA insert? It is possible that some of the transformants contain only non-recombinant plasmids that still have the ampicillin resistance gene! To address this question, plasmids were further engineered with a streptomycin resistance gene . But this antibiotic resistance gene was also engineered to contain restriction enzyme sites in the middle of the gene. Thus, inserting a cDNA in this plasmid would disrupt and inactivate the gene. Here is how this second bit of genetic engineering enabled growth only of cells transformed with a recombinant plasmid containing a cDNA insert. We can tell transformants containing recombinant plasmids apart from those containing non-recombinant plasmids by the technique of replica plating shown (illustrated below). After colonies grow on the ampicillin agar plate, lay a filter over the plate. The filter will pick up a few cells from each colony, in effect becoming a replica (mirror image) of the colonies on the plate. Place the replica filter on a new agar plate containing streptomycin; the new colonies that grow on the filter must be streptomycin-resistant, containing only non-recombinant plasmids. Colonies containing recombinant plasmids, those that did not grow in streptomycin are easily identified on the original ampicillin agar plate. In practice, highly efficient recombination and transformation procedures typically reveal very streptomycinresistant cells (i.e., colonies) after replica plating. In this case, ampicillin-resistant cells constitute a good cDNA library, ready for screening. 4. Identifying Colonies Containing Plasmids with Inserts of Interest The next step is to screen the colonies from the cDNA library for those containing the specific cDNA that you’re after. This typically begins preparing multiple replica filters like the one above. Remember, these filters are replicas of bacterial cells containing recombinant plasmids that grow on ampicillin but not streptomycin. The number of replica filters that must be screened can be calculated from assumptions and formulas for estimating how many colonies must be screened to represent an entire transcriptome (i.e., the number of different mRNAs in the original cellular mRNA source). Once the requisite number of replica filters are made, they are subjected to in situ lysis to disrupt cell walls and membranes. The result is that the cell contents are released and the DNA is denatured (i.e., becomes single-stranded). The DNA then adheres to the filter in place ( in situ , where the colonies were). The result of in situ lysis is a filter with faint traces of the original colony (below). Next, a molecular probe is used to identify DNA containing the sequence of interest. The probe is often a synthetic oligonucleotide whose sequence was inferred from known amino acid sequences. These oligonucleotides are made radioactive and placed in a bag with the filter(s). DNA from cells that contained recombinant plasmids with a cDNA of interest will bind the complementary probe. The results of in situ lysis and hybridization of a radioactive probe to a replica filter are shown below. 264 Probing a Replica Plate Filter The filters are rinsed to remove un-bound radioactive oligomer probe, and then placed on X-ray film. After a period of exposure, the film is developed. Black spots will form on the film from radioactive exposure, creating an autoradiograph of the filter. The black spots in the autoradiograph correspond to colonies on a filter that contain a recombinant plasmid with your target cDNA sequence (below). Once a positive clone is identified on the film, the corresponding recombinant colony is located on the original plate. This colony is grown up in a liquid culture and the plasmid DNA is isolated. At that point, the cloned plasmid DNA can be sequenced and the amino acid sequence encoded by its cDNA can be inferred from the genetic code dictionary to verify that the cDNA insert in fact encodes the protein of interest. Once verified as the sequence of interest, a cloned plasmid cDNA can be made radioactive or fluorescent, and used to - probe for the genes from which they originated. - identify and quantitate the mRNA even locate the transcripts in the cells. - quantitatively measure amounts of specific mRNAs. Isolated plasmid cDNAs can even be expressed in suitable cells to make the encoded protein. These days, diabetics no longer receive pig insulin, but get synthetic human insulin human made from expressed human cDNAs. Moreover, while the introduction of the polymerase chain reaction ( PCR , see below) has superseded some uses of cDNAs, they still play a role in genome-level and transcriptome-level studies.	libretexts	2025-03-17T22:27:35.485069	2021-01-03T20:12:31	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/11%3A_Amplifying_and_Manipulating_DNA_Fragments/11.08%3A_Make_and_Screen_a_cDNA_Library", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "11.8: Make and Screen a cDNA Library", "author": "Gerald Bergtrom" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/11%3A_Amplifying_and_Manipulating_DNA_Fragments/11.09%3A_DNA_Sequencing	11.9: DNA Sequencing - - Last updated - Save as PDF A. A Brief History of DNA Sequencing RNA sequencing came first, when Robert Holley sequenced a tRNA in 1965. The direct sequencing of tRNAs was possible because tRNAs are small, short nucleic acids, and because many of the bases in tRNAs are chemically modified after transcription. An early method for DNA sequencing developed by Walter Gilbert and colleagues involved DNA fragmentation, sequencing of the small fragments of DNA, and then aligning the overlapping sequences of the short fragments to assemble longer sequences. Another method, the DNA synthesis-based ‘dideoxy’ DNA sequencing technique, was developed by Frederick Sanger in England. Sanger and Gilbert both won a Nobel Prize in Chemistry in 1983 for their DNA sequencing efforts. However, because of its simplicity, Sanger’s method quickly became the standard for sequencing all manner of cloned DNAs. The first complete genome to be sequenced was that of a bacteriophage (bacterial virus) called φX174. At the same time as the advances in sequencing technology were occurring, so were some of the early developments in recombinant DNA technology. Together these led to more efficient and rapid cloning and sequencing of DNA from increasingly diverse sources. The first focus was of course on genes and genomes of important model organisms, such as E. coli , C. elegans , yeast ( S. cerevisiae )…, and of course us! By 1995, Craig Venter and colleagues at the Institute for Genomic Research had completed the sequence of an entire bacterial genome ( Haemophilus influenzae ) and by 2001, Venter’s private group along with Frances Collins and colleagues at the NIH had published a first draft of the sequence of the human genome. Venter had proven the efficacy of a whole-genome sequencing approach called shotgun sequencing , which was much faster than the gene-by-gene, fragment-by-fragment ‘linear’ sequencing strategy being used by other investigators (more later!). Since Sanger’s dideoxynucleotide DNA sequencing method remains a common and economical methodology, let’s consider the basics of the protocol. B. Details of DiDeoxy Sequencing Given a template DNA (e.g., a plasmid cDNA), Sanger used in vitro replication protocols to demonstrate that he could: - Replicate DNA under conditions that randomly stopped nucleotide addition at every possible position in growing strands. - Separate and then detect these DNA fragments of replicated DNA. Recall that DNA polymerases catalyze the formation of phosphodiester bonds by linking the $\alpha $ phosphate of a nucleotide triphosphate to the free 3’ OH of a deoxynucleotide at the end of a growing DNA strand. Recall also that the ribose sugar in the deoxynucleotide precursors of replication lack a 2’ OH (hydroxyl) group. Sanger’s trick was to add dideoxynucleotide triphosphates to his in vitro replication mix. The ribose on a dideoxynucleotide triphosphate (ddNTP) lacks a 3’ OH, in addition to the 2’ OH group (as shown below). Adding a dideoxynucleotide to a growing DNA strand stops replication. No further nucleotides can add to the 3’-end of the replicating DNA strand because the 3’–OH necessary for the dehydration synthesis of the next phosphodiester bond is absent! Because they can stop replication in actively growing cells, ddNTPs such as dideoxyadenosine (tradename, cordycepin ) are anti-cancer chemotherapeutic drugs. 266 Treating Cancer with Dideoxynucleosides A look at a manual DNA sequencing protocol reveals what is going on in the sequencing reactions. Four reaction tubes are set up, each containing the template DNA to be sequenced, a primer of known sequence and the four required deoxynucleotide precursors necessary for replication. The set-up for manual DNA sequencing is shown below. A different ddNTP, (ddATP, ddCTP, ddGTP or ddTTP) is added to each of the four tubes. Finally, DNA polymerase is added to each tube to start the DNA synthesis reaction. During DNA synthesis, different length fragments of new DNA accumulate as the ddNTPs incorporate randomly, opposite complementary bases in the template DNA being sequenced. The expectations of the didieoxy sequencing reactions in the four tubes are illustrated below. A short time after adding the DNA polymerase to begin the reactions, the mixture is heated to separate the DNA strands and fresh DNA polymerase is added to repeat the synthesis reactions. These sequencing reactions are repeated as many as 30 times in order to produce enough radioactive DNA fragments to be detected. When the heat-stable Taq DNA polymerase from the thermophilic bacterium Thermus aquaticus became available ( more later!), it was no longer necessary to add fresh DNA polymerase after each replication cycle. The many heating and cooling cycles required for what became known as chain-termination DNA sequencing were soon automated using inexpensive programmable thermocyclers . Since a small amount of a radioactive deoxynucleotide (usually 32P-labeled ATP) was present in each reaction tube, the newly made DNA fragments are radioactive. After electrophoresis to separate the new DNA fragments in each tube, autoradiography of the electrophoretic gel reveals the position of each terminated fragment. The DNA sequence can then be read from the gel as illustrated in the simulated autoradiograph below. As shown in the cartoon, the DNA sequence can be read by reading the bases from the bottom of the gel, starting with the C at the bottom of the C lane. Try reading the sequence yourself! The first semi-automated DNA sequencing method was invented in Leroy Hood’s California lab in 1986. Though still Sanger sequencing, the four dideoxynucleotides in the sequencing reaction were tagged for detection with a fluorescent dyes instead radioactive phosphate-tagged nucleotides. After the sequencing reactions, the reaction products are electrophoresed on an ‘automated DNA sequencer’. UV light excites the migrating dye-terminated DNA fragments as they pass through a detector. The color of their fluorescence is detected, processed and sent to a computer, generating color-coded graph like the one below, showing the order (and therefore length) of fragments passing the detector and thus, the sequence of the strand. A most useful feature of this sequencing method is that a template DNA could be sequenced in a single tube, containing all the required components, including all four dideoxynucleotides! That’s because the fluorescence detector in the sequencing machine separately sees all the short ddNTP-terminated fragments as they move through the electrophoretic gel. Hood’s innovations were quickly commercialized making major sequencing projects possible, including whole genome sequencing. The rapidity of automated DNA sequencing led to the creation of large sequence databases in the U.S. and Europe. The NCBI (National Center for Biological Information) maintains the U.S. database. Despite its location, the NCBI archives virtually all DNA sequences determined worldwide. New ‘tiny’ DNA sequencers have made sequencing DNA so portable that in 2016, one was even used in the International Space Station . Expanding databases and new tools and protocols (some are described below) to find, compare and analyze DNA sequences have also grown rapidly. C. Large Scale Sequencing Large-scale sequencing targets entire prokaryotic, and typically much larger eukaryotic genomes. The latter require strategies that either sequence long DNA fragments and/or sequencing DNA fragments more quickly. We already noted the shotgun sequencing used by Venter to sequence smaller and larger genomes (including our own… or more accurately, his own!). In shotgun sequencing, cloned DNA fragments 1000 base pairs or longer are broken down at random into smaller, more easily sequenced fragments. The fragments are themselves cloned and sequenced and non-redundant sequences are assembled by aligning overlapping regions of sequence. Today’s computer software is quite adept at rapid overlapping sequence alignment as well as connecting and displaying long contiguous DNA sequences. Shotgun sequencing is summarized below. Sequence gaps that remain after shotgun sequencing can be filled in by primer walking , in which a known sequence near the gap is the basis of creating a sequencing primer to “walk” into the gap region on an intact DNA that has not been fragmented. Another ‘gap-filling’ technique involves PCR (the Polymerase Chain Reaction, to be described shortly). Briefly, two oligonucleotides are synthesized based on sequence information on either side of a gap. Then PCR is used to synthesize the missing fragment, and the fragment is sequenced to fill in the gap.	libretexts	2025-03-17T22:27:35.551191	2021-01-03T20:12:32	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/11%3A_Amplifying_and_Manipulating_DNA_Fragments/11.09%3A_DNA_Sequencing", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "11.9: DNA Sequencing", "author": "Gerald Bergtrom" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/11%3A_Amplifying_and_Manipulating_DNA_Fragments/11.10%3A_Genomic_Libraries	11.10: Genomic Libraries - - Last updated - Save as PDF A genomic library might be a tube full of recombinant bacteriophage . Each phage DNA molecule contains a fragmentary insert of cellular DNA from a foreign organism. The library is made to contain a representation of all of possible fragments of that genome. Bacteriophage are often used to clone genomic DNA fragments because: - phage genomes are bigger than plasmids and can be engineered to remove a large amount of DNA that is not needed for infection and replication in bacterial host cells. - the missing DNA can thus be replaced by foreign insert DNA fragments as long as 18- 20kbp (kilobase pairs), nearly 20X as long as typical cDNA inserts in plasmids. - purified phage coat proteins can be mixed with the recombined phage DNA to make infectious phage particles that would infect host bacteria, replicate lots of new recombinant phage, and then lyse the cells to release the phage. The need for vectors like bacteriophage that can accommodate long inserts becomes obvious from the following bit of math. A typical mammalian genome consists of more than 2 billion base pairs . Inserts in plasmids are very short, rarely exceeding 1000 base pairs. Dividing 2,000,000,000 by 1000 , you get 2 million , a minimum number of phage clones that must be screened to find a sequence of interest. In fact, you would need many more than this number of clones to find a gene (or parts of one!). Of course, part of the solution to this “needle in a haystack” dilemma is to clone larger DNA inserts in more accommodating vectors. From this brief description, you may recognize the common strategy for genetically engineering a cloning vector: determine the minimum properties that your vector must have and remove non-essential DNA sequences. Consider the Yeast Artificial Chromosome ( YAC ), hosted by (replicated in) yeast cells. YACs can accept humongous foreign DNA inserts! This is because to be a chromosome that will replicate in a yeast cell requires one centromere and two telomeres … and little else! Recall that telomeres are needed in replication to keep the chromosome from shortening during replication of the DNA. The centromere is needed to attach chromatids to spindle fibers so that they can separate during anaphase in mitosis (and meiosis ). So along with a centromere and two telomeres, just include restriction sites to enable recombination with inserts as long as 2000 Kbp. That’s a YAC! The tough part of course is keeping a 2000Kbp long DNA fragment intact long enough to get it into the YAC. However a vector is engineered and chosen, sequencing its insert can tell us many things. They can show us how a gene is regulated by revealing known and uncovering new regulatory DNA sequences. They can tell us what other genes are nearby, and where genes are on chromosomes. Genomic DNA sequences from one species can probe for similar sequences in other species and comparative sequence analysis can then tell us a great deal about gene evolution and the evolution of species. One early surprise from gene sequencing studies was that we share many common genes and DNA sequences with other species, from yeast to worms to flies… and of course vertebrates and our more closely related mammal friends. You may already know that the chimpanzee’s and our genomes are 99% similar. Moreover, we have already seen comparative sequence analysis showing how proteins with different functions nevertheless share structural domains. Let’s look at cloning a genomic library in phage. As you will see, the principles are similar to cloning a foreign DNA into a plasmid, or in fact any other vector, but the numbers and details used here exemplify cloning in phage. A. Preparing Genomic DNA of a Specific Length for Cloning To begin with, high molecular weight (i.e., long molecules of) the desired genomic DNA are isolated, purified and then digested with a restriction enzyme. Usually, the digest is partial, aiming to generate overlapping DNA fragments of random length. When the digest is electrophoresed on agarose gels, the DNA (stained with ethidium bromide, a fluorescent dye that binds to DNA) looks like a bright smear on the gel. All of the DNA could be recombined with suitably digested vector DNA. But, to further reduce the number of clones to be screened for a sequence of interest, early cloners would generate a Southern blot (named after Edward Southern, the inventor of the technique) to determine the size of genomic DNA fragments most likely to contain a desired gene. Beginning with a gel of genomic DNA restriction digests, the Southern blot protocol is illustrated below To summarize the steps: a) Digest genomic DNA with one or more restriction endonucleases. b) Run the digest products on an agarose gel to separate fragments by size (length). The DNA appears as a smear when stained with a fluorescent dye. c) Place a filter on the gel. The DNA transfers (blots) to the filter for e.g., 24 hours d) Remove the blotted filter and place it in a bag containing a solution that can denature the DNA. e) Add radioactive probe (e.g., cDNA) containing the gene or sequence of interest. The probe hybridizes (bind) to complementary genomic sequences on the filter f) Prepare an autoradiograph of the filter and see a ‘band’ representing the size of genomic fragments of DNA that include the sequence of interest. Once you know the size (or size range) of restriction digest fragments that contain the DNA you want to study, you are ready to: a) run another gel of digested genomic DNA. b) cut out the piece of gel containing the fragments that ‘lit up’ with your probe in the autoradiograph. c) remove (elute) the DNA from the gel piece into a suitable buffer d) prepare the DNA for insertion into (recombination with) a genomic cloning vector B. Recombining Size-Restricted Genomic DNA with Phage DNA After elution of restriction digested DNA fragments of the right size range from the gels, the DNA is mixed with compatibly digested phage DNA at concentrations that favor the formation of H-bonds between the ends of the phage DNA and the genomic fragments. Addition of DNA ligase covalently links the recombined DNA molecules. These steps are abbreviated in the illustration below. The recombinant phage that are made next will contain sequences that become the genomic library. C. Creating Infectious Viral Particles with Recombinant Phage DNA The next step is to package the recombined phage DNA with added purified viral coat proteins to make infectious phage particles (below) 269 Genomic Libraries: Make and Package Recombinant Phage DNA Packaged phage are added to a culture tube full of host bacteria (typically E. coli ). After infection, the recombinant DNA enters the cells where it replicates and directs the production of new phage that eventually lyse the host cell (illustrated below). The recombined vector can also be introduced directly into the host cells by transduction (which is to phage DNA what transformation is to plasmid DNA). Whether by infection or transduction , the recombinant phage DNA ends up in host cells which produce new phage that eventually lyse the host cell. The released phages go on to infect more host cells until all cells have lysed. What remains is a tube full of lysate containing cell debris and lots of recombinant phage particles. 270 Infect Host with Recombinant Phage to Make a Genomic Library D. A Note About Some Other Vectors We’ve seen that phage vectors accommodate larger foreign DNA inserts than plasmid vectors, and YACs even more…, and that for larger genomes, the goal is to choose a vector able to house larger fragments of ‘foreign’ DNA so that you end up screening fewer clones. Given a large enough eukaryotic genome, it may be necessary to screen more than a hundred thousand clones in a phage-based genomic library. Apart from size-selection of genomic fragments before inserting them into a vector, selecting the appropriate vector is just as important. The table below lists commonly used vectors and the sizes of inserts they will accept. \| Vector Type \| Insert Size (thousands of bases) \| \| Plasmids \| up to 15 \| \| Phage Lambda ($\lambda $) \| up to 25 \| \| Cosmids \| up to 45 \| \| Bacteriophage P1 \| 70 to 100 \| \| P1 artificial chromosomes (PACs) \| 130 to 150 \| \| Bacterial artificial chromosomes (BACs) \| 120 to 300 \| \| Yeast artificial chromosomes (YACs) \| 250 to 2000 \| Click on the links to these vectors to learn more about them. We will continue this example by screening a phage lysate genomic library for a recombinant phage with a genomic sequence of interest. E. Screening a Genomic Library; Titering Recombinant Phage Clones A phage lysate is titered on a bacterial lawn to determine how many virus particles are present. A bacterial lawn is made by plating so many bacteria on the agar plate that they simply grow together rather than as separate colonies. In a typical titration , a lysate might be diluted 10-fold with a suitable medium and this dilution is further diluted 10-fold… and so on. Such serial 10X dilutions are then spread over bacterial (e.g., E. coli) lawns. What happens on such a culture plate? Let’s say that when 10 μl of one of the dilutions are spread on the bacterial lawn, they infect 500 E. coli cells on the bacterial lawn. After a day or so, there will be small clearings in the lawn called plaques …, 500 of them in this example. These are 500 tiny clear spaces on the bacterial lawn created by the lysis of first one infected cell, and then progressively more and more cells neighboring the original infected cell. Each plaque is thus a clone of a single virus, and each virus particle in a plaque contains a copy of the same recombinant phage DNA molecule (below). If you actually counted 500 plaques on the agar plate, then there must have been 500 virus particles in the 10 μl seeded onto the lawn. And, if this plate was the fourth dilution in a 10-fold serial dilution protocol, there must have been 2000 (4 X 500) phage particles in 10 μl of the original undiluted lysate. F. Screening a Genomic Library; Probing the Genomic Library In order to represent a complete genomic library , it is likely that many plates of such a dilution (~500 plaques per plate) will have to be created and then screened for a plaque containing a gene of interest. But, if only size-selected fragments were inserted into the phage vectors in the first place, the plaques represent only a partial genomic library, requiring screening fewer clones to find the sequence of interest. For either kind of library, the next step is to make replica filters of the plaques. Replica plating of plaques is similar to making a replica filter bacterial colonies. While much of the phage DNA in a plaque is encased in viral proteins, there will also be DNA on the plaque replicas that were never packaged into viral particles. The filters can be treated to denature the latter DNA and then directly hybridized to a probe with a known sequence. In the early days of cloning, probes for screening a genomic library were usually an already isolated and sequenced cDNA clone, either from the same species as the genomic library, or from a cDNA library of a related species. After soaking the filters in a radioactively labeled probe, X-Ray film is placed over the filter, exposed and developed. Black spots will form where the film lay over a plaque containing genomic DNA complementary to the radioactive probe. In the example illustrated below, a globin cDNA might have been used to probe the genomic library (globin genes were among the first to be cloned!). G. Isolating a Gene for Further Study Cloned genomic DNA fragments are much longer than any gene of interest, and always longer than any cDNA from a cDNA library. They are also embedded in a genome that is thousands of times as long as the gene itself, making the selection of an appropriate vector necessary. If the genome can be screened among a reasonable number of cloned phage (~100,000 plaques for instance), the one plaque producing a positive signal on the autoradiograph would be further studied. This plaque should contain the gene of interest. The next step is to find the gene within a genomic clone that can be as much a 20kbp long. The traditional strategy is to purify the cloned DNA, subject it to restriction endonuclease digestion, and separate of the digest particles by agarose gel electrophoresis . Using Southern Blotting , the separated DNA fragments are denatured and blotted to a nylon filter. The filter is then probed with the same tagged probe used to find the positive clone (plaque). The smallest DNA fragment containing the gene of interest can itself be subcloned in a suitable vector, and grown to provide enough DNA for further study of the gene.	libretexts	2025-03-17T22:27:35.627704	2021-01-03T20:12:32	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/11%3A_Amplifying_and_Manipulating_DNA_Fragments/11.10%3A_Genomic_Libraries", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "11.10: Genomic Libraries", "author": "Gerald Bergtrom" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/11%3A_Amplifying_and_Manipulating_DNA_Fragments/11.11%3A_The_Polymerase_Chain_Reaction_(PCR)	11.11: The Polymerase Chain Reaction (PCR) - - Last updated - Save as PDF The polymerase chain reaction (PCR) can amplify a region of DNA from any source, even from a single cell’s worth of DNA or from fragments of DNA obtained from a fossil. This amplification usually takes just a few hours, generating millions of copies of the desired target DNA sequence. The effect is to purify the DNA from surrounding sequences in a single reaction! Kary B. Mullis was awarded a Nobel Prize in 1993 for his development of PCR, which is now the basis of innumerable research studies of gene structure, function and evolution as well as applications in criminal forensics, medical diagnostics and other commercial uses. PCR is described in detail below. A. PCR - the Basic Process Typical PCR relies on knowing two bits of DNA sequence that will be used to design and synthesize short oligonucleotide sequences ( oligomers ) in the laboratory. The two oligomers are chosen to be complementary to sequences opposite strands of double-stranded DNA containing the gene to be studied. We say that the two oligomers face, or oppose each other. That just means that the 3’ end of one oligomer faces the 3’ end of the opposing oligomer. This way the two oligomers can serve as primers for the elongation replication of both strands of a double stranded target DNA sequence. Check out the link below for further explanation. 272 PCR: Design and Synthesize Opposing Oligonucleotide Primers The first step in PCR is to add oligomer primers to the target DNA from which a gene (or other genomic sequence) is to be amplified. The mixture is then heated to denature the target DNA. The mixture is cooled to allow the primers to H-bond to complementary target DNA strands. Next, the four deoxynucleotide precursors to DNA (dATP, dCTP, dTTP and dGTP) are added along with a small amount of a DNA polymerase. New DNA strands will now lengthen from the oligonucleotide primers on the template DNAs. To make lots of the PCR product, this reaction cycle must be repeated many times. Therefore, after allowing elongation, the mixture is heated to denature (separate) all the DNA strands. When the mixture is again cooled, the oligomers again find complementary sequences with which to H-bond. Early versions of PCR originally relied on an E. coli DNA polymerase, which is inactivated by heating, and so had to be re-added to the PCR mixture for each elongation cycle. Just as with DNA sequencing, researchers very quickly switched to the heat-stable Taq polymerase , of Thermus aquaticus . The enzymes of T. aquaticus remain active at the very high temperatures at which these organisms live. Since heating does not destroy the Taq polymerase in vitro , PCR, like DNA sequencing reactions, could be automated with programmable thermocylers that raised and lowered temperature required by the PCR reactions. There was no longer a need to replenish a DNA polymerase once the reaction cycles were begun. Thermocyling in a typical PCR amplification is illustrated below for the first two PCR cycles, the second of which, produces the first strands of DNA that will actually be amplified exponentially. You can see from the illustration that the second cycle of PCR has generated the two DNA strands that will be templates for doubling and re-doubling the desired product after each subsequent cycle. A typical PCR reaction might involve 30 PCR cycles, resulting in a nearly exponential amplification of the desired sequence. 273 PCR: The Amplification Process Challenge: Starting with a pair of complementary target DNA molecules (after the 3rd PCR cycle), how many double stranded PCR products should you theoretically have at the end of all 30 PCR cycles? The amplified products of PCR amplification products are in such abundance that they can easily be seen under fluorescent illumination on an ethidium bromide-stained agarose gel (below). In this gel, the first lane (on the left) contains a DNA ladder , a mixture of DNAs of known lengths that can be used to estimate the size of the PCR fragments in the 3 rd and 4th lanes (the gel lane next to the ladder is empty). The two bright bands in lanes 3 and 4 are PCR products generated with two different oligomer primer pairs. PCRamplified DNAs can be sequenced and used in many subsequent studies. B. The Many Uses of PCR PCR-amplified products can be labeled with radioactive or fluorescent tags to serve as hybridization probes for - screening cDNA or genomic libraries and isolation of clones. - determining migration position on a Southern blot. - determining migration position on a northern blot (a fanciful name for RNAs that are separated by size on gels and blotted to filter). - and more! 1. Quantitative PCR We noted above that PCR has wide applications to research, medicine and other practical applications. A major advance was Quantitative PCR , applied to studies of differential gene expression and gene regulation. In Quantitative PCR, initial cDNAs are amplified to detect not only the presence, but also the relative amounts of specific transcripts being made in cells. 2. Forensics Another application of PCR is in forensic science, to identify a person or organism by comparing its DNA to some standard, or control DNA. An example of one of these acrylamide gel DNA fingerprints is shown below. Using this technology, it is now possible to detect genetic relationships between near and distant relatives (as well as to exclude such relationships), determine paternity, demonstrate evolutionary relationships between organisms, and on many occasions, solve recent and even ‘cold-case’ crimes. Click Sir Alec Jeffries to learn about the origins of DNA fingerprinting in real life …and on all those TV CSI programs! Check out here for a brief history of the birth of DNA fingerprinting, and to see how analysis of changes in gene activity that occur after death may even help ID criminals. For a video on DNA fingerprinting, click Alu and DNA fingerprinting . Alu is a highly repeated ~300bp DNA sequence found throughout the human genome. Alu sequences are short interspersed elements , or SINES , a retrotransposon we saw earlier. DNA fingerprinting is possible in part because each of us has a unique number and distribution of Alu SINEs in our genome. To read more about Alu sequences and human diversity, click Alu Sequences and Human Diversity. Intriguing examples of the use of PCR for identification include establishing the identities of Egyptian mummies, the Russian Tsar deposed and killed during the Russian revolution (along with his family members), and the recently unearthed body of King Richard the 3rd of England. Variant PCR protocols and applications are manifold and often quite inventive! For a list, click Variations on Basic PCR. 3. Who are your Ancestors? Tracing your ethnic, racial and regional ancestry is related to DNA fingerprinting, in that it relies on PCR amplification of genes and other DNA regions and comparison of these your sequences to distinguishing DNA markers in large sequence databases. The price of these services have come down, and as a result, their popularity has gone up in recent years. Typically, you provide spit or a salivary (buccal) swab to the service and they amplify and sequence the DNA in your samples. The analysis compares your DNA sequences to database sequences looking for patterns of ethnic and regional markers that you might share with the database(s). Based on these comparisons, you are provided with a (more…, or less) accurate map of your DNA-based ancestry. Folks who are spending around $100.00 (less when on sale!) often ask just how accurate are these analyses, and what do they actually mean. For example, what does it mean if your DNA says you are 5% native American? In fact, different services can sometimes give you different results! You can get some answers and explanations DNA Ancestry Testing.	libretexts	2025-03-17T22:27:35.693939	2021-01-03T20:12:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/11%3A_Amplifying_and_Manipulating_DNA_Fragments/11.11%3A_The_Polymerase_Chain_Reaction_(PCR)", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "11.11: The Polymerase Chain Reaction (PCR)", "author": "Gerald Bergtrom" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/12%3A_Evaluating_Recombinant_DNA/12.01%3A_Cloning_DNA_-_Plasmid_Vectors	12.1: Cloning DNA - Plasmid Vectors - - Last updated - Save as PDF Plasmids are Naturally Present in Some Bacteria Many bacteria contain extra-chromosomal DNA elements called plasmids . These are usually small (a few 1000 bp), circular, double stranded molecules that replicate independently of the chromosome and can be present in high copy numbers within a cell. In the wild, plasmids can be transferred between individuals during bacterial mating and are sometimes even transferred between different species. Plasmids are particularly important in medicine because they often carry genes for pathogenicity and drug-resistance. In the lab, plasmids can be inserted into bacteria in a process called transformation. Using Plasmids as Cloning Vectors To insert a DNA fragment into a plasmid, both the fragment and the circular plasmid are cut using a restriction enzyme that produces compatible ends (Figure $\PageIndex{1}$). Given the large number of restriction enzymes that are currently available, it is usually not too difficult to find an enzyme for which corresponding recognition sequences are present in both the plasmid and the DNA fragment, particularly because most plasmid vectors used in molecular biology have been engineered to contain recognition sites for a large number of restriction endonucleases. After restriction digestion, the desired fragments may be further purified or selected before they are mixed together with ligase to join them together. Following a short incubation, the newly ligated plasmids, containing the gene of interest are transformed into E. coli . Transformation is accomplished by mixing the ligated DNA with E. coli cells that have been specially prepared (i.e. made competent ) to uptake DNA. Competent cells can be made by exposure to compounds such as CaCl 2 or to electrical fields ( electroporation ). Because only a small fraction of cells that are mixed with DNA will actually be transformed, a selectable marker , such as a gene for antibiotic resistance, is usually also present on the plasmid. After transformation (combining DNA with competent cells), bacteria are spread on a bacterial agar plate containing an appropriate antibiotic so that only those cells that have actually incorporated the plasmid will be able to grow and form colonies. This can then be picked and used for further study. Molecular biologists use plasmids as vectors to contain, amplify, transfer, and sometimes express genes of interest that are present in the cloned DNA. Often, the first step in a molecular biology experiment is to clone (i.e. copy) a gene into a plasmid, then transform this recombinant plasmid back into bacteria so that essentially unlimited copies of the gene (and the plasmid that carries it) can be made as the bacteria reproduce. This is a practical necessity for further manipulations of the DNA, since most techniques of molecular biology are not sensitive enough to work with just a single molecule at a time. Many molecular cloning and recombination experiments are therefore iterative processes in which: - a DNA fragment (usually isolated by PCR and/or restriction digestion) is cloned into a plasmid cut with a compatible restriction enzyme - the recombinant plasmid is transformed into bacteria - the bacteria are allowed to multiply, usually in liquid culture - a large quantity of the recombinant plasmid DNA is isolated from the bacterial culture - further manipulations (such as site directed mutagenesis or the introduction of another piece of DNA) are conducted on the recombinant plasmid - the modified plasmid is again transformed into bacteria, prior to further manipulations, or for expression An Application of Molecular Cloning: Production of Recombinant Insulin Purified insulin protein is critical to the treatment of diabetes. Prior to ~1980, insulin for clinical use was isolated from human cadavers or from slaughtered animals such as pigs. Human-derived insulin generally had better pharmacological properties, but was in limited supply and carried risks of disease transmission. By cloning the human insulin gene and expressing it in E. coli , large quantities of insulin identical to the human hormone could be produced in fermenters, safely and efficiently. Production of recombinant insulin also allows specialized variants of the protein to be produced: for example, by changing a few amino acids, longer-acting forms of the hormone can be made. The active insulin hormone contains two peptide fragments of 21 and 30 amino acids, respectively. Today, essentially all insulin is produced from recombinant sources (Figure $\PageIndex{2}$), i.e. human genes and their derivatives expressed in bacteria or yeast.	libretexts	2025-03-17T22:27:35.782780	2021-01-03T20:12:35	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/12%3A_Evaluating_Recombinant_DNA/12.01%3A_Cloning_DNA_-_Plasmid_Vectors", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "12.1: Cloning DNA - Plasmid Vectors", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/12%3A_Evaluating_Recombinant_DNA/12.02%3A__DNA_Analysis_-_Gel_Electrophoresis	12.2: DNA Analysis - Gel Electrophoresis selected template will load here This action is not available. A solution of DNA is colorless, and except for being viscous at high concentrations, is visually indistinguishable from water. Therefore, techniques such as gel electrophoresis have been developed to detect and analyze DNA (Figure $\PageIndex{11}$). This analysis starts when a solution of DNA is deposited at one end of a gel slab. This gel is made from polymers such as agarose , which is a polysaccharide isolated from seaweed. The DNA is then forced through the gel by an electrical current, with DNA molecules moving toward the positive electrode (Figure $\PageIndex{12}$). As it migrates, each piece of DNA threads its way through the pores, which form between the polymers in the gel. Because shorter pieces can move through these pores faster than longer pieces, gel electrophoresis separates molecules based on their size (length), with smaller DNA pieces moving faster than long ones. DNA molecules of a similar size migrate to a similar location in each gel, called a band . This feature makes it easy to see DNA after staining the DNA with a fluorescent dye such as ethidium bromide (Figure $\PageIndex{13}$). By separating a mixture of DNA molecules of known size ( size markers ) in adjacent lanes on the same gel, the length of an uncharacterized DNA fragment can be estimated. Gel segments containing the DNA bands can also be cut out of the gel, and the size-selected DNA extracted and used in other types of reactions, such as sequencing and cloning.	libretexts	2025-03-17T22:27:35.847568	2021-01-03T20:12:35	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/12%3A_Evaluating_Recombinant_DNA/12.02%3A__DNA_Analysis_-_Gel_Electrophoresis", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "12.2: DNA Analysis - Gel Electrophoresis", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/13%3A_Detecting_Genes_and_Gene_Products/13.01%3A__DNA_Analysis-_Blotting_and_Hybridization	13.1: DNA Analysis- Blotting and Hybridization - - Last updated - Save as PDF Bands of DNA in an electrophoretic gel form only if most of the DNA molecules are of the same size, such as following a PCR reaction, or restriction digestion of a plasmid. In other situations, such as after restriction digestion of chromosomal (genomic) DNA, there will be a large number of variable size fragments in the digest and it will appear as a continuous smear of DNA, rather than distinct bands. In this case, it is necessary to use additional techniques to detect the presence of a specific DNA sequence within the smear of DNA separated on an electrophoretic gel. This can be done using a “Southern Blot”. Southern Blots A Southern blot (also called a Southern Transfer ) is named after Ed Southern, its inventor. In the first step, DNA is digested with restriction enzymes and separated by gel electrophoresis (as discussed above). Then a sheet or membrane of nylon or similar material is laid under the gel and the DNA, in its separated position (bands or smear), is transferred to the membrane by drawing the liquid out of the gel, in a process called blotting (Figure $\PageIndex{1}$). The blotted DNA is usually covalently attached to the nylon membrane by briefly exposing the blot to UV light. Transferring the DNA to the sturdy membrane is necessary because the fragile gel would fall apart during the next two steps in the process. Next, the membrane is bathed in a solution to denature (double stranded made single stranded) the attached DNA. Then a hybridization solution containing a small amount of single-stranded probe DNA that is complementary in sequence to a target molecule on the membrane. This probe DNA is labeled using fluorescent or radioactive molecules, and if the hybridization is performed properly, the probe DNA will form a stable duplex only with those DNA molecules on the membrane that are exactly complementary to it. Then, the unhybridized probe is washed off and remaining radioactive or fluorescent signal will appear in a distinct band when appropriately detected. The band represents the presence of a particular DNA sequence within the mixture of DNA fragments. The probe is sequence specific (requires complementarity). However, variation in hybridization temperature and washing solutions can alter the stringency of the probe. At maximum stringency (higher temperature) hybridization conditions, probes will only hybridize with the exact target sequences that are perfectly complementary (maximum number of hydrogen bonds). At lower temperatures, probes will be able to hybridize to targets to which they do not match exactly, but only are roughly complementary for part of the sequence. Southern blotting is useful not only for detecting the presence of a DNA sequence within a mixture of DNA molecules, but also for determining the size of a restriction fragment in a DNA sample. Southern blots are useful for detecting fragments larger than those normally amplified by PCR, and when trying to detect fragments that may be only distantly related to a known sequence. Applications of Southern blotting will be discussed further in the context of molecular markers in a subsequent chapter. Southern blotting was invented before PCR, but PCR has replaced blotting in many applications because of its simplicity, speed, and convenience. Following the development of the Southern blot, other types of blotting techniques were invented. Northern Blots The Northern blot involves the size separation of RNA in gels like that of DNA. Because we wish to determine the native size of the RNA transcript (and because RNA is single stranded) no restriction enzymes are ever used . Because most RNA is single stranded and can fold into various conformations thorough intra-molecular base pairing, the electrophoresis separation is more haphazard and the bands are often less sharp, compared to that of double stranded DNA. Western Blots In a Western blot, protein is size separated on a gel (usually an acrylamide gel) before transferring to a membrane, which is then probed with an antibody that specifically binds to an antigenic site on the target protein. This antibody is then detected by other antibodies with some fluorescent or color production marker system. It will also give bands proportional to the amount and size of the target protein (Figure $\PageIndex{2}$). A comparison of all three blotting methods is shown in Figure $\PageIndex{3}$.	libretexts	2025-03-17T22:27:35.970961	2021-01-03T20:12:38	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/13%3A_Detecting_Genes_and_Gene_Products/13.01%3A__DNA_Analysis-_Blotting_and_Hybridization", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "13.1: DNA Analysis- Blotting and Hybridization", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/14%3A_DNA_Replication/14.01%3A_DNA_Structure	14.1: DNA Structure - - Last updated - Save as PDF A. Early Clues and Ongoing Misconceptions By 1878, a substance in the pus of wounded soldiers derived from cell nuclei (called nuclein ) was shown to be composed of 5 bases (the familiar ones of DNA and RNA). The four bases known to make up DNA (as part of nucleotides) were thought to be connected through the phosphate groups in short repeating chains of four nucleotides. By the 1940s, we knew that DNA was a long polymer. Nevertheless, it was still considered too simple to account for genes (see above). After the Hershey and Chase experiments, only a few holdouts would not accept DNA as the genetic material. So, the question remaining was how such a “simple” molecule could account for all the genes, even in so simple an organism as a bacterium. The answer to this question was to lie at least in part in an understanding of the physical structure of DNA, made possible by the advent of X-Ray Crystallography . If a substance can be crystallized, the crystal will diffract X-rays at angles revealing regular (repeating) structures of the crystal. William Astbury demonstrated that high molecular weight DNA had just such a regular structure. His crystallographs suggested DNA to be a linear polymer of stacked bases (nucleotides), each nucleotide separated from the next by 0.34 nm. Astbury is also remembered for coining the term “ molecular biology ” to describe his studies. The term now covers as all aspects of biomolecular structure, as well as molecular functions (e.g. replication, transcription, translation, gene regulation…). In an irony of history, the Russian biologist Nikolai Koltsov had already intuited in 1927 that the basis of genetic transfer of traits would be a "giant hereditary molecule" made up of "two mirror strands that would replicate in a semi-conservative fashion using each strand as a template". A pretty fantastic inference if you think about it since it was proposed long before Watson and Crick and their colleagues worked out the structure of the DNA double-helix! B. Wilkins, Franklin, Watson & Crick Maurice Wilkins, an English biochemist, was the first to isolate highly pure, high molecular weight DNA. Working in Wilkins laboratory, Rosalind Franklin was able to crystalize this DNA and produce very high-resolution X-Ray diffraction images of the DNA crystals. Franklin’s most famous (and definitive) crystallography was “Photo 51”, shown below. This image confirmed Astbury’s 0.34 nm repeat dimension and revealed two more numbers, 3.4 nm and 2 nm , reflecting additional repeat structures in the DNA crystal. When James Watson and Francis Crick got hold of these numbers, they used them along with other data to build DNA models out of nuts, bolts and plumbing. Their models eventually revealed DNA to be a pair of antiparallel complementary of nucleic acid polymers…, shades of Koltsov’s mirror-image macromolecules! Each strand is a string of nucleotides linked by phosphodiester bonds , the two strands held together in a double helix by complementary H-bond interactions. Let’s look at the evidence for these conclusions and as we do, refer to the two illustrations of the double helix below. Recalling that Astbury’s 0.34 nm dimension was the distance between successive nucleotides in a DNA strand, Watson and Crick surmised that the 3.4 nm repeat was a structurally meaningful 10-fold multiple of Astbury’s number. When they began building their DNA models, they realized from the bond angles connecting the nucleotides that the strand was forming a helix, from which they concluded that the 3.4 nm repeat was the pitch of the helix, i.e., the distance of one complete turn of the helix. This meant that there were 10 bases per turn of the helix. They further reasoned that the 2.0 nm number might reflect the diameter of helix. When their scale model of a single stranded DNA helix predicted a helical diameter much less than 2.0 nm, they were able to model a double helix that more nearly met the 2.0 nm diameter requirement. In building their double helix, Watson and Crick realized that bases in opposing strands would come together to form H-bonds, holding the helix together. However, for their double helix to have a constant diameter of 2.0 nm, they also realized that the smaller pyrimidine bases, Thymine ( T ) and Cytosine ( C ), would have to H-bond to the larger purine bases, Adenine ( A ) and Guanosine ( G ). Now to the question of how a “simple” DNA molecule could have the structural diversity needed to encode thousands of different polypeptides and proteins. In early studies, purified E. coli DNA was chemically hydrolyzed down to nucleotide monomers. The hydrolysis products contained nearly equal amounts of each base, reinforcing the notion that DNA was that simple molecule that could not encode genes. But Watson and Crick had private access to revealing data from Erwin Chargaff. Chargaff had determined the base composition of DNA isolated from different species, including E. coli . He found that the base composition of DNA from different species was not always equimolar , meaning that for some species, the DNA was not composed of equal amounts of each of the four bases (see some of this data below). The mere fact that DNA from some species could have base compositions that deviated from equimolarity put to rest the argument that DNA had to be a very simple sequence. Finally, it was safe to accept that to accept the obvious, namely that DNA was indeed the “stuff of genes”. Chargaff’s data also showed a unique pattern of base ratios. Although base compositions could vary between species, the A/T and G/C ratio was always one, for every species. Likewise the ratio of (A+C)/(G+T) and (A+G)/(C+T) . From this information, Watson and Crick inferred that A (a purine) would H-bond with T (a pyrimidine) , and G (a purine) would H-bond with C (a pyrimidine) in the double helix. When building their model with this new information, they also found H-bonding between the complementary bases would be maximal only if the two DNA strands were antiparallel , leading to the most stable structure of the double helix. Watson and Crick published their conclusions about the structure of DNA in 1953 (Click here to read their seminal article: Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid . Their article is also famous for predicting a semi-conservative mechanism of replication, something that had been predicted by Koltsov 26 years earlier, albeit based on intuition… and much less evidence! Watson, Crick and Wilkins shared a Nobel Prize in 1962 for their work on DNA structure. Unfortunately, Franklin died in 1958 and Nobel prizes were not awarded posthumously. There is still controversy about why Franklin did not get appropriate credit for her role in the work. But she has been getting well-deserved, long-delayed recognition, including a university in Chicago named in her honor! 169 Unraveling the Structure of DNA Confirmation of Watson & Crick’s suggestion of semiconservative replication came from Meselson and Stahl’s very elegant experiment, which tested the three possible models of replication shown below. In their experiment, E. coli cells were grown for in medium containing 15N, a ‘heavy’ nitrogen isotope. After many generations, all of the DNA in the cells had become labeled with the heavy isotope. At that point, the 15N-tagged cells were placed back in medium containing the more common, ‘light’ 14N isotope and allowed to grow for exactly one generation. Meselson and Stahl’s predictions and their experimental design are shown below. Meselson and Stahl knew that 14N-labeled and 15N-labeled DNA would form separate bands after centrifugation on CsCl chloride density gradients . They tested their predictions by purifying and centrifuging the DNA from the 15N-labeled cells grown in 14N medium for one generation. They found that this DNA formed a single band with a density between that of 15N-labeled DNA and 14N-labeled DNA. This result eliminated a conservative model of DNA replication (as Watson and Crick also predicted. That left two possibilities: replication was either semiconservative or dispersive. The dispersive model was eliminated when DNA isolated from cells grown for a 2nd generation on 14N were shown to contain two bands of DNA on the CsCl density gradients. Chromosomes We understood from the start of the 20th century that chromosomes contained genes. Therefore, it becomes necessary to understand the relationship between chromosomes, chromatin, DNA and genes. As noted earlier, chromosomes are a specialized, condensed version of chromatin, with key structural features shown below. We now know that the compact structure of a chromosome prevents damage to the DNA during cell division. This damage can occur when forces on centromeres generated by mitotic or meiotic spindle fibers pull chromatids apart. As the nucleus breaks down during mitosis or meiosis, late 19th century microscopists saw chromosomes condense from the dispersed cytoplasmic background. These chromosomes remained visible as they separated, moving to opposite poles of the cell during cell division. Such observations of chromosome behavior during cell division pointed to their role in heredity. A computer- colorized cell in mitosis is shown below. It is possible to distinguish one chromosome from another by karyotyping . When cells in metaphase of mitosis are placed under pressure, they burst and the chromosomes spread apart. Such a chromosome spread is shown below. By the early 1900s, the number, sizes and shapes of chromosomes were shown to be species-specific. What’s more, a close look at chromosome spreads revealed that chromosomes came in morphologically matched pairs. This was so reminiscent of Gregor Mendel’s paired hereditary factors that chromosomes were then widely accepted as the structural seat of genetic inheritance. Cutting apart micrographs like the one above and pairing the chromosomes by their morphology generates a karyotype . Paired human homologs are easily identified in the colorized micrograph below. Captured in mitosis, all dividing human cells contain 23 pairs of homologous chromosomes. The karyotype is from a female; note the pair of homologous sex (“X”) chromosomes (lower right of the inset). X and Y chromosomes in males are not truly homologous. Chromosomes in the original spread and in the aligned karyotype stained with fluorescent antibodies to chromosome-specific DNA sequences, ‘light up’ the different chromosomes.	libretexts	2025-03-17T22:27:36.144772	2021-01-03T20:12:40	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/14%3A_DNA_Replication/14.01%3A_DNA_Structure", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "14.1: DNA Structure", "author": "Gerald Bergtrom" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/14%3A_DNA_Replication/14.02%3A_Genes_and_Chromatin_in_Eukaryotes	14.2: Genes and Chromatin in Eukaryotes - - Last updated - Save as PDF Chromosomes and chromatin are a uniquely eukaryotic association of DNA with more or less protein. Bacterial DNA (and prokaryotic DNA generally) is relatively ‘naked’ – not visibly associated with protein. The electron micrograph of an interphase cell (below) reveals that the chromatin can itself exist in various states of condensation. Chromatin is maximally condensed during mitosis, forming chromosomes. During interphase, chromatin exists in more or less condensed forms, called Heterochromatin and euchromatin respectively. Transition between these chromatin forms involve changes in the amounts and types of proteins bound to the chromatin, and can that can occur during gene regulation, i.e., when genes are turned on or off. Active genes tend to be in the more dispersed euchromatin so that enzymes of replication and transcription have easier access to the DNA. Genes that are inactive in transcription are heterochromatic, obscured by additional chromatin proteins present in heterochromatin. We’ll be looking at some experiments that demonstrate this in a later chapter. We can define three levels of chromatin organization in general terms: 1. DNA wrapped around histone proteins form nucleosomes in a "beads on a string" structure. 2. Multiple nucleosomes coil (condense), forming 30 nm fiber (solenoid) structures. 3. Higher-order packing of the 30 nm fiber leads to formation of metaphase chromosomes seen in mitosis & meiosis. The levels of chromatin structure were determined in part by selective isolation and extraction of interphase cell chromatin, followed by selective chemical extraction of chromatin components. The steps are: · Nuclei are first isolated from the cells. · The nuclear envelope gently ruptured so as not to physically disrupt chromatin structure. · the chromatin can be gently extracted by one of several different chemical treatments (high salt, low salt, acid...). The levels of chromatin structure are illustrated below. Salt extraction dissociates most of the proteins from the chromatin. When a low salt extract is centrifuged and the pellet resuspended, the remaining chromatin looks like beads on a string . DNA-wrapped nucleosomes are the beads, which are in turn linked by uniform lengths of metaphorical DNA “string’ ( # 1 in the illustration above). A high salt chromatin extract appears as a coil of nucleosomes, or 30 nm solenoid fiber (# 2 above). Other extraction protocols revealed other aspects of chromatin structure shown in #s 3 and 4 above. Chromosomes seen in metaphase of mitosis are the ‘highest order’, most condensed form of chromatin. The 10 nm filament of nucleosome ‘beads-on-a-string’ remaining after a low salt extraction can be seen in an electron microscope as shown below. When these nucleosome necklaces were digested with the enzyme deoxyribonuclease ( DNAse ), the DNA between the ‘beads’ was degraded, leaving behind shortened 10nm filaments after a short digest period, or just single beads the beads after a longer digestion (below). Roger Kornberg (son of Nobel Laureate Arthur Kornberg who discovered the first DNA polymerase enzyme of replication) participated in the discovery and characterization of nucleosomes while he was still a post-doc! Electrophoresis of DNA extracted from these digests revealed nucleosomes separated by a “linker” DNA stretch of about 80 base pairs. DNA extracted from the nucleosomes was about 147 base pairs long. This is the DNA that had been wrapped around the proteins of the nucleosome. After separating all of the proteins from nucleosomal DNA, five proteins were identified (illustrated below). Histones are basic proteins containing many lysine and arginine amino acids. Their positively charged side chains enable these amino acids bind the acidic, negatively charged phosphodiester backbone of double helical DNA. The DNA wraps around an octamer of histones (2 each of 4 of the histone proteins) to form the nucleosome . About a gram of histones is associated with each gram of DNA. After a high salt chromatin extraction, the structure visible in the electron microscope is the 30nm solenoid, the coil of nucleosomes modeled in the figure below. As shown above, simply increasing the salt concentration of an already extracted nucleosome preparation will cause the ‘necklace’ to fold into the 30nm solenoid structure. 173 Chromatin Structure: Dissecting Chromatin As you might guess, an acidic extraction of chromatin should selectively remove the basic histone proteins, leaving behind an association of DNA with non-histone proteins. This proved to be the case. An electron micrograph of the chromatin remnant after an acid extraction of metaphase chromosomes is shown on the next page. DNA freed of the regularly spaced histone-based nucleosomes, loops out, away from the long axis of the chromatin. Dark material along this axis is a protein scaffolding that makes up what’s left after histone extraction. Much of this protein is topoisomerase , an enzyme that prevents DNA from breaking apart under the strain of replication.	libretexts	2025-03-17T22:27:36.209738	2021-01-03T20:12:40	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/14%3A_DNA_Replication/14.02%3A_Genes_and_Chromatin_in_Eukaryotes", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "14.2: Genes and Chromatin in Eukaryotes", "author": "Gerald Bergtrom" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/14%3A_DNA_Replication/14.03%3A_Introduction	14.3: Introduction - - Last updated - Save as PDF Replication begins at one or more origins of replication along DNA, where helicase enzymes catalyze unwinding of the double helix. DNA unwinding creates replicating bubbles, or replicons , with replication forks at either end. Making a new DNA strand starts with making an RNA primer with RNA nucleotides and primase enzymes. DNA nucleotides are then added to the 3’-ends of primers by one a DNA polymerase . Later, other DNA polymerases catalyze removal of the RNA primers and replacement of the hydrolyzed ribonucleotides with new deoxyribonucleotides. Finally, DNA ligases stitch together the fragments of new DNA synthesized at the replication forks. This complex mechanism is common to the replication of ‘naked’ prokaryotic DNA and of chromatinencased eukaryotic DNA, and must therefore have arisen early in the evolution of replication biochemistry. In this chapter, we look at the details of replication and the differences in detail between prokaryotic and eukaryotic replication that arise because of differences in DNA packing. As with any complex process with many moving parts, replication is error-prone. Therefore, we will also look at how the overall fidelity of replication relies mechanisms of DNA repair that target specific kinds of replication mistakes, or mutations . At the same time, lest we think that uncorrected errors in replication are always a bad thing, they usually do not have bad outcomes. Instead, they leave behind the very mutations that allow natural selection and the evolution of diversity . Learning Objectives - Explain how Cairns interpreted his theta ($\Theta $) images. - Compare and contrast the activities of enzymes required for replication. - Describe the order of events at an origin of replication and at each replication fork. - Compare unidirectional and bidirectional DNA synthesis from an origin of replication. - Outline the basic synthesis and proofreading functions of DNA polymerase. - Identify the major players and their roles in the initiation of replication. - Explain how Okazaki’s experimental results were not entirely consistent with how both strands of DNA replicate - List the major molecular players (enzymes, etc.) that elongate a growing DNA strand. - List the non-enzymatic players in replication and describe their functions. - Describe how the structure of telomerase enables proper replication. - Compare the activities of topoisomerases 1 and 2. - Explain the reasoning behind the hypothesis of processive replication. - Compare and contrast the impacts of germline and somatic mutations. - Describe common forms of DNA damage. - List enzymes of replication that were adapted to tasks of DNA repair. - Explain why a DNA glycosylase is useful in DNA repair. - Explain why the connection between 'breast cancer genes' and DNA repair.	libretexts	2025-03-17T22:27:36.268389	2021-01-03T20:12:41	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/14%3A_DNA_Replication/14.03%3A_Introduction", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "14.3: Introduction", "author": "Gerald Bergtrom" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/14%3A_DNA_Replication/14.04%3A_DNA_Replication	14.4: DNA Replication - - Last updated - Save as PDF As we’ve seen, DNA strands have directionality, with a 5’ nucleotide-phosphate and a 3’ deoxyribose hydroxyl end. This is even true for circular bacterial chromosomes…, if the circle is broken! Because the strands of the double helix are antiparallel , the 5’ end of one strand aligns with the 3’end of the other at both ends of the double helix. The complementary pairing of bases in DNA means that the base sequence of one strand can be used as a template to make a new complementary strand. As we’ll see, this structure of DNA created some interesting dilemmas for understanding the biochemistry of replication. The puzzlement surrounding how replication proceeds begins with experiments that visualize replicating DNA. A. Visualizing Replication and Replication Forks Recall the phenomenon of bacterial conjugation allowed a demonstration bacterial chromosomes were circular. In 1963, John Cairns confirmed this fact by direct visualization of bacterial DNA. He cultured E. coli cells for long periods on 3Hthymidine (3H-T) to make all of their cellular DNA radioactive. He then disrupted the cells gently to minimize damage to the DNA. The DNA released was allowed to settle and adhere to membranes. A sensitive film was placed over the membrane and time was allowed for the radiation to expose the film. After Cairns developed the autoradiographs, he examined the results in the electron microscope. He saw tracks of silver grains in the autoradiographs (the same kind of silver grains that create an image on film in old-fashioned photography). Look at the two drawings of his autoradiographs on the next page. Cairns measured the length of the “silver” tracks, which usually consisted of three possible closed loops, or circles. The circumferences of two of these circles were always equal, their length closely predicted by the DNA content of a single, nondividing cell. Cairns therefore interpreted these images to be bacterial DNA in the process of replication. Cairns’ autoradiographs and the measurements that led him to conclude that he was looking at images of bacterial circular chromosomes are illustrated below. He arranged his autoradiograph images in a sequence (below) to make his point. Because the replicating chromosomes looked (vaguely!) like the Greek letter $\theta $ , Cairns called them theta images . He inferred that replication starts at a single origin of replication on the bacterial chromosome, proceeding around the circle to completion. Subsequent experiments by David Prescott demonstrated bidirectional replication …, that replication did indeed begin at an origin of replication, after which the double helix was unwound and replicated in both directions, away from the origins, forming two replication forks (illustrated below). 176 Semiconservative Bidrectional Replication From Two RFs Bacterial cells can divide every hour (or even less); the rate of bacterial DNA synthesis is about 2 X 106 base pairs per hour. A typical eukaryotic cell nucleus contains thousands of times as much DNA as a bacterium, and typical eukaryotic cells double every 15-20 hours. Even a small chromosome can contain hundreds or thousands of times as much DNA as a bacterium. It appeared that eukaryotic cells could not afford to double their DNA at a bacterial rate of replication! Eukaryotes solved this problem not by evolving a faster biochemistry of replication, but by using multiple origins of replication from which DNA synthesis proceeds in both directions. This results in the creation of multiple replicons . Each replicon enlarges, eventually meeting other growing replicons on either side to replicate most of each linear chromosome, suggested in the illustration below. Before we consider the biochemical events at replication forks in detail, let's look at the role of DNA polymerase enzymes in the process. B. DNA Polymerases Catalyze Replication The first of these enzymes was discovered in E. coli by Arthur Kornberg, for which he received the 1959 Nobel Prize in Chemistry. Thomas Kornberg, one of Arthur’s sons later found two more of DNA polymerases! All DNA polymerases require a template strand against which to synthesize a new complementary strand. They all grow new DNA by adding to the 3’ end of the growing DNA chain in successive condensation reactions. And finally, all DNA polymerases also have the odd property that they can only add to a pre-existing strand of nucleic acid, raising the question of where the ‘preexisting’ strand comes from! DNA polymerases catalyze the formation of a phosphodiester linkage between the end of a growing strand and the incoming nucleotide complementary to the template strand. The energy for the formation of the phosphodiester linkage comes in part from the hydrolysis of two phosphates (pyrophosphate) from the incoming nucleotide during the reaction. While replication requires the participation of many nuclear proteins in both prokaryotes and eukaryotes, DNA polymerases perform the basic steps of replication, as shown in the illustration below. 178 DNA Polymerases & Their Activities Although DNA polymerases replicate DNA with high fidelity with as few as one error per 107 nucleotides, mistakes do occur. The proofreading ability of some DNA polymerases corrects many of these mistakes. The polymerase can sense a mismatched base pair, slow down and then catalyze repeated hydrolyses of nucleotides until it reaches the mismatched base pair. This basic proofreading by DNA polymerase is shown below. After mismatch repair, DNA polymerase resumes forward movement. Of course, not all mistakes are caught by this or other repair mechanisms (see DNA Repair , below). Mutations in the eukaryotic germ line cells that elude correction can cause genetic diseases. However, most are the mutations that fuel evolution. Without mutations in germ line cells (egg and sperm), there would be no mutations and no evolution, and without evolution, life itself would have reached a quick dead end! Other replication mistakes can generate mutations somatic cells. If these somatic mutations escape correction, they can have serious consequences, including the generation of tumors and cancers. C. The Process of Replication DNA replication is a sequence of repeated condensation (dehydration synthesis) reactions linking nucleotide monomers into a DNA polymer. Like all biological polymerizations, replication proceeds in three enzymatically catalyzed and coordinated steps: initiation , elongation and termination . 1. Initiation As we have seen, DNA synthesis starts at one or more origins or replication. These are DNA sequences targeted by initiator proteins in E. coli (below). After breaking hydrogen bonds at the origin of replication, the DNA double helix is progressively unzipped in both directions (i.e., by bidirectional replication ). The separated DNA strands serve as templates for new DNA synthesis. Sequences at replication origins that bind to initiation proteins tend to be rich in adenine and thymine bases. This is because A-T base pairs have two hydrogen (H-) bonds that require less energy to break than the three H-bonds holding G-C pairs together. Once initiation proteins loosen H-bonds at a replication origin, DNA helicase uses the energy of ATP hydrolysis to unwind the double helix. DNA polymerase III is the main enzyme that then elongates new DNA. Once initiated, a replication bubble (replicon) forms as repeated cycles of elongation proceed at opposite replication forks. 179 Replication Initiation in E. coli Recalling that new nucleotides can only be added to the free 3' hydroxyl group of a pre-existing nucleic acid strand. Since no known DNA polymerase can start synthesizing new DNA strands from scratch, this is a problem! The action of DNA polymerases therefore requires a primer , a nucleic acid strand to which to add nucleotides. The questions were…, what is the primer and where does it come from? Since RNA polymerases (enzymes that catalyze RNA synthesis) are the only nucleotide polymerase that can grow a new nucleic acid strand against a DNA template from scratch (i.e., from the first base), it was suggested that RNA might be the primer, After synthesis of a short RNA primer, new deoxynucleotides would be added to its 3’ end by DNA polymerase. The discovery of short stretches of RNA nucleotides at the 5’ end of Okazaki fragments confirmed the notion of RNA primers. We now know that cells use primase , a special RNA polymerase active during replication, to make those RNA primers against DNA templates before a DNA polymerase can grow the DNA strands at replication forks. As we will see now, the requirement for RNA primers is nowhere more in evidence in events at a replication fork. 2. Elongation Looking at elongation at one replication fork (below), we see another problem: One of the two new DNA strands can grow continuously towards the replication fork as the double helix unwinds. But what about the other strand? Either this other strand must grow in pieces in the opposite direction, or it must wait to begin synthesis until the double helix is fully unwound. If one strand of DNA must be replicated in fragments, then those fragments would have to be stitched (i.e., ligated) together. The problem is illustrated below. According to this hypothesis, a new leading strand of DNA is lengthened continuously by sequential addition of nucleotides to its 3’ end against its leading strand template . The other strand however, would be made in pieces that would be joined in phosphodiester linkages in a subsequent reaction. Because of the extra step and presumably extra time it takes to make and join these new DNA fragments, this new DNA is called the lagging strand , making its template the lagging strand template . Reiji Okazaki and his colleagues were studying mutants of T4 phage that grew slowly in their E. coli host cells. They graphed the growth rates of wild-type and mutant T4 phage and demonstrated that slow growth was due to a deficient DNA ligase enzyme, already known to catalyze the circularization of linear phage DNA molecules being replicated in infected host cells. The graph below summarizes their results. Okazaki’s hypothesis was that the deficient DNA ligase in the mutant phage not only slowed down circularization of replicating T4 phage DNA, but would also be slow at joining phage DNA fragments replicated against at least one of the two template DNA strands. When the hypothesis was tested, the Okazakis found that short DNA fragments did indeed accumulate in E. coli cells infected with ligasedeficient mutants, but not in cells infected with wild type phage. The lagging strand fragments are now called Okazaki fragments . 180 Okazaki Experiments & Fragments - Solving a Problem at an RF 181 Okazaki Fragments are Made Beginning with RNA Primers You can check out Okazaki’s original research at this link. Each Okazaki fragment would have to begin with a 5’ RNA primer, creating yet another dilemma! The RNA primer must be replaced with deoxynucleotides before stitching the fragments together. This in fact happens, and the process illustrated below Removal of RNA primer nucleotides from Okazali fragments requires the action of DNA polymerase I, an enzyme that can also catalyze hydrolysis of the phosphodiester bonds between the RNA (or DNA) nucleotides from the 5’-end of a nucleic acid strand. Flap Endonuclease 1 ( FEN 1 ) also plays a role in removing ‘flaps’ of nucleic acid from the 5’ ends of the fragments often displaced by polymerase as it replaces the replication primer. At the same time as the RNA nucleotides are removed, DNA polymerase I catalyzes their replacement by the appropriate deoxynucleotides. Finally, when a fragment is entirely DNA, DNA ligase links it to the rest of the already assembled lagging strand DNA. Because of its 5’ exonuclease activity (not found in other DNA polymerases), DNA polymerase 1 also plays unique roles in DNA repair (discussed further below). As Cairn’s suggested and others demonstrated, replication proceeds in two directions from the origin to form a replicon with its two replication forks (RFs). Each RF has a primase associated with replication of Okazaki fragments along lagging strand templates. As Cairn’s suggested and others demonstrated, replication proceeds in two directions from the origin to form a replicon with its two replication forks (RFs). Each RF has a primase associated with replication of Okazaki fragments along lagging strand templates. The requirement for primases at replication forks is shown below. Now we can ask what happens when replicons reach the ends of linear chromosomes in eukaryotes. 3. Termination In prokaryotes, replication is complete when two replication forks meet after replicating their portion of the circular DNA molecule. In eukaryotes, many replicons fuse to become larger replicons, eventually reaching the ends of the chromosomes…, where there is yet another problem (below)! When a replicon nears the end of a chromosome (i.e. a double-stranded DNA molecule), the strand synthesized continuously stops when it reaches the 5’ end of its template DNA. In theory, synthesis of a last Okazaki fragment can be primed from the 3’ end of the lagging template strand. The illustration above implies removal of a primer from the penultimate Okazaki fragment and DNA polymerase catalyzed replacement with DNA nucleotides. But what about the last Okazaki fragment? Would its primer be hydrolyzed? Moreover, without a free 3’ end to add to, how are those RNA nucleotides replaced with DNA nucleotides? The problem here is that every time a cell replicates, one strand of new DNA (likely both) would get shorter and shorter. Of course, this would not do…, and does not happen! Eukaryotic replication undergoes a termination process involving extending the length of one of the two strands by the enzyme telomerase . The action of telomerase is summarized in the illustration below Telomerase consists of several proteins and an RNA. From the drawing,, the RNA component serves as a template for 5’-> 3’ extension of the problematic DNA strand. The protein with the requisite reverse transcriptase activity is called Te lomerase R everse T ranscriptase , or TERT . The Te lomerase R NA C omponent is called TERC . Carol Greider, Jack Szostak and Elizabeth Blackburn shared the 2009 Nobel Prize in Physiology or Medicine for discovering telomerase. 183 Telomerase Replication Prevents Chromosome Shortening https ://youtu.be/M4dmfrxGKKU One of the more interesting recent observations was that differentiated, nondividing cells no longer produce the telomerase enzyme. On the other hand, the telomerase genes are active in normal dividing cells (e.g., stem cells) and cancer cells, which contain abundant telomerase. 4. Is Replication Processive? Drawings of replicons and replication forks suggest separate events on each DNA strand. Yet events at replication forks seem to be coordinated. Replication may be processive , meaning both new DNA strands are replicated in the same direction at the same time, smoothing out the process. How might this be possible? The drawing below shows lagging strand template DNA bending, so that it faces in the same direction as the leading strand at the replication fork. The replisome structure cartooned at the replication fork consists of clamp proteins , primase, helicase, DNA polymerase and single-stranded binding proteins among others. Newer techniques of visualizing replication by real-time fluorescence videography call the processive model into question, suggesting that the replication process is anything but smooth! Are lagging and leading strand replication not in fact coordinated? Alternatively, is the jerky movement of DNA elongation in the video an artifact, so that the model of smooth, coordinated replication integrated at a replisome still valid? Or is coordination defined and achieved in some other way? Check out the video yourself in the article here. 5. One More Problem with Replication Cairns recorded many images of E.coli of the sort shown below. The coiled, twisted appearance of the replicating circles were interpreted to be a natural consequence of trying to pull apart helically intertwined strands of DNA… or intertwined strands of any material! As the strands continued to unwind, the DNA should twist into a supercoil of DNA. Increased DNA unwinding would cause the phosphodiester bonds in the DNA to rupture, fragmenting the DNA. Obviously, this does not happen. Experiments were devised to demonstrate supercoiling, and to test hypotheses explaining how cells relax the supercoils during replication. Testing these hypotheses revealed the topoisomerase enzymes. These enzymes bind and hold on to DNA, catalyze hydrolysis of phosphodiester bonds, control unwinding of the double helix, and finally catalyze the re-formation of the phosphodiester linkages. It is important to note that the topoisomerases are not part of a replisome, but can act far from a replication fork, probably responding to the tensions in overwound DNA. Recall that topoisomerases comprise much of the protein lying along eukaryotic chromatin. 185 Topoisomerases Relieve Supercoiling During Replication We have considered most of the molecular players in replication. Below is a list of the key replication proteins and their functions (from here). \| Enzyme \| Function in DNA Replication \| \| DNA Helicase \| Also known as helix destabilizing enzyme. Unwinds the DNA double helix at the Replication Fork. \| \| DNA Polymerase \| Builds a new duplex DNA strand by adding nucleotides in the 5' to 3' direction. Also performs proof-reading and error correction. \| \| DNA clamp \| A protein which prevents DNA polymerase III from dissociating from the DNA parent strand. \| \| Single-Strand Binding (SSB) Proteins \| Bind to ssDNA and preven the DNA double helix from re-annealing after DNA helicase unwinds it thus maintaining the strand separation. \| \| Topoisomerase \| Relaxes the DNA from its super-coiled nature. \| \| DNA Gyrase \| Relieves strain of unwinding by DNA helicase; this is a specific type of topoisomerase \| \| DNA Ligase \| Re-anneals the semi-conservative strands and joins Okazaki Fragments of the lagging strand \| \| Primase \| Provides a starting point of RNA (or DNA) for DNA polymerase to begin synthesis of the new DNA strand. \| \| Telomerase \| Lengthens telomeric DNA by adding repetitive nucleotide sequences to the ends of eukaryotic chromosomes. \|	libretexts	2025-03-17T22:27:36.354143	2021-01-03T20:12:42	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/14%3A_DNA_Replication/14.04%3A_DNA_Replication", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "14.4: DNA Replication", "author": "Gerald Bergtrom" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/15%3A_Transcription/15.01%3A_Introduction	15.1: Introduction - - Last updated - Save as PDF Transcription, the synthesis of RNA based on a DNA template, is the central step of the Central Dogma proposed by Crick in 1958. The basic steps of transcription are the same as for replication: initiation, elongation and termination. Differences between transcription in prokaryotes and eukaryotes are in the details. - E.coli uses a single RNA polymerase enzyme to transcribe all kinds of RNAs while eukaryotic cells use different RNA polymerases to catalyze ribosomal RNA (rRNA), transfer RNA (tRNA) and messenger RNA (mRNA) synthesis. - In contrast to eukaryotes, some bacterial genes are part of operons whose mRNAs encode multiple polypeptides. - Bacterial mRNAs are typically translated as they are being transcribed. - Most RNA transcripts in prokaryotes emerge from transcription ready to use - Eukaryotic transcripts synthesized as longer precursors undergo processing by trimming , splicing , or both! - DNA in bacteria is virtually ‘naked’ in the cytoplasm while eukaryotic DNA is wrapped up in chromatin proteins in a nucleus. - In bacterial cells the association of ribosomes with mRNA and the translation of a polypeptide can begin even before the transcript is finished. This is because these cells have no nucleus. In our cells, RNAs must exit the nucleus before they encounter ribosomes in the cytoplasm. In this chapter, you will meet bacterial polycistronic mRNAs (transcripts of operons that encode more than one polypeptide) and the split genes of eukaryotes (with their introns and exons ). We will look at some details of transcription of the three major classes of RNA and then at how eukaryotes process precursor transcripts into mature, functional RNAs. Along the way, we will see one example of how protein structure has evolved to interact with DNA. Learning Objectives 1. Discriminate between the three steps of transcription in pro- and eukaryotes, and the factors involved in each. 2. State an hypothesis for why eukaryotes evolved complex RNA processing steps. 3. Speculate on why any cell in its right mind would have genes containing introns and exons so that their transcripts would have to be processed by splicing . 4. Articulate the differences between RNA vs. DNA structure . 5. Explain the need for sigma factors in bacteria. 6. Speculate on why eukaryotes do not have operons. 7. List structural features of proteins that bind and recognize specific DNA sequences. 8. Explain how proteins that do not bind specific DNA sequences can still bind to specific regions of the genome. 9. Formulate an hypothesis for why bacteria do not polyadenylate their mRNAs as much as eukaryotes do. 10. Formulate an hypothesis for why bacteria do not cap their mRNAs.	libretexts	2025-03-17T22:27:36.480770	2021-01-03T20:12:44	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/15%3A_Transcription/15.01%3A_Introduction", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "15.1: Introduction", "author": "Gerald Bergtrom" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/15%3A_Transcription/15.02%3A_Overview_of_Transcription	15.2: Overview of Transcription - - Last updated - Save as PDF A. The Major Types of Cellular RNA All cells make three main kinds of RNA: ribosomal RNA ( rRNA ), transfer RNA ( tRNA ) and messenger RNA ( mRNA ). rRNA is a structural as well as enzymatic component of ribosomes, the protein-synthesizing machine in the cell. Quantitatively, rRNAs are by far the most abundant RNAs in the cell and mRNAs, the least. Three rRNAs and about 50 ribosomal proteins make up the two subunits of a bacterial ribosome, as illustrated below. tRNAs are the decoding devices used in protein synthesis ( translation ) to convert nucleic acid sequence information into the amino acid sequences of polypeptides. tRNAs attached to amino acids are positioned on ribosomes based on codonanticodon recognition, as shown below. During translation, tRNAs decode base sequences in messenger RNA ( mRNAs ) into amino acid sequences of polypeptides. 186 Transcription Overview: Ribosomes and Ribosomal RNAs 187 Transcription Overview: Demonstrating the Major RNAs In 2009, Venkatraman Ramakrishnan, Thomas A. Steitz and ADA Yonath received the Nobel Prize in Chemistry their studies on the structure and molecular biology of the ribosome. Dr. Yonath is one of five women to receive a Nobel Prize – the others were Marie Curie, Irène Joliot-Curie, Dorothy Hodgkin and Barbara Mclintock. The fact that genes are inside the eukaryotic nucleus and that the synthesis of polypeptides encoded by those genes happens in the cytoplasm led to the proposal that there must be a messenger RNA (mRNA. Sidney Brenner eventually confirmed the existence of mRNAs. Check out his classic experiment in Brenner S (1961, An unstable intermediate carrying information from genes to ribosomes for protein synthesis . Nature 190:576-581). Recall polypeptide synthesis by the formation of polyribosomes (polysomes) along a single mRNA, as illustrated below. While mRNA is a small fraction of total cellular RNA, there are still smaller amounts of other RNAs such as the transient primers that we saw in DNA replication. We’ll encounter still other kinds of low-abundance RNAs later. B. Key Steps of Transcription In transcription, an RNA polymerase uses the template DNA strand of a gene to catalyze synthesis of a complementary, antiparallel RNA strand. RNA polymerases use ribose nucleotide triphosphate (NTP) precursors, in contrast to DNA polymerases, which use deoxyribose nucleotide (dNTP) precursors. In addition, RNAs incorporate uracil (U) nucleotides into RNA strands instead of the thymine (T) nucleotides that end up in new DNA. Another contrast to replication - RNA synthesis does not require a primer. With the help of transcription initiation factors, RNA polymerase locates the transcription start site of a gene and begins synthesis of a new RNA strand from scratch. Finally, like replication, transcription is error-prone. The basic steps of transcription are summarized on the next page. Here we can identify several of the DNA sequences that characterize a gene. The promoter is the binding site for RNA polymerase. It usually lies 5’ to, or upstream of the transcription start site (the bent arrow). Binding of the RNA polymerase positions the enzyme to near the transcription start site, where it will start unwinding the double helix and begin synthesizing new RNA. The transcribed grey DNA region in each of the three panels are the transcription unit of the gene. Termination sites are typically 3’ to, or downstream from the transcribed region of the gene. By convention, upstream refers to DNA 5’ to a given reference point on the DNA (e.g., the transcription start-site of a gene). Downstream then, refers to DNA 3’ to a given reference point on the DNA. 188 Transcription Overview: The Basic Mechanism of RNA Synthesis In bacteria, some transcription units encode more than one kind of RNA. Bacterial operons are an example of this phenomenon. The resulting mRNAs can be translated into multiple polypeptides at the same time. In the illustration below, RNA polymerase is transcribing a single mRNA molecule encoding three separate polypeptides. Bacterial transcription of the different RNAs requires only one RNA polymerase. Different RNA polymerases catalyze rRNA, mRNA and tRNA transcription in eukaryotes. Roger Kornberg received the Nobel Prize in Medicine in 2006 for his discovery of the role of RNA polymerase II and other proteins involved in eukaryotic messenger RNA transcription (like father-like son!!). 189 RNA Polymerases in Prokaryotes and Eukaryotes While mRNAs, rRNAs and tRNAs are most of what cells transcribe, a growing number of other RNAs (e.g., siRNAs, miRNAs, lncRNAs …) are also transcribed. Some functions of these transcripts (including control of gene expression or other transcript use) are discussed elsewhere. C. RNAs are Extensively Processed After Transcription in Eukaryotes Many eukaryotic RNAs are processed (trimmed, chemically modified) from large precursor RNAs to mature, functional RNAs. These precursor RNAs (pre-RNAs, or primary transcripts) contain in their sequences the information necessary for their function in the cell. Processing of the three major types of transcripts in eukaryotes is shown below. To summarize the illustration: - Many eukaryotic genes are ‘split’ into coding regions (exons) and non-coding intervening regions (introns). - Transcription of split genes generates a primary mRNA transcript (pre-mRNA). - Primary transcripts are spliced to remove the introns from the exons; exons are then ligated into a continuous mRNA. In some cases, the same pre-mRNA is spliced into alternate mRNAs encoding related but not identical polypeptides! - Pre-rRNA is cleaved and/or trimmed (not spliced!) to make shorter mature rRNAs. - Pre-tRNAs are trimmed, some bases within the transcript are modified and 3 bases (not encoded by the tRNA gene) are enzymatically added to the 3’-end. 190 Post Transcriptional Processing Overview The details of transcription and processing differ substantially in prokaryotes and eukaryotes. Let’s focus first on details of transcription itself, and then RNA processing.	libretexts	2025-03-17T22:27:36.545851	2021-01-03T20:12:45	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/15%3A_Transcription/15.02%3A_Overview_of_Transcription", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "15.2: Overview of Transcription", "author": "Gerald Bergtrom" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/15%3A_Transcription/15.03%3A_Details_of_Transcription	15.3: Details of Transcription - - Last updated - Save as PDF Find a well-written summary of transcription in prokaryotes and eukaryotes at an NIH website (Transcription in Prokaryotes and Eukaryotes). Here (and at this link), you will encounter proteins that bind DNA. Some proteins bind DNA to regulate transcription, inducing or silencing transcription of a gene. We will discuss their role in the regulation of gene expression later. Other proteins interact with DNA simply to allow transcription. These include one or more that, along with RNA polymerase itself, that must bind to the gene promoter to initiate transcription. We will look at bacterial transcription first. A. Transcription in Prokaryotes In E. coli, a single RNA polymerase transcribes all kinds of RNA, associating with one of several sigma factor proteins ( $\sigma $ -factors ) to initiate transcription. It turns out that different promoter sequences and corresponding $\sigma $ -factors play roles in the transcription of different genes (illustrated below). In the absence of the $\sigma $-factor, the E. coli RNA polymerase can transcribe RNA, but does so at a high rate, and from random sequences in the chromosome. In contrast, when the $\sigma $-factor is bound to the RNA polymerase, the complex seems to scan the DNA, recognize and then bind to the promoter sequence of a gene. In this case, the overall transcription rate is slower, but only genes are transcribed, rather than random bits of the bacterial genome! The Pribnow box, named for its discoverer, was the first promoter sequence characterized. Elongation is the successive addition of nucleotides complementary to their DNA templates, forming phosphodiester linkages. The enzymatic reactions of elongation are similar to the DNA polymerase-catalyzed elongation during replication. There are two ways that bacterial RNA polymerase ‘knows’ when it has reached the end of a transcription unit. In one case, as the RNA polymerase nears the 3’ end of the nascent transcript, it transcribes a 72 base, C-rich region. At this point, a termination factor called the rho protein binds to the nascent RNA strand. Rho is an ATP-dependent helicase that breaks the H-bonds between the RNA and the template DNA strand, thereby preventing further transcription. Rho-dependent termination is illustrated below. In the other mechanism of termination, the polymerase transcribes RNA whose termination signal assumes a secondary hairpin loop structure that causes the dissociation of the RNA polymerase, template DNA and the new RNA transcript. The role of the hairpin loop in rho-independent termination is illustrated below. B. Transcription in Eukaryotes Whereas bacteria rely on a single RNA polymerase for their transcription needs, eukaryotes use three different RNA polymerases to synthesize the three major different kinds of RNA, as shown below. Note that catalysis of the synthesis of most of the RNA in a eukaryotic cell (rRNAs) is by RNA polymerase I. With the help of initiation proteins, each RNA polymerase initiates transcription at a promoter sequence. Once initiated, the RNA polymerases then catalyze the successive formation of phosphodiester bonds to elongate the transcript. Recall that mRNAs are the least abundant in eukaryotes as they are in bacterial cells. Unfortunately, the details of the termination of transcription in eukaryotes are not as well understood as they are in bacteria. Therefore, we will focus on initiation, and then consider the processing of different eukaryotic RNAs into ready-to-use molecules. 1. Eukaryotic mRNA Transcription The multiple steps of eukaryotic mRNA transcription are shown on the next page. Transcription of eukaryotic mRNAs by RNA polymerase II begins with the sequential assembly of a eukaryotic initiation complex at a gene promoter. The typical eukaryotic promoter for a protein-encoding gene contains a TATA box DNA sequence motif as well as additional short upstream sequences. TATA-binding protein (TBP) first binds to the TATA box along with TFIID ( transcription initiation factor IID ). This intermediate recruits TFIIA and TFIIB . Next, TFGIIE , TFIIF and TFIIH , several other initiation factors and RNA polymerase II bind to form the transcription initiation complex . Phosphorylation adds several phosphates to the aminoterminus of the RNA polymerase, after which some of the TF’s dissociate from the initiation complex. The remaining RNA polymerase-TF complex can now start making the mRNA. Unlike prokaryotic RNA polymerase, eukaryotic RNA Polymerase II does not have an inherent helicase activity. For this, eukaryotic gene transcription relies on the multi-subunit TFIIH protein, in which two subunits have helicase activity. Consistent with the closer relationship of archaea to eukaryotes (rather to prokaryotes), archaeal mRNA transcription initiation resembles that of eukaryotes, albeit requiring fewer initiation factors during formation of an initiation complex. 192 Eukaryotic mRNA Transcription A significant difference between prokaryotic and eukaryotic transcription is that RNA polymerase and other proteins involved at a gene promoter do not see naked DNA. Instead, they must recognize specific DNA sequences through chromatin proteins. On the other hand, all proteins that interact with DNA have in common a need to recognize the DNA sequences to which they must bind…, within the double helix. In other words, they must see the bases within the helix, and not on its uniformly electronegative phosphate backbone surface. To this end, they must penetrate the DNA, usually through the major groove of the double helix. We will see that DNA regulatory proteins face the same problems in achieving specific shape-based interactions! 2. Eukaryotic tRNA and 5SRNA Transcription Transcription of 5S rRNA and tRNAs by RNA Polymerase III is unusual in that the promoter sequence to which it binds (with the help of initiation factors) is not upstream of the transcribed sequence, but lies within the transcribed sequence. After binding to this internal promoter, the polymerase re-positions itself to transcribe the RNA from the transcription start site so that the final transcript thus contains the promoter sequence! 5S rRNA by RNA polymerase III is shown below. 3. Transcription of the Other Eukaryotic rRNAs tRNAs are also transcribed by RNA polymerase III in much the same way as the 5S rRNA. The other rRNAs are transcribed by RNA polymerase I, which binds to an upstream promoter along with transcription initiation factors. We know less of the details of this process compared to our understanding of mRNA transcription. We’ll explore what we do know next. As already noted, transcription termination is not as well understood in eukaryotes as in prokaryotes. Coupled termination and polyadenylation steps common to most prokaryotic mRNAs are discussed in more detail below, with a useful summary at the NIH-NCBI website here.	libretexts	2025-03-17T22:27:36.612058	2021-01-03T20:12:45	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/15%3A_Transcription/15.03%3A_Details_of_Transcription", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "15.3: Details of Transcription", "author": "Gerald Bergtrom" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/16%3A_Transcriptional_Regulation_(prokaryotes)/16.01%3A_The_lac_Operon	16.1: The lac Operon - - Last updated - Save as PDF Early insights into mechanisms of transcriptional regulation came from studies of E. coli by researchers Francois Jacob & Jacques Monod. In E. coli, and many other bacteria, genes encoding several different proteins may be located on a single transcription unit called an operon . The genes in an operon share the same transcriptional regulation, but are translated individually. Eukaryotes generally do not group genes together as operons (exception is C. elegans and a few other species). Basic lac Operon structure E. coli encounters many different sugars in its environment. These sugars, such as lactose and glucose , require different enzymes for their metabolism. Three of the enzymes for lactose metabolism are grouped in the lac operon : lacZ , lacY , and lacA (Figure $\PageIndex{1}$). LacZ encodes an enzyme called β-galactosidase , which digests lactose into its two constituent sugars: glucose and galactose. lacY is a permease that helps to transfer lactose into the cell. Finally, lacA is a trans-acetylase ; the relevance of which in lactose metabolism is not entirely clear. Transcription of the lac operon normally occurs only when lactose is available for it to digest. Presumably, this avoids wasting energy in the synthesis of enzymes for which no substrate is present. A single mRNA transcript includes all three enzyme-coding sequences and is called polycistronic. A cistron is equivalent to a gene. cis- and trans Regulators In addition to the three protein-coding genes, the lac operon contains short DNA sequences that do not encode proteins, but are instead binding sites for proteins involved in transcriptional regulation of the operon. In the lac operon, these sequences are called P (promoter) , O (operator) , and CBS (CAP-binding site) . Collectively, sequence elements such as these are called cis -elements because they must be located on the same piece of DNA as the genes they regulate. On the other hand, the proteins that bind to these cis -elements are called trans -regulators because (as diffusible molecules) they do not necessarily need to be encoded on the same piece of DNA as the genes they regulate. lacI is an allosterically regulated repressor One of the major trans -regulators of the lac operon is encoded by lacI . Four identical molecules of lacI proteins assemble together to form a homotetramer called a repressor (Figure $\PageIndex{2}$). This repressor binds to two operator sequences adjacent to the promoter of the lac operon. Binding of the repressor prevents RNA polymerase from binding to the promoter (Figure $\PageIndex{3}$). Therefore, the operon will not be transcribed when the operator is occupied by a repressor. Besides its ability to bind to specific DNA sequences at the operator, another important property of the lacI protein is its ability to bind to lactose. When lactose is bound to lacI , the shape of the protein changes in a way that prevents it from binding to the operator. Therefore, in the presence of lactose, RNA polymerase is able to bind to the promoter and transcribe the lac operon, leading to a moderate level of expression of the lacZ , lacY , and lacA genes. Proteins such as lacI that change their shape and functional properties after binding to a ligand are said to be regulated through an allosteric mechanism. The role of lacI in regulating the lac operon is summarized in Figure $\PageIndex{4}$. CAP is an allosteric activator of the lac operon A second aspect of lac operon regulation is conferred by a trans -factor called cAMP binding protein ( CAP , Figure $\PageIndex{4}$). CAP is another example of an allosterically regulated trans -factor. Only when the CAP protein is bound to cAMP can another part of the protein bind to a specific cis -element within the lac promoter called the CAP binding sequence (CBS) . CBS is located very close to the promoter (P). When CAP is bound to at CBS, RNA polymerase is better able to bind to the promoter and initiate transcription. Thus, the presence of cAMP ultimately leads to a further increase in lac operon transcription. The physiological significance of regulation by cAMP becomes more obvious in the context of the following information. The concentration of cAMP is inversely proportional to the abundance of glucose: when glucose concentrations are low, an enzyme called adenylate cyclase is able to produce cAMP from ATP. Evidently, E. coli prefers glucose over lactose, and so expresses the lac operon at high levels only when glucose is absent and lactose is present. This provides another layer of logical control of lac operon expression: only in the presence of lactose, and in the absence of glucose is the operon expressed at its highest levels.	libretexts	2025-03-17T22:27:36.705166	2021-01-03T20:12:49	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/16%3A_Transcriptional_Regulation_(prokaryotes)/16.01%3A_The_lac_Operon", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "16.1: The lac Operon", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/16%3A_Transcriptional_Regulation_(prokaryotes)/16.02%3A_The_Use_of_Mutants_to_Study_the_lac_Operon	16.2: The Use of Mutants to Study the lac Operon - - Last updated - Save as PDF Single mutants of the lac operon The lac operon and its regulators were first characterized by studying mutants of E. coli that exhibited various abnormalities in lactose metabolism. Some mutants expressed the lac operon genes constitutively, meaning the operon was expressed whether or not lactose was present in the medium. Such mutant are called constitutive mutants. The operator locus ( lacO ) - One example is O c , in which a mutation in an operator sequence and reduces or precludes the repressor (the lacI gene product) from recognizing and binding to the operator sequence. Thus, in O c mutants, lacZ , lacY , and lacA are expressed whether or not lactose is present. The lacI locus – One type of mutant allele of lacI (callled I - ) prevents either the production of a repressor polypeptide or produces a polypeptide that cannot bind to the operator sequence. This is also a constitutive expresser of the lac operon because absence of repressor binding permits transcription. Another type of mutant of lacI called I s prevents the repressor polypeptide from binding lactose, and thus will bind to the operator and be non-inducible.. This mutant constitutively represses the lac operon whether lactose is present or not. The lac operon is not expressed and this mutant is called a “super-suppressor”. The F-factor and two lac operons in a single cell – partial diploid in E.coli More can be learned about the regulation of the lac operon when two different copies are present in one cell. This can be accomplished by using the F-factor to carry one copy, while the other is on the genomic E. coli chromosome. This results in a partial diploid in E. coli. The F-factor is an episome that is capable of being either a free plasmid or integrated into the host bacterial chromosome. This switching is accomplished by IS elements where unequal crossing over can recombine the F-factor and adjacent DNA sequences (genes) in and out of the host chromosome. Researchers have used this genetic tool to create partial diploids (merozygotes) that allow them to test the regulation with different combinations of different mutations in one cell. For example, the F-factor copy may have a I S mutation while the genomic copy might have an O C mutation. How would this cell respond to the presence/absence of lactose (or glucose)? This partial diploid can be used to determine that I S is dominant to I + , which in turn is dominant to I - . It can also be used to show the O C mutation only acts in cis - while the lacI mutation can act in trans - .	libretexts	2025-03-17T22:27:36.766581	2021-01-03T20:12:50	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/16%3A_Transcriptional_Regulation_(prokaryotes)/16.02%3A_The_Use_of_Mutants_to_Study_the_lac_Operon", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "16.2: The Use of Mutants to Study the lac Operon", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/16%3A_Transcriptional_Regulation_(prokaryotes)/16.03%3A_Gene_Regulation_in_Prokaryotes	16.3: Gene Regulation in Prokaryotes - - Last updated - Save as PDF Many prokaryotic genes are organized in operons , linked genes transcribed into a single mRNA encoding two or more proteins. Operons usually encode proteins with related functions. Regulating the activity of an operon (rather than multiple single genes encoding single proteins) allows better coordination of the synthesis of several proteins at once. In E. coli , the regulated lac operon encodes three enzymes involved in the metabolism of lactose (an alternative nutrient to glucose). Regulation of an operon (or of a single gene for that matter) can be by repression or by induction . When a small metabolite in a cell binds to a regulatory repressor or inducer protein, the protein undergoes an allosteric change that allows it to bind to a regulatory DNA sequence…, or to un-bind from the DNA. We will see examples of such regulation in the lac and trp operons. Lac operon gene regulation is an example of gene repression as well as induction . Trp (tryptophan) operon regulation is by gene repression . In both the operons, changes in levels of intracellular metabolites reflect the metabolic status of the cell and elicit appropriate changes in gene transcription. We will look at the regulation of both operons. 216 Overview of Prokaryotic Gene Regulation The mRNA transcribed from the lac operon is simultaneously translated into those three enzymes, as shown below. A. Mechanisms of Control of the Lac Operon In the animal digestive tract (including ours), genes of the E. coli lac operon regulate the use of lactose as an alternative nutrient to glucose. Think cheese instead of chocolate! The operon consists of lacZ, lacY, and lacA genes that were called structural genes . By definition, structural genes encode proteins that participate in cell structure and metabolic function. As already noted, the lac operon is transcribed into an mRNA encoding the Z, Y and A proteins. Let’s take a closer look at the structure of the lac operon and the function of the Y, Z and A proteins (below). The lacZ gene encodes β-galactosidase , the enzyme that breaks lactose (a disaccharide) into galactose and glucose. The lacY gene encodes lactose permease , a membrane protein that facilitates lactose entry into the cells. The role of the lacA gene (a transacetylase ) in lactose energy metabolism is not well understood. The I gene to the left of the lac Z gene is a regulatory gene (to distinguish it from structural genes). Regulatory genes encode proteins that interact with regulatory DNA sequences associated with a gene to control transcription. The operator sequence separating the I and Z genes is a transcription regulatory DNA sequence. The E. coli lac operon is usually silent (repressed) because these cells prefer glucose as an energy and carbon source. In the presence of sufficient glucose, a repressor protein (the I gene product) is bound to the operator , blocking transcription of the lac operon. Even if lactose is available, cells will not be use it as an alternative energy and carbon source when glucose levels adequate. However, when glucose levels drop, the lac operon is active and the three enzyme products are translated. We will see how limiting glucose levels induce maximal lac operon transcription by both derepression and direct induction , leading to maximal transcription of the lac genes only when necessary (i.e., in the presence of lactose and absence of glucose). Let’s look at some of the classic experiments that led to our understanding of E. coli gene regulation in general, and of the lac operon in particular. In the late 1950s and early 1960s, Francois Jacob and Jacques Monod were studying the use of different sugars as carbon sources by E. coli . They knew that wild type E. coli would not make the $\beta $-galactosidase, $\beta $-galactoside permease or $\beta $-galactoside transacetylase proteins when grown on glucose. Of course, they also knew that the cells would switch to lactose for growth and reproduction if they were deprived of glucose! They then searched for and isolated different E. coli mutants that could not grow on lactose, even when there was no glucose in the growth medium. Here are some of the mutants they studied: - One mutant failed to make active $\beta $-galactosidase enzyme but made permease. - One mutant failed to make active permease but made normal amounts of $\beta $-galactosidase. - Another mutant failed to make transacetylase..., but could still metabolize lactose in the absence of glucose. Hence the uncertainty of its role in lactose metabolism. - Curiosly, one mutant strain failed to make any of the three enzymes! Since double mutants are very rare and triple mutants even rarer, Jacob and Monod inferred that the activation of all three genes in the presence of lactose were controlled together in some way. In fact, it was this discovery that defined the operon as a set of genes transcribed as a single mRNA, whose expression could therefore be effectively coordinated. They later characterized the repressor protein produced by the lacI gene. Jacob, Monod and Andre Lwoff shared the Nobel Prize in Medicine in 1965 for their work on bacterial gene regulation. We now know that negative and positive regulation of the lac operon (described below) depend on two regulatory proteins that together, control the rate of lactose metabolism. 1. Negative Regulation of the Lac Operon by Lactose Refer to the illustration below to identify the players in lac operon derepression. The repressor protein product of the I gene is always made and present in E. coli cells. I gene expression is not regulated! In the absence of lactose in the growth medium, the repressor protein binds tightly to the operator DNA. While RNA polymerase is bound to the promoter and ready to transcribe the operon, the presence of the repressor bound to the operator sequence close to the Z gene physically blocks its forward movement. Under these conditions, little or no transcript is made. If cells are grown in the presence of lactose, the lactose entering the cells is converted to allolactose . Allolactose binds to the repressor sitting on the operator DNA to form a 2-part complex, as shown below. The allosterically altered repressor dissociates from the operator and RNA polymerase can transcribe the lac operon genes as illustrated below 2. Positive Regulation of the Lac Operon; Induction by Catabolite Activation The second control mechanism regulating lac operon expression is mediated by CAP (cAMP-bound catabolite activator protein or cAMP receptor protein). When glucose is available, cellular levels of cAMP are low in the cells and CAP is in an inactive conformation. On the other hand, if glucose levels are low, cAMP levels rise and bind to the CAP, activating it. If lactose levels are also low, the cAMP-bound CAP will have no effect. If lactose is present and glucose levels are low, then allolactose binds the lac repressor causing it to dissociate from the operator region. Under these conditions, the cAMP-bound CAP can bind to the operator in lieu of the repressor protein. In this case, rather than blocking the RNA polymerase, the activated Camp-bound CAP induces even more efficient lac operon transcription. The result is synthesis of higher levels of lac enzymes that facilitate efficient cellular use of lactose as an alternative to glucose as an energy source. Maximal activation of the lac operon in high lactose and low glucose is shown below. 217 Regulation of the Lac Operon cAMP-bound CAP is an inducer of transcription. It does this by forcing the DNA in the promoter-operator region to bend. And since bending the double helix loosens H-bonds, it becomes easier for RNA polymerase to find and bind the promoter on the DNA strand to be transcribed…, and for transcription to begin. cAMP-CAPinduced bending of DNA is illustrated below. 3. Lac Operon Regulation by Inducer Exclusion and Multiple Operators In recent years, additional layers of lac operon regulation have been uncovered. In one case, the ability of lac permease to transport lactose across the cell membrane is regulated. In another, additional operator sequences have been discovered to interact with a multimeric repressor to control lac gene expression. a) Regulation of Lactose use by Inducer Exclusion When glucose levels are high (even in the presence of lactose), phosphate is consumed to phosphorylate glycolytic intermediates, keeping cytoplasmic phosphate levels low. Under these conditions, unphosphorylated EIIAGlc binds to the lactose permease enzyme in the cell membrane, preventing it from bringing lactose into the cell. The role of phosphorylated and unphosphorylated EIIA Glc in regulating the lac operon are shown below. High glucose levels block lactose entry into the cells, effectively preventing allolactose formation and the derepression of the lac operon. Inducer exclusion is thus a logical way for the cells to handle an abundance of glucose, whether or not lactose is present. On the other hand, if glucose levels are low in the growth medium, phosphate concentrations in the cells rise sufficiently for a specific kinase to phosphorylate the EIIAGlc. Phosphorylated EIIAGlc then undergoes an allosteric change and dissociates from the lactose permease, making it active so that more lactose can enter the cell. In other words, the inducer is not “excluded” under these conditions! The kinase that phosphorylates EIIA Glc is part of a phosphoenolpyruvate (PEP)- dependent phosphotransferase system (PTS) cascade. When extracellular glucose levels are low, the cell activates the PTS system in an effort to bring whatever glucose is around into the cell. But the last enzyme in the PTS phosphorylation cascade is the kinase that phosphorylates EIIA Glc . Phosphorylated EIIA Glc dissociates from the lactose permease, re-activating it, bringing available lactose into the cell from the medium. b) Repressor Protein Structure and Additional Operator Sequences The lac repressor is a tetramer of identical subunits (below). Each subunit contains a helix-turn-helix motif capable of binding to DNA. However, the operator DNA sequence downstream of the promoter in the operon consists of a pair of inverted repeats spaced apart in such a way that they can only interact two of the repressor subunits, leaving the function of the other two subunits unknown… that is, until recently! Two more operator regions were recently characterized in the lac operon. One, called O 2 , is within the lac z gene itself and the other, called O 3 , lies near the end of, but within the lac I gene. Apart from their unusual location within actual genes, these operators, which interact with the remaining two repressor subunits, went undetected at first because mutations in the O2 or the O3 region individually do not contribute substantially to the effect of lactose in derepressing the lac operon. Only mutating both regions at the same time results in a substantial reduction in binding of the repressor to the operon. B. Mechanism of Control of the Tryptophan Operon If ample tryptophan ( trp ) is available, the tryptophan synthesis pathway can be inhibited in two ways. First, recall how feedback inhibition by excess trp can allosterically inhibit the trp synthesis pathway. A rapid response occurs when tryptophan is present in excess, resulting in rapid feedback inhibition by blocking the first of five enzymes in the trp synthesis pathway. The trp operon encodes polypeptides that make up two of these enzymes. Enzyme 1 is a multimeric protein, made from polypeptides encoded by the trp5 and trp4 genes. The trp1 and trp2 gene products make up Enzyme 3 . If cellular tryptophan levels drop because the amino acid is rapidly consumed (e.g., due to demands for proteins during rapid growth), E. coli cells will continue to synthesize the amino acid, as illustrated below. On the other hand, if tryptophan consumption slows down, tryptophan accumulates in the cytoplasm. Excess tryptophan will bind to the trp repressor. The trp-bound repressor then binds to the trp operator, blocking RNA polymerase from transcribing the operon. The repression of the trp operon by trp is shown below. In this scenario, tryptophan is a co-repressor . The function of a co-repressor is to bind to a repressor protein and change its conformation so that it can bind to the operator.	libretexts	2025-03-17T22:27:36.841666	2021-01-03T20:12:50	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/16%3A_Transcriptional_Regulation_(prokaryotes)/16.03%3A_Gene_Regulation_in_Prokaryotes", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "16.3: Gene Regulation in Prokaryotes", "author": "Gerald Bergtrom" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/17%3A_RNA_processing/17.01%3A_Details_of_Eukaryotic_mRNA_Processing	17.1: Details of Eukaryotic mRNA Processing - - Last updated - Save as PDF Eukaryotic mRNA primary transcripts undergo extensive processing, including splicing, capping and, polyadenylation . The steps described here are considered in order of (sometimes overlapping!) occurrence. We begin with splicing, an mRNA phenomenon. A. Spliceosomal Introns Bacterial gene coding regions are continuous. The discovery of eukaryotic split genes with introns and exons came as quite a surprise. Not only did it seem incongruous for evolution to have stuck irrelevant DNA in the middle of coding DNA, no one could have dreamt up such a thing! For their discovery of split genes, by Richard J. Roberts and Phillip A. Sharp shared the Nobel Prize for Physiology in 1993. In fact, all but a few eukaryotic genes are split, and some have one, two (or more than 30-50!) introns separating bits of coding DNA, the exons . Splicing is summarized below. Splicing involves a number of small ribonuclear proteins ( snRNPs ). snRNPs are particles composed of RNA and proteins. They bind to specific sites in an mRNA and then direct a sequential series of cuts and ligations (the splicing ) necessary to process the mRNAs. The role of snRNPs in splicing pre-mRNAs is illustrated below. snRNP binding to a pair of splice sites flanking an intron in a pre-mRNA forms the spliceosome that completes the splicing, including removal of the lariat (the intermediate structure of the intron). The last step is to ligate exons into a continuous mRNA with all its codons intact and ready for translation. Spliceosome action is summarized below. 194 The Discovery of Split Genes B. Specific Nuclear bodies and their associated proteins facilitate the assembly and function of the SnRNPs Recall the organization of nuclei facilitated by nuclear bodies. Cajal bodies ( CBs ) and Gems are nuclear bodies that are similar in size and have related functions in assembling spliceosomal SnRNPs. Some splicing defects correlate with mutations in the coil protein that associate with Cajal bodies; others correlate with mutations in SMN proteins normally associated with Gems. An hypothesis was that CBs and Gems interact in SnRNP and spliceosome assembly…, but how? Consider the results of an experiment in which antibodies to coilin and the SMN protein were localized in undifferentiated and differentiated neuroblastoma cells. A and C are undifferentiated cells in culture; B and D are cells that were stimulated to differentiate. In the fluorescence micrographs at the right, arrows point to fluorescent nuclear bodies. The coilin protein is associated with CBs and SMN is found in Gems. Therefore, we expect that fluorescent antibodies to coilin (green) will localize to CBs and antibodies to SMN protein (red) will bind to Gems. This is what happens in the nuclei of undifferentiated cells (panel C). But in panel D, the two antibodies colocalize, suggesting that the CBs and Gems aggregate in the differentiated cells. This would explain the need for both functional coilin and SMN protein to produce functional SnRNPs. The CBs and Gems may be aggregating in differentiated cells due to an observed increase in expression of the SMN protein. This could lead to more active Gems more able to associate with the CBs. This and similar experiments demonstrate that different nuclear bodies do have specific functions. They are not random structural artifacts, have evolved to organize nuclear activities in time and space in ways that are essential to the cell. C. Group I and Group II Self-Splicing Introns While Eukaryotic Spliceosomal introns are spliced using snRNPs as described above, Group I or Group II introns are removed by different mechanisms. Group I introns interrupt mRNA and tRNA genes in bacteria and in mitochondrial and chloroplast genes. They are occasionally found in bacteriophage genes, but rarely in nuclear genes, and then only in lower eukaryotes. Group I introns are self-splicing ! Thus, they are ribozymes that do not require snRNPs or other proteins. Instead, they fold into a secondary stem-loop structure that positions catalytic nucleotides at appropriate splice sites, excise themselves, and re-ligate the exons. Group II introns in chloroplast and mitochondrial rRNA, mRNA, tRNA and some bacterial mRNAs can be quite long, form complex stem-loop tertiary structures, and self-splice, at least in a test tube! However, Group II introns encode proteins required for their own splicing in vivo . Like spliceosomal introns, they form a lariat structure at an A residue branch site. All this suggests that the mechanism of spliceosomal intron splicing evolved from that of Group II introns. D. So, Why Splicing? The puzzle implied by the question of course is why higher organisms have split genes in the first place. While the following discussion can apply to all splicing, it will reference mainly spliceosomal introns. Here are some answers to the question “Why splicing?” - Introns in nuclear genes are typically longer (often much longer!) than exons. Since they are non-coding, they are large targets for mutation. In effect, noncoding DNA, including introns can buffer the ill effects of random mutations . - You may recall that gene duplication on one chromosome (and loss of a copy from its homolog) arise from unequal recombination (non-homologous crossing over). It occurs when similar DNA sequences align during synapsis of meiosis. In an organism that inherits a chromosome with both gene copies, the duplicate can accumulate mutations as long as the other retains original function. The diverging gene then becomes part of a pool of selectable DNA, the grist of evolution, in the descendants of organisms that inherit the duplicated genes, increasing species diversity. Unequal recombination can also occur between similar sequences (e.g., in introns) in the same or different genes. Introns can also enable the sharing of exons between genes. After unequal recombination between introns flanking an exon, one gene will acquire another exon while the other will lose it. Once again, as long as an organism retains a copy of the participating genes with original function, the organism can make the required protein and survive. Meanwhile, the gene with the extra exon may produce the same protein, but one with a new structural domain and function. Like a complete duplicate gene, one with a new exon and added function is in the pool of selectable DNA. Thus, this phenomenon of exon shuffling increases species diversity ! The evidence indicates that exon shuffling has occurred, creating proteins with different overall functions that nonetheless share at least one domain and one common function. An example discussed earlier involves calcium-binding proteins that regulate many cellular processes. Structurally related calcium (Ca++) binding domains are common to many otherwise structurally and functionally unrelated proteins. Consider exon shuffling in the unequal crossover (non-homologous recombination) illustrated below. In this example, regions of strong similarity in different (non-homologous) introns in the same gene align during synapsis of meiosis. Unequal crossing over between the genes inserts exon C in one of the genes. The other gene loses the exon (not shown in the illustration). In sum, introns are buffers against deleterious mutations , and equally valuable, are potential targets for gene duplication and exon shuffling. This makes introns key players in creating genetic diversity, the hallmark of evolution . E. Capping A methyl guanosine cap added 5’-to-5’ to an mRNA functions in part to help mRNAs leave the nucleus and associate with ribosomes. The cap is added to an exposed 5’ end, even as transcription and splicing are still in progress. A capping enzyme places a methylated guanosine residue at the 5’-end of the mature mRNA. The 5’ cap structure is shown below (check marks are 5’-3’ linked nucleotides). F. Polyadenylation After transcription termination, poly(A) polymerase catalyzes the addition of multiple AMP residues (several hundred in some cases) to the 3’ terminus by the enzyme. The enzyme binds to an AAUAA sequence near the 3’ end of an mRNA and begins to catalyze the addition of the adenosine monophosphates. The AAUAA poly(A) recognition site is indicated in red in the illustration of polyadenylation shown below. The result of polyadenylation is a 3’ poly (A) tail whose functions include assisting in the transit of mRNAs from the nucleus and regulating the half-life of mRNAs in the cytoplasm. The poly (A) tail shortens each time a ribosome completes translating the mRNA.	libretexts	2025-03-17T22:27:36.937482	2021-01-03T20:12:47	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/17%3A_RNA_processing/17.01%3A_Details_of_Eukaryotic_mRNA_Processing", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "17.1: Details of Eukaryotic mRNA Processing", "author": "Gerald Bergtrom" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/18%3A_Transcriptional_Regulation_(eukaryotes)/18.01%3A_Eukaryotic_Gene_Regulation	18.1: Eukaryotic Gene Regulation - - Last updated - Save as PDF Like prokaryotes, transcriptional regulation in eukaryotes involves both cis -elements and trans -factors, only there are more of them and they interact in a more complex way. A diagram of a typical eukaryotic gene, including several types of cis -elements, is shown in Figure $\PageIndex{7}$. Proximal Regulatory Sequences As in prokaryotes the RNA polymerase binds to the gene at its promoter to begin transcription. In eukaryotes, however, RNApol is part of a large protein complex that includes additional proteins that bind to one or more specific cis -elements in the promoter region, including GC-rich boxes , CAAT boxes , and TATA boxes . High levels of transcription require both the presence of this protein complex at the promoter, as well as their interaction with other trans -factors described below. The approximate position of these elements relative to the transcription start site ( + 1) is shown in Figure $\PageIndex{7}$, but it should be emphasized that the distance between any of these elements and the transcription start site can vary, but are typically within ~200 base pairs of the start of transcription. This contrasts the next set of elements. Distal Regulatory elements Even more variation is observed in the position and orientation of the second major type of cis -regulatory element in eukaryotes, which are called enhancer elements . Regulatory trans -factor proteins called transcription factors bind to enhancer sequences, then, while still bound to DNA, these proteins interact with RNApol and other proteins at the promoter to enhance the rate of transcription. There are a wide variety of different transcription factors and each recognizes a specific DNA sequence (enhancer element) to promote gene expression in the adjacent gene under specific circumstances. Because DNA is a flexible molecule, enhancers can be located near (~100s of bp) or far (~10K of bp), and either upstream or downstream, from the promoter (Figures $\PageIndex{7}$ and $\PageIndex{8}$). Example 1: Drosophila yellow gene The yellow gene of Drosophila provides an example of the modular nature of enhancers. This gene encodes an enzyme in the pathway that produces a dark pigment in the insect’s exoskeleton. Mutants have a yellow cuticle rather than the wild type darker pigmented cuticle. (Why call the gene “yellow”: recall that genes are often named after their mutant phenotype.) Figure $\PageIndex{9}$ shows three enhancer elements (left side - wing, body, mouth), each binds a different tissue specific transcription factor to enhancer transcription of yellow + in that tissue and makes the pigment. So, the wing cells will have a transcription factor that binds to the wing enhancer to drive expression; likewise in the body and mouth cells. Thus, specific combinations of cis -elements and trans -factors control the differential, tissue-specific expression of genes. This type of combinatorial action of enhancers is typical of the transcriptional activation of most eukaryotic genes: specific transcription factors activate the transcription of target genes under specific conditions. While enhancer sequences promote expression, there is an oppositely acting type of element, called silencers . These elements function in much the same manner, with transcription factors that bind to DNA sequences, but they act to silence or reduce transcription from the adjacent gene. Again, a gene’s expression profile (transcription level, tissue specific, temporal specific) is a combination of various enhancer and silencer elements. Example 2: Gal4-UAS system from yeast – a genetic tool Genetic researchers have taken advantage of a yeast distal enhancer sequence to make the GAL4-UAS system , a powerful technique for studying the expression of genes in other eukaryotes. It relies on two parts: a “ driver ” and a “ responder ” (Figure $\PageIndex{10}$). The driver part is a gene encoding a yeast transcriptional activator protein called Gal4. It is separate from the responder part, which contains the enhancer sequence, or upstream activation sequence (UAS, as it is called in yeast) to which the Gal4 protein specifically binds. This UAS is placed upstream (using genetic engineering) from a promoter transcribing a reporter gene, or other gene of interest, such as GFP (green fluorescent protein). Both parts must be present in the same cell for the system to express the responder gene. If the driver is absent, the responder product will not be expressed. However, both are in the same cell (or organism) the pattern of expression of the driver part will induce the responder part’s expression in the same pattern. This system works is a variety of eukaryotes, including humans. It has been especially well exploited in Drosophila where 100’s (1,000’s ? ) of differently expressing driver lines are available. These lines permit the tissue specific expression of any responder gene to examine its effect on development.	libretexts	2025-03-17T22:27:37.137765	2021-01-03T20:12:52	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/18%3A_Transcriptional_Regulation_(eukaryotes)/18.01%3A_Eukaryotic_Gene_Regulation", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "18.1: Eukaryotic Gene Regulation", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/18%3A_Transcriptional_Regulation_(eukaryotes)/18.02%3A_Regulatory_Elements_in_Evolution	18.2: Regulatory Elements in Evolution - - Last updated - Save as PDF Mutations can occur in both cis -elements and trans -factors; both can result in altered patterns of gene expression. If an altered pattern of gene expression results in a selective advantage (or at least do not produce a major disadvantage), they may be selected and maintained in future populations. They may even contribute to the evolution of new species. An example of a sequence change in an enhancer is found in the Pitx gene. Example: Pitx expression in Stickleback The three-spined stickleback (Figure $\PageIndex{11}$) provides an example of natural selection of a mutation in a cis -regulatory element. This fish occurs in two forms: (1) populations that inhabit deep, open water and have a spiny pelvic fin that deters larger predator fish from feeding on them; (2) populations from shallow water environments and lack this spiny pelvic fin. In shallow water, it appears that a long, spiny pelvic fin would be a disadvantage because it frequently contacts the sediment at the bottom of the pond and allows parasitic insects in the sediment to invade the stickleback. Researchers compared gene sequences of individuals from both deep and shallow water environments as shown in Figure $\PageIndex{11}$. They observed that in embryos from the deep-water population, a gene called Pitx was expressed in several groups of cells, including those that developed into the pelvic fin. Embryos from the shallow-water population expressed Pitx in the same groups of cells as the other population, with an important exception: Pitx was not expressed in the pelvic fin primordium in the shallow-water population. Further genetic analysis showed that the absence of Pitx gene expression from the developing pelvic fin of shallow-water stickleback was due to the absence (mutation) of a particular enhancer element upstream of Pitx . Example: Hemoglobin expression in placental mammals. Hemoglobin is the oxygen-carrying component of red blood cells (erythrocytes). Hemoglobin usually exists as tetramers of four non-covalently bound hemoglobin molecules (Fig 12.12). Each hemoglobin molecule consists of a globin polypeptide with a covalently attached heme molecule. Heme is made through a specialized metabolic pathway and is then bound to globin polypeptide through post-translational modification . The composition of the tetramers changes during development (Figure $\PageIndex{13}$). From early childhood onward, most tetramers are of the type $\mathbf{\alpha}$ 2 $\mathbf{\beta}$ 2 , which means they contain of two copies of each of two slightly different globin proteins named $\alpha$ and $\beta$. A small amount of adult hemoglobin is $\alpha$ 2 $\delta$ 2 , which has $\delta$ globin instead of the more common $\beta$ globin. Other tetrameric combinations predominate before birth: $\zeta$ 2 $\varepsilon$ 2 is most abundant in embryos, and $\alpha$ 2 $\gamma$ 2 is most abundant in fetuses. Although the six globin proteins ($\alpha$ = alpha, $\beta$ = beta , $\gamma$ = gamma, $\delta$ =delta, $\varepsilon$ =epsilon , $\zeta$ = zeta) are very similar to each other, they do have slightly different functional properties. For example, fetal hemoglobin has a higher oxygen affinity than adult hemoglobin, allowing the fetus to more effectively extract oxygen from maternal blood. The specialized $\gamma$ globin genes that are characteristic of fetal hemoglobin are found only in placental mammals. Each of these globin polypeptides is encoded by a different gene. In humans, globin genes are located in clusters on two chromosomes (Figure $\PageIndex{14}$). We can infer that these clusters arose through a series of duplications of an ancestral globin gene. Gene duplication events can occur through rare errors in processes such as DNA replication, meiosis, or transposition. The duplicated genes can accumulate mutations independently of each other. Mutations can occur in either the regulatory regions (e.g. promoter regions), or in the coding regions, or both. In this way, the promoters of globin genes have evolved to be expressed at different phases of development, and to produce proteins optimized for the prenatal environment. Of course, not all mutations are beneficial: some mutations can lead to inactivation of one or more of the products of a gene duplication. This can produce what is called a pseudogene . Examples of pseudogenes ($\psi$) are also found in the globin clusters. Pseudogenes have mutations that prevent them from being expressed at all. The globin genes provide an example of how gene duplication and mutation, followed by selection, allows genes to evolve specialized expression patterns and functions. Many genes have evolved as gene families in this way, although they are not always clustered together as are the globins.	libretexts	2025-03-17T22:27:37.197760	2021-01-03T20:12:53	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/18%3A_Transcriptional_Regulation_(eukaryotes)/18.02%3A_Regulatory_Elements_in_Evolution", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "18.2: Regulatory Elements in Evolution", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/18%3A_Transcriptional_Regulation_(eukaryotes)/18.03%3A_Additional_Levels_of_Regulating_Transcription	18.3: Additional Levels of Regulating Transcription - - Last updated - Save as PDF Eukaryotes regulate transcription via promoter sequences close to the transcription unit (as in prokaryotes) and also use more distant enhancer sequences to provide more variation in the timing, level, and location of transcription, however, there are still additional levels of genetic control. This consists of two major mechanism: (1) large-scale changes in chromatin structure, and (2) modification of bases in the DNA sequence. These two are often inter-connected. Chromatin Dynamics Despite the simplified way in which we often represent DNA in figures such as those in this chapter, DNA is almost always associated with various chromatin proteins. For example, histones remain associated with the DNA even during transcription. Thus the rate of transcription is also controlled by the accessibility of DNA to RNApol and regulatory proteins. So, in regions were the chromatin is highly compacted, it is unlikely that any gene will be transcribed, even if all the necessary cis - and trans - factors are present in the nucleus. The extent of chromatin compaction in various regions is regulated through the action of chromatin remodeling proteins. These protein complexes include enzymes that add or remove chemical tags, such as methyl or acetyl groups, to various DNA bound proteins. These modifications alter the local chromatin density and thus the availability for transcription. Acetylated histones, for example, tend to be associated with actively transcribed genes, whereas deacetylated histone are associated with genes that are silenced (Figure $\PageIndex{15}$). Likewise, methylation of DNA itself is also associated with transcription regulation. Cytosine bases, particularly when followed by a guanine ( CpG sites ) are important targets for DNA methylation (Figure $\PageIndex{16}$). Methylated cytosine within clusters of CpG sites is often associated with transcriptionally inactive DNA. The modification of DNA and its associated proteins is enzymatically reversible (acetylation/deacetylation; methylation/demethylation) and thus a cyclical activity. Regulation of this provides another layer through which eukaryotic cells control the transcription of specific genes.	libretexts	2025-03-17T22:27:37.255468	2021-01-03T20:12:53	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/18%3A_Transcriptional_Regulation_(eukaryotes)/18.03%3A_Additional_Levels_of_Regulating_Transcription", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "18.3: Additional Levels of Regulating Transcription", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/18%3A_Transcriptional_Regulation_(eukaryotes)/18.04%3A_Gene_Regulation_in_Eukaryotes	18.4: Gene Regulation in Eukaryotes - - Last updated - Save as PDF A. The Difference between Eukaryotic and Prokaryotic Gene Regulation Let's Recall an experiment described earlier and illustrated below. Results of this experiment provided the evidence that even very different cells of an organism contain the same genes. In fact, in any multicellular eukaryotic organism, every cell contains the same DNA (genes). Therefore, the different cell types in an organism differ not in which genes they contain, but which sets of genes they express! Looked at another way, cells differentiate when they turn on new genes and turn off old ones. Thus, gene regulation produces different sets of gene products during differentiation, leading to cells that look and function differently in the organism. 220 An Experiment: All of an Organism's Cells Have the Same Genome Compared to prokaryotes, many steps in eukaryotes lie between transcription of an mRNA and the accumulation of a polypeptide end product. Eleven of these steps are shown in the pathway from gene to protein below. Theoretically, cells could turn on, turn off, speed up or slow down any of the steps in this pathway, changing the steady state concentration of a polypeptide in the cells. While regulation of any of these steps is possible, the expression of a single gene is typically controlled at only one or a few steps. A common form of gene regulation is at the level of transcription initiation, similar to transcriptional control in bacteria, in principle if not in detail. B. Complexities of Eukaryotic Gene Regulation Gene regulation in eukaryotes is more complex than in prokaryotes. This is in part because their genomes are larger and because they encode more genes. For example, the E. coli genome houses about 5,000 genes, compared to around 25,000 genes in humans. Furthermore, eukaryotes can produce even more than 25,000 proteins by alternative splicing of mRNAs and in at least a few cases, by initiating transcription from alternative start sites in the same gene. And of course, the activity of many more genes must be coordinated without the benefit of multigene operons! Finally, eukaryotic gene regulation is made more complicated because all nuclear DNA is wrapped in protein in the form of chromatin. All organisms control gene activity with transcription factors that bind to specific DNA sequences ( cis regulatory elements ). In eukaryotes, these elements can be proximal to (near) the promoter of a gene, or distal to (quite far from) the gene they regulate. A eukaryotic map showing the components of a typical gene and its associated cis-acting regulatory elements is shown below. Enhancers are typical distal cis elements that recognize and bind transcription factors to increase the rate of transcription of a gene. Oddly enough, these short DNA elements can be in the 5’ or 3’ non-translated region of the gene, or even within introns, and can lie thousands of base pairs away from the genes they control. Note that enhancer elements are even in introns can also be very far from the start-site of transcription of a gene. Upstream regulatory regions of eukaryotic genes (to the left of a gene promoter as shown above) often have distal binding sites for more than a few transcription factors, some with positive ( enhancing ) and others with negative ( silencing ) effects. Of course, which of these DNA regions are active in controlling a gene depends on which transcription factor(s) are present in the nucleus. Sets of positive regulators will work together to coordinate and maximize gene expression when needed, and sets of negative regulators will bind negative regulatory elements to silence a gene. 222 Transcription Factors Bind DNA Near and Far We saw that in eukaryotes, the initiation of transcription involves many transcription factors and RNA polymerase II acting at a gene promoter to form a transcription preinitiation complex . TFIID , or TATA binding protein is one of the first factors to bend, causing the DNA in the promoter region to bend, much like the CAP protein in bacteria. TFIID also recruits other transcription factors to the promoter. As in bacteria, bending the DNA loosens H-bonds between bases, facilitating unwinding the double helix near the gene. Bending eukaryotic DNA also brings distal regulatory proteins bound to enhancer sequences far from the promoter together with the proteins bound to more proximal regulatory elements, as shown in the drawing below. Nucleotide methyation sites may facilitate regulatory protein-enhancer binding. When such regulatory proteins, here called activators (i.e., of transcription), bind to their enhancers, they acquire an affinity for protein cofactors that enable recognition and binding to other proteins in the transcription initiation complex. This attraction stabilizes the bend in the DNA that then makes it easier for RNA polymerase II to initiate transcription 223 Assembling a Eukaryotic Transcription Initiation Complex It is worth reminding ourselves that it is shape and allosteric change that allow DNAprotein interactions (in fact, any interactions of macromolecules). The lac repressor we saw earlier is a transcription factor with helix-turn-helix DNA binding motifs. This motif and two others ( zinc finger , and leucine zipper ) characterize DNA binding proteins are illustrated below. DNA-binding motifs in each regulatory protein shown here bind one or more regulatory elements ‘visible’ to the transcription factor in the major groove of the double helix. 224 Transcription Factor Domains/Motifs Bind Specific DNA Sequences We will look next at some common ways in which eukaryotic cells are signaled to turn genes on or off, or to increase or decrease their rates of transcription. As we describe these models, remember that eukaryotic cells regulate gene expression in response to changes in extracellular environments. These can be unscheduled, unpredictable changes in blood or extracellular fluid composition (ions, small metabolites), or dictated by changes in a long-term genetic program of differentiation and development. Changes in gene expression even obey circadian (daily) rhythms, the ticking of a clock. In eukaryotes, changes in gene expression, expected or not, are usually mediated by the timely release of chemical signals from specialized cells (e.g., hormones, cytokines, growth factors, etc.). We will focus on some betterunderstood models of gene regulation by these chemical signals. C. Regulation of Gene Expression by Hormones that enter Cells and Those That Don't Gene-regulatory (cis) elements in DNA and the transcription factors that bind to them have co-evolved. But not only that! Eukaryotic organisms have evolved complete pathways that respond to environmental or programmed developmental cues and lead to an appropriate cellular response. Chemicals that regulate genes in prokaryotes are not usually signals communicated by other cells. In eukaryotes, chemicals released by some cells signal other cells to respond, thus coordinating the activity of the whole organism. Hormones released by cells in endocrine glands are well-understood signal molecules; hormones affect target cells elsewhere in the body. 225 Chemicals That Control Gene Expression 1. How Steroid Hormones Regulate Transcription Steroid hormones cross the cell membranes to have their effects. Common steroid hormones include testosterone, estrogens, progesterone, glucocorticoids and mineral corticoids. Once in target cell, such hormones bind to a steroid hormone receptor protein to form a steroid hormone-receptor complex . The receptor may be in the cytoplasm or in the nucleus, but in the end, the hormone-receptor complex must bind to DNA regulatory elements of a gene to either enhance or silence transcription. Therefore, a steroid hormone must cross the plasma membrane, and may also need to cross the nuclear envelope. Follow the Binding of a steroid hormone to a cytoplasmic receptor below. Here the hormone (the triangle) enters the cell. An allosteric change in the receptor releases a protein subunit called Hsp90 (the black rectangle in the illustration). The remaining hormone-bound receptor enters the nucleus. The fascinating thing about Hsp90 is that it was first discovered in cells subjected to heat stress. When the temperature gets high enough, cells shut down most transcription and instead transcribe Hsp 90 and/or other special heat shock genes. The resulting heat shock proteins seem to protect the cells against metabolic damage until temperatures return to normal. Since most cells never experience such high temperatures, the evolutionary significance of this protective mechanism is unclear. As we now know, heat shock proteins have critical cellular functions, in this case blocking the DNA-binding site of a hormone receptor until a specific steroid hormone binds to it. Back to hormone action! No longer associated with the Hsp90 protein, the receptor bound to its hormone cofactor binds to a cis-acting transcription control element in the DNA, turning transcription of a gene on or off. The hormone receptors for some steroid hormones are already in the nucleus of the cell, so the hormone must cross not only the plasma membrane, but also the nuclear envelope in order to access the receptor. As for steroid hormone functions, we already saw that glucocorticoids turn on the genes of gluconeogenesis. Steroid hormones also control sexual development and reproductive cycling in females, salt and mineral homeostasis in the blood, metamorphosis in arthropods, etc., all by regulating gene expression. 2. How Protein Hormones Regulate Transcription Protein hormones are of course large and soluble, with highly charged surfaces. Other hormones might be relatively small (e.g., adrenalin), but are charged. Large or highly charged signal molecules cannot get across the phospholipid barrier of the plasma membrane. To have any effect at all, they must bind to receptors on the surface of cells. These receptors are typically membrane glycoproteins. The information (signals) carried by protein hormones must be conveyed into the cell indirectly, by a process called signal transduction . There are two well-known pathways of signal transduction, each of which involves activating pathways of protein phosphorylation in cytoplasm. The phosphorylation cascade that results activates a transcription factor that binds to regulatory DNA, turning a gene on or off. Binding of a hormone to a cell surface receptor leads to an allosteric change in the receptor. This in turn activates other proteins either in the plasma membrane or in the cytoplasm, leading to the synthesis of a cytoplasmic second messenger . The second messenger typically binds to a protein kinase in the cytoplasm, launching a series of protein phosphorylations, or a phosphorylation cascade. The last in the series of proteins to be phosphorylated is an activated transcription factor that will bind to a cis-regulatory DNA sequence. cAMP was the first second messenger metabolite to be discovered. It mediates many hormonal responses, controlling both gene activity and enzyme activity. cAMP forms when the hormone-receptor in the membrane binds to and activates a membrane-bound adenylate cyclase enzyme. The cAMP produced then binds to a protein kinase, the first of several in a phosphorylation cascade. Signal transduction mediated by cAMP is summarized in the illustration below. A different kind of signal transduction involves a hormone receptor that is itself the protein kinase. The role of enzyme-linked hormone receptors in signal transduction is summarized below. Binding of the signal protein (e.g. hormone) to the enzyme-linked receptor causes an allosteric change that activates the receptor kinase, starting phosphorylation cascade resulting in an active transcription factor. We look at signal transduction in more detail in another chapter. D. Regulating Eukaryotic Genes Means Contending with Chromatin Consider again the illustration of the different levels of chromatin structure (below). Transcription factors bind specific DNA sequences by detecting them through the grooves (mainly the major groove) in the double helix. The drawing above reminds us however, that unlike the nearly naked DNA of bacteria, eukaryotic (nuclear) DNA is coated with proteins that, in aggregate are by mass, greater than the mass of DNA that they cover. The protein-DNA complex of the genome is of course, chromatin. Again, as a reminder, DNA coated with histone proteins forms the 9 nm diameter beads-on-a-string structure in which the beads are the nucleosomes . The association of specific non-histone proteins causes the nucleosomes to fold over on themselves to form the 30 nm solenoid . As we saw earlier, it is possible to selectively extract chromatin. Take a second look at the results of typical extractions of chromatin from isolated nuclei below. Further accretion of non-histone proteins leads to more folding and the formation of euchromatin and heterochromatin characteristic of non-dividing cells. In dividing cells, the chromatin further condenses to form the chromosomes that separate during either mitosis or meiosis . Recall that biochemical analysis of the 10 nm filament extract revealed that the DNA wraps around histone protein octamers, the nucleosomes or beads in this beads-on-astring structure. Histone proteins are highly conserved in the eukaryotic evolution (they are not found in prokaryotes). They are also very basic (many lysine and arginine residues) and therefore very positively charged. This explains why they are able to arrange themselves uniformly along DNA, binding to the negatively charged phosphodiester backbone of DNA in the double helix. Since the DNA in euchromatin is less tightly packed than it is in heterochromatin, perhaps active genes are to be found in euchromatin and not in heterochromatin. Experiments in which total nuclear chromatin extracts were isolated and treated with the enzyme deoxyribonuclease (DNAse) revealed that the DNA in active genes was degraded more rapidly than non-transcribed DNA. More detail on these experiments can be found in the two links below. 228 Question: Is Euchromatic DNA Transcribed? 229 Experiment and Answer: Euchromatin is Transcribed The results of such experiments are consistent with the suggestion that active genes are more accessible to DNAse because they are in less coiled, or less condensed chromatin. DNA in more condensed chromatin is surrounded by more proteins, and thus is less accessible to, and protected from DNAse attack. When packed up in chromosomes during mitosis or meiosis, all genes are largely inactive. Regulating gene transcription must occur in non-dividing cells or during the interphase of cells, where changing the shape of chromatin ( chromatin remodeling ) in order to silence some and activate other genes is possible. Changing chromatin conformation involves chemical modification of chromatin proteins and DNA. For example, chromatin can be modified by histone acetylation, de-acetylation, methylation and phosphorylation, reactions catalyzed by histone acetyltransferases (HAT enzymes), de-acetylases, methyl transferases and kinases, respectively. For example, acetylation of lysines near the amino end of histones H2B and H4 tends to unwind nucleosomes and open the underlying DNA for transcription. De-acetylation then, promotes condensation of the chromatin in the affected regions of DNA. Likewise, methylation of lysines or arginines (the basic amino acids that characterize histones!) of H3 and H4 can open DNA for transcription, while demethylation has the opposite effect. In one case, di-methylation of a lysine in H3 can suppress transcription. These chemical modifications affect recruitment of other proteins that alter chromatin conformation and ultimately activate or block transcription. This reversible and its effect on chromatin are illustrated below. Nucleosomes themselves can be moved, slid and otherwise repositioned by complexes that hydrolyze ATP for energy to accomplish the physical shifts. Some cancers are associated with mutations in genes for proteins involved in chromatin remodeling. This is no doubt, because failures of normal remodeling could adversely affect normal cell cycling and normal replication. In fact, a single, specific pattern of methylation may mark DNA in multiple cancer types (check out Five Cancers with the Same Genomic Signature - Implications). E. Regulating all the Genes on a Chromosome at Once Recall that X chromosomes in human female somatic cells is inactivated, visible in the nucleus as a Barr body. One of the two X chromosomes in female fruit flies is also inactivates. However, both males and females of Drosophila (presumably also us!) require X chromosome gene expression during embryogenesis. Given the difference in X chromosome gene dosage between males and females, do males get by with fewer X chromosome gene products than females? Experiments looking at the expression of X chromosome gene in male and female flies revealed similar levels of gene products. It turns out that the activity of a nuclear body called HLB ( H istone L ocus B ody) is required for increase in X chromosome gene transcription. A protein, called CLAMP ( C hromatin- L inked A daptor for M ale-specific lethal (MSL) P rotein), was shown to bind to GAGA nucleotide repeats lying between the genes for histones 3 and 4. As there are about 100 repeats of the fivegene histone locus on X chromosomes, and thus about 100 repeats of the GAGA repeats. Therefore, many CLAMP proteins bind to the HLBs, where they recruit many MSL proteins. The MSL protein complexes that form then globally increase male X chromosome gene expression, compensating for the lower X gene dosage in males. Read the original research here (L.E. Reider et al. (2018) Genes & Development 31:1-15). And finally, there is emerging evidence that the HLB action may also be involved in inactivation of an entire female X chromosome later in embryogenesis in females!	libretexts	2025-03-17T22:27:37.333387	2021-01-03T20:12:54	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/18%3A_Transcriptional_Regulation_(eukaryotes)/18.04%3A_Gene_Regulation_in_Eukaryotes", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "18.4: Gene Regulation in Eukaryotes", "author": "Gerald Bergtrom" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/18%3A_Transcriptional_Regulation_(eukaryotes)/18.05%3A__Transgenic_organisms	18.5: Transgenic organisms - - Last updated - Save as PDF General principles of transgenesis Transgenic organisms contain foreign DNA that has been introduced using biotechnology. Foreign DNA (the transgene ) is defined here as DNA from another species, or else recombinant DNA from the same species that has been manipulated in the laboratory then reintroduced. The terms transgenic organism and genetically modified organism ( GMO ) are generally synonymous. The process of creating transgenic organisms or cells to be come whole organisms with a permanent change to their germline has been called either transformation or transfection . (Unfortunately, both words have alternate meanings. Transformation also refers to the process of mammalian cell becoming cancerous, while transfection also refers to the process of introducing DNA into cells in culture, either bacterial or eukaryote, for a temporary use, not germ line changes.) Transgenic organisms are important research tools, and are often used when exploring a gene’s function. Transgenesis is also related to the medical practice of gene therapy, in which DNA is transferred into a patient’s cells to treat disease. Transgenic organisms are widespread in agriculture. Approximately 90% of canola, cotton, corn, soybean, and sugar beets grown in North America are transgenic. No other transgenic livestock or crops (except some squash, papaya, and alfalfa) are currently produced in North America. To make a transgenic cell, DNA must first be transferred across the cell membrane, (and, if present, across the cell wall), without destroying the cell. In some cases, naked DNA (meaning plasmid or linear DNA that is not bound to any type of carrier ) may be transferred into the cell by adding DNA to the medium and temporarily increasing the porosity of the membrane, for example by electroporation . When working with larger cells, naked DNA can also be microinjected into a cell using a specialized needle. Other methods use vectors to transport DNA across the membrane. Note that the word “vector” as used here refers to any type of carrier, and not just plasmid vectors. Vectors for transformation/transfection include vesicles made of lipids or other polymers that surround DNA; various types of particles that carry DNA on their surface; and infectious viruses and bacteria that naturally transfer their own DNA into a host cell, but which have been engineered to transfer any DNA molecule of interest. Usually the foreign DNA is a complete expression unit that includes its own cis-regulators (e.g. promoter) as well as the gene that is to be transcribed. When the objective of an experiment is to produce a stable (i.e. heritable) transgenic eukaryote, the foreign DNA must be incorporated into the host’s chromosomes. For this to occur, the foreign DNA must enter the host’s nucleus, and recombine with one of the host’s chromatids. In some species, the foreign DNA is inserted at a random location in a chromatid, probably wherever strand breakage and non-homologous end joining happen to occur. In other species, the foreign DNA can be targeted to a particular locus, by flanking the foreign DNA with DNA that is homologous to the host’s DNA at that locus. The foreign DNA is then incorporated into the host’s chromosomes through homologous recombination. Furthermore, to produce multicellular organisms in which all cells are transgenic and the transgene is stably inherited, the cell that was originally transformed must be either a gamete or must develop into tissues that produce gametes. Transgenic gametes can eventually be mated to produce homozygous, transgenic offspring. The presence of the transgene in the offspring is typically confirmed using PCR or Southern blotting, and the expression of the transgene can be measured using reverse-transcription PCR (RT-PCR), RNA blotting, and Western (protein blotting). The rate of transcription of a transgene is highly dependent on the state of the chromatin into which it is inserted (i.e. position effects ), as well as other factors. Therefore, researchers often generate several independently transformed/transfected lines with the same transgene, and then screen for the lines with the highest expression. It is also good practice to clone and sequence the transgenic locus from a newly generated transgenic organism, since errors (truncations, rearrangements, and other mutations) can be introduced during transformation/transfection. Producing a transgenic plant The most common method for producing transgenic plants is Agrobacterium-mediated transformation (Figure $\PageIndex{1}$). Agrobacterium tumifaciens is a soil bacterium that, as part of its natural pathogenesis, injects its own tumor-inducing ( T i ) plasmid into cells of a host plant. The natural T i plasmid encodes growth-promoting genes that cause a gall (i.e. tumor) to form on the plant, which also provides an environment for the pathogen to proliferate. Molecular biologists have engineered the T i plasmid by removing the tumor-inducing genes and adding restriction sites that make it convenient to insert any DNA of interest. This engineered version is called a T-DNA (transfer-DNA) plasmid; the bacterium transfers a linear fragment of this plasmid that includes the conserved “left-border (LB)”, and right-border (RB)” DNA sequences, and anything in between them (up to about 10 kb). The linear T-DNA fragment is transported into the nucleus, where it recombines with the host-DNA, probably wherever random breakages occur in the host’s chromosomes. In Arabidopsis and a few other species, flowers can simply be dipped in a suspension of Agrobacterium, and ~1% of the resulting seeds will be transformed. In most other plant species, cells are induced by hormones to form a mass of undifferentiated tissues called a callus. The Agrobacterium is applied to a callus and a few cells are transformed, which can then be induced by other hormones to regenerate whole plants (Figure $\PageIndex{2}$). Some plant species are resistant (i.e. “ recalcitrant ”) to transformation by Agrobacterium. In these situations, other techniques must be used such as particle bombardment , whereby DNA is non-covalently attached to small metallic particles, which are accelerated by compressed air into callus tissue, from which complete transgenic plants can sometimes be regenerated. In all transformation methods, the presence of a selectable marker (e.g. a gene that confers antibiotic resistance or herbicide resistance) is useful for distinguishing transgenic cells from non-transgenic cells at an early stage of the transformation process. Producing a transgenic mouse In a commonly used method for producing a transgenic mouse, stem cells are removed from a mouse embryo, and a transgenic DNA construct is transferred into the stem cells using electroporation, and some of this transgenic DNA enters the nucleus, where it may undergo homologous recombination (Figure $\PageIndex{3}$). The transgenic DNA construct contains DNA homologous to either side of a locus that is to be targeted for replacement. If the objective of the experiment is simply to delete (“ knock-out ”) the targeted locus, the host’s DNA can simply be replaced by selectable marker, as shown. It is also possible to replace the host’s DNA at this locus with a different version of the same gene, or a completely different gene, depending on how the transgenic construct is made. Cells that have been transfected and express the selectable marker (i.e. resistance to the antibiotic neomycin resistance, neoR, in this example) are distinguished from unsuccessfully transfected cells by their ability to survive in the presence of the selective agent (e.g. an antibiotic). Transfected cells are then injected into early stage embryos, and then are transferred to a foster mother. The resulting pups are chimeras, meaning that only some of their cells are transgenic. Some of the chimeras will produce gametes that are transgenic, which when mated with a wild-type gamete, will produce mice that are hemizygous for the transgene. Unlike the chimeras, these hemizygotes carry the transgene in all of their cells. Through further breeding, mice that are homozygous for the transgene can be obtained. Human gene therapy Many different strategies for human gene therapy are under development. In theory, either the germline or somatic cells may be targeted for transfection, but most research has focused on somatic cell transfection, because of risks and ethical issues associated with germline transformation. Gene therapy approaches may be further classified as either ex vivo or in vivo , with the former meaning that cells (e.g. stem cells) are transfected in isolation before being introduced to the body, where they replace defective cells. Ex vivo gene therapies for several blood disorders (e.g. immunodeficiencies, thalassemias) are undergoing clinical trials. For in vivo therapies, the transfection occurs within the patient. The objective may be either stable integration, or non-integrative transfection. As described above, stable transfection involves integration into the host genome. In the clinical context, stable integration may not be necessary, and carries with it higher risk of inducing mutations in either the transgene or host genome). In contrast, transient transfection does not involve integration into the host genome and the transgene may therefore be delivered to the cell as either RNA or DNA. Advantages of RNA delivery include that no promoter is needed to drive expression of the transgene. Besides mRNA transgenes, which could provide a functional version of a mutant protein, there is great interest in delivery of siRNA (small-inhibitory RNAs), which can be used to silence specific genes in the host cell’s genome. Vectors for in vivo gene therapy must be capable of delivering DNA or RNA to a large proportion of the targeted cells, without inducing a significant immune response, or having any toxic effects. Ideally, the vectors should also have high specificity for the targeted cell type. Vectors based on viruses (e.g. lentiviruses ) are being developed for in both in vivo and ex vivo gene therapies. Other, non-viral vectors (e.g. vesicles and nanoparticles) are also being developed for gene therapy as well.	libretexts	2025-03-17T22:27:37.401681	2021-01-03T20:12:55	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/18%3A_Transcriptional_Regulation_(eukaryotes)/18.05%3A__Transgenic_organisms", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "18.5: Transgenic organisms", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/19%3A_Small_RNAs/19.01%3A_Post-transcriptional_Control_of_Gene_Expression	19.1: Post-transcriptional Control of Gene Expression - - Last updated - Save as PDF Not too long ago we thought that very little of the eukaryotic genome was ever transcribed. We also thought that the only non-coding RNAs were tRNAs and rRNAs. Now we know that other RNAs play roles in gene regulation and the degradation of spent cellular DNA or unwanted foreign DNA. These are discussed in detail below. A. Ribosomes The riboswitches is a bacterial transcription mechanism for regulating gene expression. While this mechanism is not specifically post-transcriptional, it is included here because the action occurs after transcription initiation and aborts completion of an mRNA. When the mRNA for an enzyme in the guanine synthesis pathway is transcribed, it folds into stem-&-loop structures. Enzyme synthesis will continue for as long as the cell needs to make guanine. But if guanine accumulates in the cell, excess guanine will bind stem-loop elements near the 5’ end of the mRNA, causing the RNA polymerase and the partially completed mRNA dissociate from the DNA, prematurely ending transcription. The basis of guanine riboswitch regulation of expression of a guanine synthesis pathway enzyme is shown below. 232 Riboswitches Interrupt Bacterial Transcription The ability to form folded, stem-loop structures at the 5’ ends of bacterial mRNAs seems to have allowed the evolution of translation regulation strategies. Whereas guanine interaction with the stem-loop structure of an emerging 5’ mRNA can abort its own transcription, similar small metabolite/mRNA and even protein/mRNA interactions can also regulate (in this case prevent) translation. As we will see shortly, 5’ mRNA folded structures also play a role in eukaryotic translation regulation. 233 Small Metabolites Also Regulate Bacterial mRNA translation B. CRISPR/Cas: RNA-Protein Complex of a Prokaryotic Adaptive Immune System In higher organisms, the immune system is adaptive. It remembers prior exposure to a pathogen, and can thus mount a response to a second exposure to the same pathogen. The discovery of an ‘ adaptive immune system ’ in many prokaryotes (bacteria, archaebacteria) was therefore something of a surprise. CRISPR ( C lustered R egularly I nterspaced S hort P alindromic R epeat) RNAs are derived from phage transcripts that have interacted with CRISPR-Associated (Cas) proteins. They make up the CRISPR/ Cas system that seems to have evolved to fight of viral infection by targeting phage DNA for destruction. When viral DNA gets into a cell during a phage infection, it can generate a CRISPR/Cas gene array in the bacterial genome, with spacer DNA sequences separating repeats of the CRISPR genes. These remnants of a phage infection are the memory of this p rokaryotic immune system . When a phage attempts to re-infect a previously exposed cell, spacer RNAs and Cas genes are transcribed. After Cas mRNA translation, the Cas protein and spacer RNAs will engage and target the incoming phage DNA for destruction to prevent infection. Thus, the CRISPR/Cas systems (there is more than one!) remember prior phage attacks, and transmit that memory to progeny cells. The CRISPR/Cas9 system in Streptococcus pyogenes is one of the simplest of these immune defense systems (illustrated below). The CRISPR/Cas gene array consists of the following components: - Cas: Genes native to host cells - CRISPR: 24-48 bp repeats native to host cells - Spacer DNA: DNA between CRISPR repeats: typically, phage DNA from prior phage infection or plasmid transformation - leader DNA: Contains promoter for CRISPR/spacer RNA transcription - tracr gene: Encodes transcription activator (tracr) RNA (not all systems) Let's look at CRISPR/Cas in action. 1. The CRISPR/Cas Immune Response Consider the mechanism of action of this prokaryotic immune system. The action begins when infectious phage DNA gets into the cell, as drawn below. Let’s summarize what has happened here: a) Incoming phage DNA was detected after phage infection. b) Then the tracr and Cas genes are transcribed along with the CRISPR/spacer region. Cas mRNAs are translated to make the Cas protein. Remember, the spacer DNAs in the CRISPR region are the legacy of a prior phage infection. c) CRISPR/spacer RNA forms hydrogen bonds with a complementary region of the tracr RNA as the two RNAs associate with Cas proteins. d) Cas protein endonucelases hydrolyze spacer RNA from CRISPR RNA sequences. The spacer RNAs remain associated with the complex while the actual, imperfectly palindromic CRISPR sequences (shown in blue in the illustration above) fall off. I n the next steps, phage-derived spacer RNAs, now called guide RNAs (or gRNAs ) ‘guide’ mature Cas9/tracrRNA/spacer RNA complexes to new incoming phage DNA resulting from a phage attack. The association of the complex with the incoming phage DNA and subsequent events are illustrated below. Once again, let’s summarize: a) Spacer (i.e., gRNA) in the complex targets incoming phage DNA. b) Cas helicase unwinds incoming phage DNA at complementary regions. c) gRNA H-bonds to incoming phage DNA. d) Cas endonucleases create a double-stranded break (hydrolytic cleavage) at specific sites in incoming phage DNA. Because precise site DNA strand cleavage is guided by RNA molecules, CRISPR/Cas endonucleases are classified as type V restriction enzymes. e) The incoming phage DNA is destroyed and a new phage infection is aborted. Check out here to learn more about how bacteria acquire spacer DNAs, and therefore how this primitive adaptive immune system ‘remembers’) in the first place 2. Using CRISPR/Cas to Edit/Engineer Genes Early studies demonstrated the reproducible cleavage of incoming phage DNA at specific nucleotides. Several labs quickly realized that it might be possible to adapt the system to cut DNA at virtually any specific nucleotide in a target DNA! It has turned out that the system works both in vivo and in vitro, allowing virtually unlimited potential for editing genes and RNAs in a test tube… or in any cell. Here is the basic process: a) Engineer gDNA with a Cas-specific DNA sequence that targets a desired target in genomic DNA. b) Fuse the gDNA to tracr DNA to make a single guide DNA (sgDNA) so that it can be made as a single guide transcript (sgRNA). c) Engineer a CRISPR/Cas9 gene array that substitutes this sgDNA for its original spacer DNAs. d) Place engineered array in a plasmid next to regulated promoters. e) Transform cells by ‘electroporation’ (works for almost any cell type!) f) Activate the promoter to transcribe the CRISPR/Cas9 genes… The applications are powerful… and controversial! 3. The Power and the Controversy The application of gene editing with CRISPR/Cas systems has already facilitated studies of gene function in vitro, in cells and in whole organisms. Click here for a description of CRISPR/Cas applications already on the market! The efficiency of specific gene editing using CRISPR/Cas systems holds great promise for understanding basic gene structure and function, for determining the genetic basis of disease, and for accelerating the search for gene therapies. Here are just a few examples of how CRISPR/Cas approaches are being applied. - One can engineer an sgRNA with desired mutations targeting specific sites in chromosomal DNA. Then clone sgRNA into the CRISPR/Cas9 array on a plasmid. After transformation of appropriate cells, the engineered CRISPR/Cas9 forms a complex with target DNA sequences. Following nicking of both strands of the target DNA, DNA repair can insert the mutated guide sequences into the target DNA. The result is loss or acquisition of DNA sequences at specific, exact sites , or Precision Gene Editing . It is the ability to do this in living cells that has excited the basic and clinical research communities. - Before transforming cells, engineer the CRISPR/Cas9 gene array on the plasmid to eliminate both endonuclease activities from the Cas protein. Upon transcription of the array in transformed cells, the CRISPR/Cas9-sgRNA still finds an sgRNA -targeted gene. However, lacking CAS protein endonuclease activities, the complex that forms just sits there blocking transcription . This technique is sometimes referred to as CRISPRi ( CRISPER interference ), by analogy to RNAi . Applied to organisms (and not just in vitro or to cells), it mimics the much more difficult knockout mutation experiments that have been used in studies of behavior of cells or organisms rendered unable to express a specific protein. - There are now several working CRISPR/Cas systems capable of Precision Gene Editing . They are exciting for their speed, precision, their prospects for rapid, targeted gene therapies to fight disease, and their possibilities to alter entire populations (called Gene Drive ). By inserting modified genes into the germline cells of target organisms, gene drive can render harmless entire malarial mosquito populations, to eliminate pesticide resistance in e.g. insects, eliminate herbicide resistance in undesirable plants, or genetically eliminate invasive species. For more information, click Gene drive; for an easy read about this process and the controversies surrounding applications of CRISPR technologies to mosquitoes in particular, check out J. Adler, (2016) A World Without Mosquitoes . Smithsonian, 47(3) 36-42, 84. - It is even possible to delete an entire chromosome from cells. This bit of global genetic engineering relies on identifying multiple unique sequences on a single chromosome and then targeting these sites for CRISPR/Cas. When the system is activated, the chromosome is cut at those sites, fragmenting it beyond the capacity of DNA repair mechanisms to fix the situation. Click here to learn more. If for no other reason than its efficiency and simplicity, precision gene editing with CRISPR/Cas techniques has raised ethical issues. Clearly, the potential exists for abuse, or even for use with no beneficial purpose at all. It is significant that, as in all discussions of biological ethics, scientists are very much engaged in the conversation. Despite the controversy, we will no doubt continue to edit genes with CRISPR/Cas, and we can look for a near future Nobel Prize for its discovery and application! If you still have qualms, maybe RNA editing will be the answer. Check out the link at Why edit RNA? for an overview of the possibilities! Finally, “mice and men” (and women and babies too) have antibodies to Cas9 proteins, suggesting prior exposure to microbial CRISPR/Cas9 antigens. This observation may limit clinical applications of the technology! See Uncertain Future of CRISPR-Cas9 Technology. C. The Small RNAs: miRNA and siRNA in Eukaryotes Micro RNAs (miRNAs) and small interfering RNAs (siRNAs) are found in C. elegans , a small nematode (roundworm) that quickly became a model for studies of cell and molecular biology and development. The particular attractions C. elegans are that (a) its genome has ~21,700 genes, comparable to the ~25,000 genes in a human genome!; (b) it uses the products of these genes to produce an adult worm consisting of just 1031 cells organized into all of the major organs found in higher organisms; (c) It is possible to trace the embryonic origins of every single cell in its body! C. elegans is illustrated below. 1. Small Interfering RNA (siRNA) siRNA was first found in plants as well as in C. elegans . However, siRNAs (and miRNAs) are common in many higher organisms. siRNAs were so-named because they interfere with the function of other RNAs foreign to the cell or organism. Their action was dubbed RNA interference ( RNAi ). For their discovery of siRNAs, A. Z. Fire and C. C. Mello shared the 2006 Nobel Prize in Physiology or Medicine. The action of siRNA targeting foreign DNA is illustrated below. When cells recognize foreign double-stranded RNAs (e.g., some viral RNA genomes) as alien, the DICER a nuclease called hydrolyzes them. The resulting short double-stranded hydrolysis products (the siRNAs ) combine with R NAi I nduced S ilencing C omplex, or RISC proteins. The antisense siRNA strand in the resulting siRNA-RISC complex binds to complementary regions of foreign RNAs, targeting them for degradation. Cellular use of RISC to control gene expression in this way may have derived from the use of RISC proteins by miRNAs as part of a cellular defense mechanism, to be discussed next. Custom-designed siRNAs have been used to disable expression of specific genes in order to study their function in vivo and in vitro . Both siRNAs and miRNAs are being investigated as possible therapeutic tools to interfere with RNAs whose expression leads to cancer or other diseases. 234 siRNA Post Transcriptional Regulation 235 Did siRNA Coopt RISC strategy to Trash Corrupt or Worn out RNA? For an example check out a Youtube video of unexpected results of an RNAi experiment at this link . In the experiment described, RNAi was used to block embryonic expression of the orthodenticle ( odt ) gene that is normally required for the growth of horns in a dung beetle. The effect of this knock-out mutation was, as expected, to prevent horn growth. What was unexpected however, was the development of an eye in the middle of the beetle’s head (‘third eye’ in the micrograph). The 3rd eye not only looks like an eye, but is a functional one. This was demonstrated by preventing normal eye development in odt -knockout mutants. The 3rd eye appeared…, and was responsive to light! Keep in mind that this was a beetle with a 3rd eye, not Drosophila ! To quote Justin Kumar from Indiana University, who though not involved in the research, stated that “…lessons learned from Drosophila may not be as generally applicable as I or other Drosophilists, would like to believe … The ability to use RNAi in non-traditional model systems is a huge advance that will probably lead to a more balanced view of development.” 2. Micro RNAs (miRNA) miRNAs target unwanted endogenous cellular RNAs for degradation. They are transcribed from genes now known to be widely distributed in eukaryotes. The pathway from pre-miRNA transcription through processing and target mRNA degradation is illustrated on the next page. As they are transcribed, pre-miRNAs fold into a stem-loop structure that is lost during cytoplasmic processing. Like SiRNAs, mature miRNAs combine with RISC proteins. The RISC protein-miRNA complex targets old or no-longer needed mRNAs or mRNAs damaged during transcription. An estimated 250 miRNAs in humans may be sufficient to H-bond to diverse target RNAs; only targets with strong complementarity to a RISC protein-miRNA complex will be degraded. D. Long Non-Coding RNAs Long non-coding RNAs ( lncRNAs ) are a yet another class of eukaryotic RNAs. They include transcripts of antisense, intronic, intergenic, pseudogene and retroposon DNA. Retroposons are one kind of transposon, or mobile DNA element; pseudogenes are recognizable genes with mutations that make them non-functional. While some lncRNAs might turn out to be incidental transcripts that the cell simply destroys, others have a role in regulating gene expression. A recently discovered lncRNA is XistAR that, along with the Xist gene product, is required to form Barr bodies . Barr bodies form in human females when one of the X chromosomes in somatic cells is inactivated. For a review of lncRNAs, see Lee, J.T. (2012. Epigenetic Regulation by Long Noncoding RNAs ; Science 338, 1435-1439). An even more recent article (at lncRNAs and smORFs) summarizes the discovery that some long non-coding RNAs contain short open reading frames ( smORFs ) that are actually translated into short peptides of 30+ amino acids! Who knows? The human genome may indeed contain more than 21,000-25,000 protein-coding genes! E. Circular RNAs (circRNA) Though discovered more than 20 years ago, circular RNAs (circRNAs) are made in different eukaryotic cell types. Click Circular RNAs (circRNA) to learn more about this peculiar result of alternative splicing. At first circRNAs were hard to isolate. When they were isolated, circRNAs contained “scrambled” exonic sequences and were therefore thought to be nonfunctional errors of mRNA splicing. In fact, circRNAs are fairly stable. Their levels can rise and fall in patterns suggesting that they are functional molecules. Levels of one circRNA, called circRims1 , rise specifically during neural development. In mice, other circRNAs accumulate during synapse formation, likely influencing how these neurons will ultimately develop and function. Thus, circRNAs do not seem to be ‘molecular mistakes’. In fact, errors in their own synthesis may be correlated with disease! Speculation on the functions of circRNAs also includes roles in gene regulation, particularly the genes or mRNAs from which they themselves are derived. F. "Junk DNA" in Perspective Not long ago, we thought that less than 5% of a eukaryotic genome was transcribed (i.e., into mRNA, rRNA and tRNA), and that much of the non-transcribed genome served a structural function… or no function at all. The latter, labeled junk DNA, included non-descript intergenic sequences, pseudogenes, ‘dead’ transposons, long stretches of intronic DNA, etc. Thus, junk DNA was DNA we could do without. Junk DNAs were thought to be accidental riders in our genomes, hitchhikers picked up on the evolutionary road. While miRNA genes are a small proportion of a eukaryotic genome, their discovery, and that of more abundant lnc RNAs suggest a far greater amount of functional DNA in the genome. Might there be in fact, no such thing as “junk DNA”? The debate about how much of our genomic DNA is a relic of past evolutionary experiments and without genetic purpose continues. Read all about it at Junk DNA - not so useless after all and Only 8.2% of human DNA is functional. Perhaps we need to re-think what it means for DNA to be “junk” or to be without “genetic purpose”. Maintenance of more than 90% of our own DNA with no known genetic purpose surely comes at an energy cost. At the same time, all of that DNA is grist for future selection, a source of the diversity required for long-term survival. The same natural selection that picks up ‘hitchhiker’ DNA sequences, as we have seen, can at some point, put them to work! G. The RNA Methylome Call this an RNA epi-transcriptome if you like! Recall that methyl groups direct cleavage of ribosomal RNAs from eukaryotic 45S pre-RNA transcripts. tRNAs among other transcripts, are also post-transcriptionally modified. Known since the 1970s, such modifications were thought to be non-functional. But are they?	libretexts	2025-03-17T22:27:37.513850	2021-01-03T20:12:57	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/19%3A_Small_RNAs/19.01%3A_Post-transcriptional_Control_of_Gene_Expression", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "19.1: Post-transcriptional Control of Gene Expression", "author": "Gerald Bergtrom" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/20%3A_The_Genetic_Code/20.01%3A_The_Function_of_Genes	20.1: The Function of Genes - - Last updated - Save as PDF Beadle and Tatum: one gene, one enzyme hypothesis Life depends on (bio)chemistry to supply energy and to produce the molecules to construct and regulate cells. In 1908, A. Garrod described “in born errors of metabolism” in humans using the congenital disorder, alkaptonuria (black urine disease), as an example of how “genetic defects” led to the lack of an enzyme in a biochemical pathway and caused a disease (phenotype). Over 40 years later, in 1941, Beadle and Tatum built on this connection between genes and metabolic pathways. Their research led to the “ one gene, one enzyme (or protein) ” hypothesis, which states that each of the enzymes that act in a biochemical pathway is encoded by a different gene. Although we now know of many exceptions to the “one gene, one enzyme (or protein)” principle, it is generally true that each different gene produces a protein that has a distinct catalytic, regulatory, or structural function. Beadle and Tatum used the fungus Neurospora crassa (a mold) for their studies because it had practical advantages as a laboratory organism. They knew that Neurospora was prototrophic , meaning that it could synthesize its own amino acids when grown on minimal medium , which lacked most nutrients except for a few minerals, simple sugars, and one vitamin (biotin). They also knew that by exposing Neurospora spores to X-rays, they could randomly damage its DNA to create mutations in genes. Each different spore exposed to X-rays potentially contained a mutation in a different gene. After genetically screening many, many spores for growth, most appeared to still be prototrophic and still able to grow on minimal medium. However, some spores had mutations that changed them into auxotrophic strains that could no longer grow on minimal medium, but did grow on complete medium supplemented with nutrients (Figure $\PageIndex{12}$). In fact, some auxotrophic mutations could grow on minimal medium with only one, single nutrient supplied, such as arginine. B&T’s 1 gene: 1 enzyme hypothesis led to Biochemical Pathway dissection using genetic screens and mutations Beadle and Tatum’s experiments are important not only for its conceptual advances in understanding genes, but also because they demonstrate the utility of screening for genetic mutants to investigate a biological process – genetic analysis . Beadle and Tatum’s results were useful to investigate biological processes, specifically the metabolic pathways that produce amino acids. For example, Srb and Horowitz in 1944 tested the ability of the amino acids to rescue auxotrophic strains. They added one of each of the amino acids to minimal medium and recorded which of these restored growth to independent mutants. For example, if the progeny of a mutagenized spore could grow on minimal medium only when it was supplemented with arginine (Arg), then the auxotroph must bear a mutation in the Arg biosynthetic pathway and was called an “arginineless” strain (arg-). Synthesis of even a relatively simple molecule such as arginine requires many steps, each with a different enzyme. Each enzyme works sequentially on a different intermediate in the pathway (Figure $\PageIndex{13}$). For arginine (Arg), two of the intermediates are ornithine (Orn) and citrulline (Cit). Thus, mutation of any one of the enzymes in this pathway could turn Neurospora into an Arg auxotroph (arg-). Srb and Horowitz extended their analysis of Arg auxotrophs by testing the intermediates of amino acid biosynthesis for the ability to restore growth of the mutants (Figure $\PageIndex{14}$). They found that some of the Arg auxotrophs could be rescued only by Arg, while others could be rescued by either Arg or Cit, and still other mutants could be rescued by Arg, Cit, or Orn (Table $\PageIndex{1}$). Based on these results, they deduced the location of each mutation in the Arg biochemical pathway, (i.e. which gene was responsible for the metabolism of which intermediate). \| \| MM + Orn \| MM + Cit \| MM + Arg \| \|---\|---\|---\|---\| \| gene A mutants \| Yes \| Yes \| Yes \| \| gene B mutants \| No \| Yes \| Yes \| \| gene C mutants \| No \| No \| Yes \|	libretexts	2025-03-17T22:27:37.609085	2021-01-03T20:12:59	{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/20%3A_The_Genetic_Code/20.01%3A_The_Function_of_Genes", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "20.1: The Function of Genes", "author": "Todd Nickle and Isabelle Barrette-Ng" }
https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/20%3A_The_Genetic_Code/20.02%3A_An_Overview_of_the_Genetic_Code	20.2: An Overview of the Genetic Code - - Last updated - Save as PDF A. The (Nearly) Universal, Degenerate Genetic Code The genetic code is the information for linking amino acids into polypeptides in an order based on the base sequence of 3-base code words ( codons ) in a gene and its messenger RNA (mRNA). With a few exceptions (some prokaryotes, mitochondria, chloroplasts), the genetic code is universal – it’s the same in all organisms from viruses and bacteria to humans. The table of the Standard Universal Genetic Code on the next page shows the RNA version of triplet codons and their corresponding amino acids. There is a single codon for two amino acids (methionine and tryptophan), but two or more codons for each of the other 18 amino acids. For the latter reason, we say that the genetic code is degenerate . The three stop codons in the Standard Genetic Code ‘tell’ ribosomes the location of the last amino acid to add to a polypeptide. The last amino acid itself can be any amino acid consistent with the function of the polypeptide being synthesized. However, evolution has selected AUG as the start codon for all polypeptides, regardless of function, as well as for the placement of methionine within a polypeptide. Thus, all polypeptides begin life with a methionine at their amino-terminal end. As we will see in more detail, the mRNA translation machine is the ribosome and the decoding device is tRNA. Each amino acid attaches to a tRNA whose short sequence contains a 3-base anticodon that is complementary to an mRNA codon. Enzymatic reactions catalyze the dehydration synthesis ( condensation ) reactions that link amino acids in peptide bonds in the order specified by codons in the mRNA. B. Comments on the Nature and Evolution of Genetic Information The near-universality of the genetic code from bacteria to humans implies that the code originated early in evolution. It is probable that portions of the code were in place even before life began. Once in place however, the genetic code was highly constrained against evolutionary change. The degeneracy of the genetic code enabled and contributed to this constraint by permitting base many base changes that do not affect the amino acid encoded in a codon. The near universality of the genetic code and its resistance to change are features of our genomes that allow us to compare gene and other DNA sequences to establish evolutionary relationships between organisms (species), groups of organisms (genus, family, order, etc.) and even individuals within a species. In addition to constraints imposed by a universal genetic code, some organisms show codon bias , a recent constraint on which universal codons an organism uses. Codon bias is seen in organisms preferably use A-T rich codons, or in organisms that favor codons richer in G and C. Interestingly, codon bias in genes often accompanies corresponding genomic nucleotide bias . An organism with an AT codon bias may also have an AT-rich genome (likewise GC-rich codons in GC-rich genomes). You can recognize genome nucleotide bias in Chargaff’s base ratios! Finally, we often think of genetic information as genes for proteins. Obvious examples of non-coding genetic information include the genes for rRNAs and tRNAs, common to all organisms. The amount of these kinds of informational DNA (i.e., genes for polypeptides, tRNAs and rRNAs) as a proportion of total DNA can range across species, although it is higher in eukaryotes prokaryotes. For example, ~88% of the E. coli circular chromosome encodes polypeptides, while that figure is less ~1.5% for humans. Some less obvious informative DNA sequences in higher organisms are transcribed (e.g., introns). Other informative DNA in the genome is never transcribed. The latter include regulatory DNA sequences, DNA sequences that support chromosome structure and other DNAs that contribute to development and phenotype. As for that amount of truly non-informative (useless) DNA in a eukaryotic genome, that amount is steadily shrinking as we sequence entire genomes, identify novel DNA sequences and discover novel RNAs (topics covered elsewhere in this text).	libretexts	2025-03-17T22:27:37.666708	2021-01-03T20:13:01	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://bio.libretexts.org/Courses/Ohio_State_University/Ohio_State_University_SP22%3A_Molecular_Genetics_4606_(Chamberlin)/20%3A_The_Genetic_Code/20.02%3A_An_Overview_of_the_Genetic_Code", "book_url": "https://commons.libretexts.org/book/bio-42776", "title": "20.2: An Overview of the Genetic Code", "author": "Gerald Bergtrom" }

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