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The current data clearly show that H4-12 3′-UTR binds the SLBP and undergoes dynamic structural rearrangements that favour U7 snRNA hybridization. Such reaction is reminiscent of U6 snRNA which displays a very compact structure in its naked form, whereas in presence of Prp24p and Lsm proteins the RNA structure is much more opened, more accessible to V1 cuts, which would facilitate pairing with U4 RNA ( 31 ). A role of RNA chaperones was proposed for Prp24p and Lsm proteins, acting in order to open the RNA structure and stabilize the active U6 snRNA structure, which would not be sufficiently stable by its own. Binding of the SLBP on H4-12 and H1t 3′-UTR induces comparable effects as Prp24p and Lsm proteins on the U6 snRNA. The histone RNA structures become more accessible to the lead, RNases, U7 snRNA and also water molecules. Indeed, spontaneous hydrolysis of the 3′-UTR at the cleavage site was stimulated after SLBP binding, suggesting that the cleavage process mediated by CPSF-73 might be water-mediated.
|
16982637_p22
|
16982637
|
DISCUSSION
| 4.455654 |
biomedical
|
Study
|
[
0.9992334842681885,
0.000480001064715907,
0.00028651198954321444
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[
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0.00042340901563875377,
0.000148470324347727
] |
en
| 0.999995 |
An interesting consequence of the SLBP role in promoting U7 binding is that 3′ end processing reaction of histone pre-mRNAs is an ordered process. The SLBP binds first, facilitates anchoring of U7 snRNP and finally the whole complex is locked by the ZFP100, which bridges the SLBP-hairpin complex to the U7 snRNP. This model implies that SLBP-binding might initiate and control pre-mRNAs processing. In the absence of the SLBP, the processing machinery would be unable to promote processing and to accumulate processing intermediate complexes (histone pre-mRNAs/U7snRNP/ZFP100). To avoid intermediate-products accumulation, it makes sense to think that SLBP is the first trans -acting factor to bind newly synthesized histone pre-mRNAs. The fact that the HDE of histone precursor mRNAs is ‘hidden’ in the RNA secondary structure from U7 snRNA anchoring might be looked at as a safety mechanism to prevent processing and therefore histone biosynthesis in non-S phase especially at the G1-S transition phase when histone transcription is increasing and SLBP is not yet available ( 32 , 33 ). SLBP is the cornerstone of histone expression, its binding on histone pre-mRNAs ‘gives a go’ to the whole process.
|
16982637_p23
|
16982637
|
DISCUSSION
| 4.636237 |
biomedical
|
Study
|
[
0.9992269277572632,
0.0004255026578903198,
0.00034753032377921045
] |
[
0.9967032074928284,
0.0016097866464406252,
0.0014539036201313138,
0.00023303246416617185
] |
en
| 0.999996 |
To conclude, the SLBP is necessary for 3′ end processing of histone pre-mRNAs. In addition, the SLBP is essential for cell-cycle regulation of histone expression. The SLBP is one of the three actually known cell cycle regulated factors that are involved in the 3′ end processing reaction of the histone pre-mRNAs ( 32 , 33 ). Together with Symplekin and CstF-64, two components of the cleavage/polyadenylation machinery ( 11 , 34 , 35 ), the SLBP regulates histone expression at the 3′-end processing level.
|
16982637_p24
|
16982637
|
DISCUSSION
| 4.288258 |
biomedical
|
Study
|
[
0.9995285272598267,
0.00019213405903428793,
0.0002794086467474699
] |
[
0.9970310926437378,
0.001492549548856914,
0.0013732397928833961,
0.00010306185140507296
] |
en
| 0.999997 |
Retrotransposition is a wide spread phenomenon occurring in eukaryotic genomes of diverse taxonomic groups. It is believed to be responsible for various important events in the genome, such as gene inactivation, transduction of genomic sequences, regulation of gene expression and genome expansion ( 1 ). It has also been implicated in human genetic diseases ( 2 ). The insertion sites of many non-long terminal repeat (LTR) retrotransposons, including human L1 are distributed throughout the genome. How these sites are selected for element insertion is not clear. An appreciation of the major factors that determine the preferred location of a retrotransposon in a genome will give us a tool to understand, predict and possibly manipulate the course of genomic evolution due to transposition events.
|
17040894_p0
|
17040894
|
INTRODUCTION
| 4.117502 |
biomedical
|
Study
|
[
0.9992851614952087,
0.0002830442099366337,
0.0004318548017181456
] |
[
0.575617790222168,
0.017858074977993965,
0.40586233139038086,
0.0006617766921408474
] |
en
| 0.999996 |
Entamoeba histolytica , a primitive eukaryote, is the third leading cause of morbidity and mortality due to parasitic disease in humans, and is estimated to be responsible for between 50 000 and 100 000 deaths every year ( 3 ). It is home to the non-LTR retrotransposons EhLINEs and EhSINEs. These together account for about 6–8% of the genome, where they are distributed in the intergenic regions ( 4 ). Being located close to protein-coding genes, they may be capable of influencing the expression of genes in their vicinity, as reported for amoebapore, a virulence factor ( 5 ). The nonpathogenic sibling species Enatmoeba dispar also contains its own set of EdLINEs/EdSINEs. However the sites occupied by these elements in their respective genomes are distinct. It is possible that the evolution of pathogenesis could be linked to diversification of transposable elements in the common ancestor of the two species.
|
17040894_p1
|
17040894
|
INTRODUCTION
| 4.347541 |
biomedical
|
Study
|
[
0.9995245933532715,
0.0002325664390809834,
0.0002428738953312859
] |
[
0.9986478686332703,
0.00040460628224536777,
0.0008663792978040874,
0.00008110652561299503
] |
en
| 0.999997 |
Target primed reverse transcription (TPRT) is thought to be the mechanism by which non-LTR retrotransposons insert in the genome ( 6 ). Since retrotransposition is initiated by the element-encoded endonuclease (EN) making a nick at the bottom strand of the site of insertion, an important determinant of target site specificity could be the preferred nucleotide sequences recognized by the EN. The ENs encoded by all known non-LTR retrotransposons belong to one of two major classes: the apurinic/apyrimidinic endonuclease (APE) and the restriction enzyme-like endonuclease (REL-ENDO) ( 7 ). In general the elements encoding APE-like domains do not insert in a sequence specific manner unlike those encoding REL-ENDO domains, although several exceptions to this generalization are known. For example, the APE class of element, R1Bm, inserts at a specific location in the 28S rRNA gene of Bombyx mori ( 8 ) and Tx1L inserts specifically into another transposon Tx1D in Xenopus laevis ( 9 ). The EN encoded by EhLINEs in E.histolytica is of the REL-ENDO type. The known members of this class either insert into specific repetitive genes (R2Bm of B.mori and R4 of Ascaris insert in the 28S rRNA gene; members of CRE clade insert in the spliced leader genes) or into TAA repeats (Dong element of B.mori , or Rex 6 in vertebrates) ( 10 , 11 ). On the other hand, EhLINEs/SINEs in the E.histolytica genome are not known to insert within any gene or specific DNA sequence.
|
17040894_p2
|
17040894
|
INTRODUCTION
| 4.627788 |
biomedical
|
Study
|
[
0.9991776347160339,
0.000392392830690369,
0.0004300067375879735
] |
[
0.9968343377113342,
0.0006379327969625592,
0.002376391552388668,
0.00015130701649468392
] |
en
| 0.999997 |
The apparent lack of targeted insertion of many non-LTR elements could be due to non sequence specific nicking by the element-encoded EN, or it may imply that these elements recognize structural features of the DNA rather than sequence alone. Do the insertion sites share conserved structural features which are recognized by the element in order for subsequent events like nicking and reverse transcription to take place? A number of methods are available which measure DNA structural features, such as bendability ( 12 , 13 ), and propeller twist ( 14 ); thermodynamic features, such as stacking energy ( 15 ), duplex stability ( 16 , 17 ) and denaturation energy ( 18 ); protein interaction measures, such as protein-induced deformability ( 19 , 20 ) and nucleosomal positioning ( 21 ). We show that these features deviate significantly at insertion hot spots of a variety of non-LTR retroelements in different organisms. Using pre insertion sites of EhLINE1/SINE1 as our model we have developed a tool (DNA SCANNER), which scans and plots a given set of parameters in a DNA sequence; this facilitates analysis of these structural features and thus indicates the potential of a given putative site for actual insertion. We have also measured the substrate specificity of EhLINE1-EN using an in vitro assay ( 22 ), to determine the contribution of the EN in target site selection. We show that although the EN is not strictly sequence-specific, it is possible to assign a consensus sequence at which the enzyme nicks preferentially. A combination of EN nicking preference and DNA structure at pre insertion loci may define insertion hot spots.
|
17040894_p3
|
17040894
|
INTRODUCTION
| 4.476753 |
biomedical
|
Study
|
[
0.9992470741271973,
0.0004569145676214248,
0.0002959688426926732
] |
[
0.9988991022109985,
0.0002859982487279922,
0.0006906213820911944,
0.00012422859435901046
] |
en
| 0.999996 |
EhLINE1-EN protein was purified as described ( 22 ) except that Escherichia coli cells were grown for 90 min after adding isopropyl-β- d -thiogalactopyranoside (IPTG). The recombinant protein was eluted with 250 mM imidazole after extensive washing with buffer containing 80 mM imidazole. The protein was immediately dialyzed against 50 mM Tris–HCl (pH 7.5), 100 mM NaCl, 10 mM MgCl 2 , 2 mM DTT and 2 mM phenylmethylsulfonyl fluoride (PMSF) at 4°C for 2 h with one change. It was stored at −80°C in aliquots.
|
17040894_p4
|
17040894
|
Expression and purification of EhLINE1-EN
| 4.134273 |
biomedical
|
Study
|
[
0.9995313882827759,
0.0002078574034385383,
0.0002608195354696363
] |
[
0.9951019287109375,
0.00427886750549078,
0.0004656666424125433,
0.0001534162147436291
] |
en
| 0.999996 |
For preparation of radiolabeled substrates by PCR, the bottom strand primer (50 pmol) was end labeled in a 20 μl reaction using 50 μCi of [γ- 32 P]ATP (Amersham pharmacia Biosciences) and T4 polynucleotide kinase (NEB). The reaction was stopped by incubating at 65°C for 20 min and the labeled primer was purified by passing through Sephadex G-25 (Amersham Pharmacia Biosciences) ( 23 ). The DNA substrates were generated by PCR with 176 bp DNA fragment as template and a combination of one end-labeled primer and the other unlabeled primer. PCR products were separated on 6–15% native polyacrylamide gels depending on the size of the products. DNA band corresponding to the full-length product was excised from the gel. DNA was recovered by the ‘crush and soak’ method ( 23 ).
|
17040894_p5
|
17040894
|
Preparation of substrates and EN assays
| 4.158287 |
biomedical
|
Study
|
[
0.9994951486587524,
0.00028867696528322995,
0.00021611031843349338
] |
[
0.9971875548362732,
0.0021890602074563503,
0.0005019610980525613,
0.00012142363266320899
] |
en
| 0.999996 |
The DNA substrate (100 ng) was incubated with 40 ng EN protein in a 10 μl reaction for 1 h at 37°C. The enzyme was inactivated by adding 25 mM EDTA. Denaturing electrophoresis was performed on 6–12% polyacrylamide gels containing 7 M urea. A 2 μl aliquot of the reaction product was mixed with 8 μl of formamide gel loading dye (95% formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol FF), boiled for 5 min and chilled on ice before loading. The parallel sequencing reaction was carried out by using Thermo Sequenase cycle Sequencing Kit (Amersham Pharmacia Biosciences). Template DNA (100–150 ng) and 1–2 pmol primer was used for each sequencing reaction. Electrophoresis was done at 45 W for 1–3 h with gel temperature being maintained at 45–50°C (Owl Separation System, S4S). The gels were fixed, dried and exposed to X-ray film. Quantitation of the reaction product was carried out with FLA 5000 imaging system (Fujifilm).
|
17040894_p6
|
17040894
|
Preparation of substrates and EN assays
| 4.190656 |
biomedical
|
Study
|
[
0.9993999004364014,
0.0003869788779411465,
0.0002131739747710526
] |
[
0.9963878393173218,
0.0029800492338836193,
0.0004896800382994115,
0.00014241634926293045
] |
en
| 0.999997 |
Synthetic substrates (32–35 bp) and the 27 bp substrate were prepared by annealing the overlapping complementary single-stranded oligonucleotides, followed by gap filling and PCR. The substrates were purified and treated with EN as described above.
|
17040894_p7
|
17040894
|
Preparation of substrates and EN assays
| 3.955081 |
biomedical
|
Study
|
[
0.9995123147964478,
0.00017729614046402276,
0.0003103938070125878
] |
[
0.9968205690383911,
0.0027791643515229225,
0.0003033453831449151,
0.00009683358075562865
] |
en
| 0.999997 |
The insertion loci were obtained by using an automated software tool ELEANALYSER that was developed for analysis of elements in a genome ( 4 ). This tool incorporates various Perl programs as filters and parsers along with BLAST ( 24 ) suite of programs. The target site duplications at the boundaries of elements were determined with pair-wise alignment. Redundant data were removed.
|
17040894_p8
|
17040894
|
Data retrieval
| 3.994383 |
biomedical
|
Study
|
[
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0.0001457425969420001,
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] |
[
0.996574878692627,
0.002995706396177411,
0.00034705529105849564,
0.00008242582407547161
] |
en
| 0.999998 |
The positive dataset consisted of 93 sequences of known insertion sites (see the Results section) while the negative dataset consisted of 100 sequences known not to permit insertion. For each of the structural properties discussed here, a graphical profile was constructed for each member of the positive or negative dataset by evaluating the said property in a sliding window of 5 bp along the sequence. The set of profiles was then averaged separately over the positive and the negative datasets. Properties, such as the propellar twist, bendability and denaturation depend on the sequence of dinucleotides or trinuclotides in the DNA. Previous studies, such as protein–DNA interactions and DNase I experiments ( 12 , 13 ) give estimates of the values of bendability, etc. for each di/trinucleotide. These data are also available at ( 25 ), as well as on our web server, . The total value of a given property in a given window is a sum of contributions from the constituent sequence of di/trinucleotides.
|
17040894_p9
|
17040894
|
Computational analysis of E.histolytica pre insertion sequences
| 4.115128 |
biomedical
|
Study
|
[
0.9993878602981567,
0.0002564671740401536,
0.0003556180454324931
] |
[
0.9994707703590393,
0.00017282979388255626,
0.0003057604481000453,
0.00005065641016699374
] |
en
| 0.999996 |
In order to measure if differences between the controls and the insertion sites were significant, we used Mann–Whitney tests using the statistical software MINITAB. Analysis of any DNA sequence with respect to the different parameters described here is possible at this website with the tool DNA SCANNER.
|
17040894_p10
|
17040894
|
Computational analysis of E.histolytica pre insertion sequences
| 2.786576 |
biomedical
|
Study
|
[
0.9980090260505676,
0.00030470328056253493,
0.001686332281678915
] |
[
0.9013228416442871,
0.09706281870603561,
0.0009705998818390071,
0.00064374681096524
] |
en
| 0.999997 |
In a similar manner, we constructed positive and negative datasets for other genomes. Namely, upstream regions for a set of known insertion sites were curated from GenBank for the organisms, such as Dictyostelium discoideum, Takifugu rubripes and Drosophila melanogaster . A negative dataset was also constructed as follows: for site specific elements, the sequences near known insertion sites were taken whereas for dispersed elements, sequences were randomly picked from the genome. For each of these sets, as discussed above, average property profiles were constructed. To determine whether a given property was sufficiently discriminatory between the positive and the negative datasets, the profiles were compared and required to have a separation of at least one SD. These profiles were also required to be distinct over a region of at least 18 nt in order that the said property could be considered useful in distinguishing between the two sets.
|
17040894_p11
|
17040894
|
Insertion site analysis in other genomes
| 4.075225 |
biomedical
|
Study
|
[
0.9994407296180725,
0.0002046157605946064,
0.000354691524989903
] |
[
0.9994114637374878,
0.00020147541363257915,
0.00034528030664660037,
0.00004185895886621438
] |
en
| 0.999999 |
According to the TPRT model of retrotransposition by non-LTR elements, the process is initiated by a nick in the bottom strand of the target site, generated by EN. An important determinant in the choice of insertion sites by EhLINEs/SINEs could, therefore, be the preferred substrate requirements of the EN. To test this we chose oligonucleotide substrates derived from a sequence in the E.histolytica genome where EhSINE1 is known to insert. We had earlier shown that a 176 bp fragment containing this insertion site was nicked specifically by EN at the bottom strand, exactly at the point of EhSINE1 insertion ( 22 ). In addition to this nicking hot spot (termed site #3), the 176 bp fragment contained other hot spots, of which site #2 and site #1 will be used as controls in subsequent experiments. Deletion analysis of the region surrounding site #3 showed that a 27 bp fragment (−11 to +16 of nick with respect to bottom strand) was sufficient as a substrate for EN activity . Next, we altered the sequences immediately surrounding the bottom strand nick in site #3 to determine their role, if any, in substrate recognition of the EN. Transition mutations were introduced using oligos with the appropriately altered sequence to PCR amplify a 117 bp fragment from the 176 bp template (position 60 to 176). The DNAs thus obtained contained a normal site #2 and a mutated site #3. The activity of EN on the mutated site #3 was quantitated using site #2 as an internal control. The results showed that changing the nucleotides downstream of the nick reduced the EN activity only marginally. However, changing the nucleotides upstream of the nick had a much larger impact (upstream A to G decreased the activity to 17% and T to C increased the activity to 178%) .
|
17040894_p12
|
17040894
|
Role of the element-encoded EN in target site selection
| 4.316969 |
biomedical
|
Study
|
[
0.9993239641189575,
0.0003588764520827681,
0.0003171607095282525
] |
[
0.9993927478790283,
0.00023472662724088877,
0.0002950765483547002,
0.00007746947085252032
] |
en
| 0.999995 |
It had earlier been observed that a G residue was frequently present 3–4 nt upstream of the nick in most sites nicked by the EN ( 22 ). This may be significant given that the E.histolytica genome is highly A + T rich. ( 26 ). Site #3 contains three G residues upstream of the nick . Using the procedure described above, the G residues were changed singly, or in pairs and activities of these substrates quantified with respect to normal site #2. The results showed that changing a single G residue alone did not affect EN activity. However, changing the first two Gs (proximal to the nick) to T or A reduced the activity very significantly, although changing them to C had less effect . The third G also contributed to the EN activity . Compared with the second G alone , the first G alone retained greater activity. The results showed that the upstream G residues did play a significant role in substrate recognition by EN. From this data the preferred recognition sequence for EN was deduced to be 5′-GGCATT-3′.
|
17040894_p13
|
17040894
|
Role of the element-encoded EN in target site selection
| 4.221298 |
biomedical
|
Study
|
[
0.9993982315063477,
0.00028709316393360496,
0.0003146679373458028
] |
[
0.9995085000991821,
0.00019244947179686278,
0.0002326836111024022,
0.00006632513395743445
] |
en
| 0.999996 |
To validate the general applicability of this sequence requirement, site #2 in the 176 bp fragment was also analyzed by mutation analysis. A fragment of 85 bp (position 92 to 176) was PCR amplified from the 176 bp template. The ‘wild-type’ sequence of this site (bottom strand) was 5′-TGCATTG-3′. In agreement with the results obtained for site #3 it was found that changing the A to G reduced activity to 37%; changing C to T only reduced the activity to 70%, but changing GC to TT reduced the activity to 8% . From this data, the preferred recognition sequence was deduced to be 5′-GCATT-3′.
|
17040894_p14
|
17040894
|
Role of the element-encoded EN in target site selection
| 4.152061 |
biomedical
|
Study
|
[
0.999417781829834,
0.0002645309432409704,
0.00031769939232617617
] |
[
0.9994949102401733,
0.00026639472343958914,
0.0001719904539640993,
0.00006668765126960352
] |
en
| 0.999998 |
Nucleotides in the vicinity of the nicking site were checked for their role in substrate recognition. A 37 bp substrate containing 15 bp upstream and 22 bp downstream of the nick in site #3 was used. Transition mutations were introduced in every alternate nucleotide, keeping the central 9 bp (GAATACCTC) unchanged . This increased the GC content of the substrate from 13.5 to 46%. Enzyme activity in the mutated substrate was comparable with wild-type showing that the nucleotide sequence at a distance from the nicking site did not influence EN activity.
|
17040894_p15
|
17040894
|
Role of the element-encoded EN in target site selection
| 4.122827 |
biomedical
|
Study
|
[
0.9993526339530945,
0.0003082608454860747,
0.0003391080826986581
] |
[
0.9994572997093201,
0.0002902369888033718,
0.00018685428949538618,
0.0000656782794976607
] |
en
| 0.999997 |
The above substrates were derived from a natural E.histolytica sequence in which EhSINE1 is known to insert. A completely artificial substrate was next tested for enzyme activity with EN. It was made AT-rich and variants containing two Cs or two Gs were also tested . The results confirmed the earlier observations with sites #2 and 3 in the 176 bp fragment: the enzyme prefers to nick between AT and TT (5′-ATT-3′), if 5′-ATT-3′ is changed to 5′-GCC-3′ the activity is reduced, inclusion of two Gs upstream of the nicking site (lower strand 5′-GGATT-3′) improves nicking efficiency, changing Ts to As at the nicking site abolishes activity and a minimum of 15 nu upstream of the nick seems to be necessary for activity.
|
17040894_p16
|
17040894
|
Role of the element-encoded EN in target site selection
| 4.156594 |
biomedical
|
Study
|
[
0.999472439289093,
0.00023396621691063046,
0.0002935360826086253
] |
[
0.999414324760437,
0.00027204849175177515,
0.0002554336388129741,
0.00005815616168547422
] |
en
| 0.999996 |
The above data shows that EhLINE1-encoded EN, while being flexible in its sequence requirement, has a strong preference for nicking the bottom strand between A and T residues located downstream of GC (5′-GCATT-3′). Sequences further upstream or downstream of this basic sequence had little or no effect on enzyme activity.
|
17040894_p17
|
17040894
|
Role of the element-encoded EN in target site selection
| 4.098845 |
biomedical
|
Study
|
[
0.9993877410888672,
0.0002266072406200692,
0.00038558701635338366
] |
[
0.9988839030265808,
0.0008465697756037116,
0.00018936801643576473,
0.0000802424328867346
] |
en
| 0.999996 |
The 176 bp fragment of E.histolytica DNA used as a substrate for EN in the above experiments has three hotspots of nicking by EN. Of these, EhSINE1 is known to insert at site #3. We tested whether site #2, which was also efficiently nicked by the EN, was used as an integration site for these elements. Primers were designed to PCR amplify the DNA surrounding site #2. These were used to amplify genomic DNA from two different E.histolytica strains (HM-1:IMSS and HK-9) ( 27 ). While primers flanking site #3 amplified a band expected of EhSINE1 integration at that site, primers from site #2 amplified only the unoccupied sequence from both strains. Thus, only a subset of EN-recognition sites appears to be utilized for integration of these elements. When the E.histolytica genome was searched for the string GGCATT (the preferred nicking sequence of EN), a total of 5754 instances were found, 3902 of which were in genic regions where no insertion of EhLINEs/SINEs has been found so far. The remaining 1852 were in intergenic regions but these were not occupied by the elements. Thus additional structure must be present in the vicinity of the GCATT motif in order that an element insert there. We have used a computational approach to determine whether target sites share some common features of DNA structure.
|
17040894_p18
|
17040894
|
The preferred EN recognition sequence alone is insufficient for element insertion
| 4.261924 |
biomedical
|
Study
|
[
0.9993255138397217,
0.00035143797867931426,
0.00032305935746990144
] |
[
0.9994843006134033,
0.00018109231314156204,
0.00026203435845673084,
0.00007258751429617405
] |
en
| 0.999997 |
We selected all the EhSINE1 insertion sites in which the 5′ end of EhSINE1 could be clearly identified. These numbered a total of 93 and were used to construct a set of pre insertion loci of EhSINE1 as follows. Each occupied EhSINE1 site was analyzed and the element, together with one of the target site duplications, was removed. The resulting sequence 40 bp upstream from insertion site to 40 bp downstream of it constituted one such locus. A negative dataset of 100 fragments was constructed which consisted of randomly chosen E.histolytica sequences of 80 bp; sequences from Borrelia burgdorferi genome (genomic A + T content similar to E.histolytica ); Entamoeba and Plasmodium genes; and randomly shuffled sequences of the positive dataset. Insertion Site Finder (ISF), a machine learning tool, was developed based upon Bayes' rule ( 28 ) and AdaBoost ( 29 ) to incorporate the characteristics of insertion sites as signals for identification and prediction (manuscript in preparation). Specificity and sensitivity of the tool were determined. Specificity is defined as percentage of strings from the negative data set rejected by ISF at a particular cut-off. The cut-off value was determined during the training process. The pre insertion loci of EhLINEs/SINEs constituted the positive dataset. Sensitivity is defined as percentage of true examples detected by ISF based on the cutoff determined above. Both specificity and sensitivity were in the range of 89–97% ( Table 1 ).
|
17040894_p19
|
17040894
|
Structural features of the insertion site of EhSINE1 as deduced from computational analysis
| 4.130003 |
biomedical
|
Study
|
[
0.9993385672569275,
0.000304677989333868,
0.00035665641189552844
] |
[
0.9995940327644348,
0.00013977526396047324,
0.0002183698961744085,
0.00004777975118486211
] |
en
| 0.999997 |
Positive and negative datasets were compared with respect to the following criteria: DNA sequence, structure, energy profiles, protein induced deformability and nucleosome location. Computation of nine measures was performed in a moving window of length 5 over each 80 bp segment, and the profile was averaged for all loci. In order to determine the significance of the results, all the positive datasets were scrambled and the parameters were estimated. There was no significant difference between scrambled positive sequences and the negative datasets. To test whether the sample size of the positive dataset (93) was sufficient, one-third of the data were randomly removed from the dataset. All the features observed with the entire dataset persisted, showing that the sample size was sufficient. Our results are summarized below. Thymine Excess: pre-insertion loci were specifically observed to be T enriched. The T content profile showed a significant peak (>75%) at position −22 bp relative to the insertion site . This enhancement was evident for all window sizes ranging from 3 to 15. In contrast, the negative dataset had an essentially flat profile for T content. The Mann–Whitney test on the difference in the average T percentages for positive and negative examples gave a P -value <0.0001, indicating that the difference was significant. Bendability Profile: the bendability profile depicts the relative flexibility of the sequences flanking the insertion point. A region of low bendability (namely increased rigidity), between −40 to −10 of the insertion site, followed by a sharp peak at position −9, was characteristic of the positive dataset . Propeller Twist Profile: the propeller twist measures the tendency of twist about the long axis of the DNA strand: this makes the two bases of a pair non-coplanar ( 14 ). The propeller twist profile delineated a rigid region (−35 to −18 bp) changing to flexible region at around −10 bp . Stacking energy: stacking energy provides a measure of the stability of a given DNA sequence ( 15 ). The peak observed between positions −35 to −19 bp upstream of the insertion site reflects a region, which would de-stack or melt easily . Free Energy Profile: the duplex stability of a DNA depends on 10 different nearest-neighbor interactions ( 16 , 17 ). Higher negative values indicate higher stability. The insertion sites were found to have higher value (−0.57 kcal/mol) at the −18 position, suggesting that this region is destabilized more easily in comparison to the controls . DNA denaturation energy: the melting of double-stranded DNA at the insertion site is necessary for retrotransposition to occur ( 18 ). A strong signal was observed in the region −35 to −11 indicating that only a relatively small amount of energy would be required to denature this region upstream of the insertion site . Protein induced deformability: the sequence-dependent deformability of DNA is considered to be important for potential interaction of DNA with proteins ( 19 , 20 ). Since retrotransposition would require such interaction this parameter has a potential to be indicative of insertion sites ( 19 ). A region of low deformability was found between −37 to −14 bp followed by a region of high deformability . Therefore, retrotransposition complex can form downstream of the low deformation area around the site of insertion. Nucleosomal related features: two different nucleosomal related features were used, namely the bending energy/persistence length ( 30 ) and nucleosomal positioning profiles ( 21 , 31 ). Both were computed as described in the Materials and Methods. Since nucleosomal density and nucleosome-induced changes in DNA can contribute to processes, such as transcription or recombination, these parameters are likely to influence retrotransposition events ( 32 , 33 ). The bending energy or persistence length profile for insertion site loci reveals a low energy region between positions −34 and −11 with significant dip of value (−16.5 nm persistence length) at position −19 . The minimum in the profile indicates that nucleosome might be positioned in the vicinity of insertion site. Similar results were obtained using nucleosomal positioning profile . A major difference between positive and negative datasets was obtained between positions −37 and −10.
|
17040894_p20
|
17040894
|
Structural features of the insertion site of EhSINE1 as deduced from computational analysis
| 4.377586 |
biomedical
|
Study
|
[
0.9992619156837463,
0.0004667427856475115,
0.0002714151924010366
] |
[
0.9990562796592712,
0.00024673945154063404,
0.0005866226856596768,
0.00011036797513952479
] |
en
| 0.999997 |
When the 93 true insertion sites were tested with the nine measures listed above, for 10 of these sites none of the measures scored positive . For the remaining 83 sites one or more of the measures scored positive, with more than half the sites scoring positive on four or more of the measures. The 5754 E.histolytica genomic sites containing the preferred EN nicking sequence were also tested in the same manner. We found that only in 8% of cases did these sites score positive on at least one of the above measures, whereas for a randomly selected set of 5754 sequences from the E.histolytica genome, 20% of the sites scored positive. This analysis suggests, therefore, that the number of unused ‘good’ sites for EhSINE1/LINE1 (namely those where the element can integrate in an efficient manner, but are presently unoccupied) in the E.histolytica genome may be small.
|
17040894_p21
|
17040894
|
Structural features of the insertion site of EhSINE1 as deduced from computational analysis
| 4.111111 |
biomedical
|
Study
|
[
0.9993122816085815,
0.0002961892168968916,
0.0003915468114428222
] |
[
0.9995583891868591,
0.0002073518407996744,
0.00018527291831560433,
0.000048979505663737655
] |
en
| 0.999998 |
Since, DNA structure at pre insertion loci of EhSINE1 was distinct, we further checked to see if structure had an influence on EhLINE1-encoded EN activity as well. The various mutated substrates used to check EN activity were analyzed for changes in DNA structure as a result of the introduced mutations. The substrates used were classified into two groups depending on whether the EN activity with the substrate was greater (group A) or lesser (group B) than 50% of the normal substrate. For both groups, the eight measures listed in the previous section were computed: all parameters (except for the T-rule) displayed significant differences (Mann–Whitney scores had P -values below 0.05). A representative graph is shown for the nucleosomal positioning measure in Figure 9 : the blue curve is for Group A, while the magenta curve is for Group B. Differences at the mutation sites (−5 to +5) are clearly visible, suggesting that change in DNA structure is responsible for the change in enzyme activity.
|
17040894_p22
|
17040894
|
Computational analysis of DNA structure adopted by mutated substrates of the EhLINE1-EN
| 4.120269 |
biomedical
|
Study
|
[
0.9993172883987427,
0.00029293072293512523,
0.0003897633287124336
] |
[
0.9996216297149658,
0.0001322414173046127,
0.0001985888957278803,
0.000047420271584996954
] |
en
| 0.999997 |
To see if the physical features listed above for EhSINE1 insertion sites were shared by retrotransposon insertion sites in other genomes as well, a few selected genomes were analyzed using DNA SCANNER ( Table 2 ). Site-specific as well as non site-specific elements were analyzed in each genome. A stretch of 40 bp upstream of each insertion point was used for this analysis. The downstream sequences were not included since they did not exhibit any novel features in E.histolytica . The elements TDD3, TRE3B and TRE3C were analyzed in D.discoideum . Almost all the features that characterize the insertion sites in E.histolytica are significant for TRE3C when compared with a carefully constructed negative dataset (randomly picked genomic sequences for non-site specific elements and genic sequences for site specific sequences), even after stringent filtering (≥18 nt). In contrast, insertion sites for the elements TRE3B and TDD3 in the same genome appeared to rely on only a subset of these properties (2 for TRE3B) or, as for TDD3, on other features. In D.melanogaster , insertion sites for R1 element scored positive for five of the properties, while Jockey was positive for six of them. Results of our analysis of several other elements are summarized in Table 2 . We also considered elements, such as TX1, DONG and REX1 in Takifugu , but the low copy number of these elements did not permit clear conclusions.
|
17040894_p23
|
17040894
|
Insertion sites of many non-LTR retrotransposons share common structural features
| 4.147452 |
biomedical
|
Study
|
[
0.9993273019790649,
0.0002969502529595047,
0.000375724455807358
] |
[
0.9994868040084839,
0.00014345468662213534,
0.0003166345413774252,
0.00005308173058438115
] |
en
| 0.999995 |
While all the measures used in the present study to detect the insertion sites of elements of E.histolytica are not universally applicable in other genomes, the present observations suggest the possibility of subsets of these properties being pertinent for different organisms. Although, the structure and intensity of the signals relevant to each genome is distinct, there is sufficient overlap in the nature of the signals. One may therefore hypothesize that out of the common pool of features examined here, retrotransposons in a range of organisms will share some of these signals at their insertion sites.
|
17040894_p24
|
17040894
|
Insertion sites of many non-LTR retrotransposons share common structural features
| 4.029667 |
biomedical
|
Study
|
[
0.9992535710334778,
0.00018409801123198122,
0.0005623538163490593
] |
[
0.9988621473312378,
0.0004809668753296137,
0.0005971862701699138,
0.00005969223639112897
] |
en
| 0.999996 |
Amongst parasitic protozoa E.histolytica is one of the few in which non-LTR retrotransposons occupy as much as 6–8% of its 23 Mb genome ( 4 , 34 ). From a phylogenetic standpoint it is important to understand whether this primitive organism shares the same mechanisms for insertion and maintenance of these elements in its genome as those adopted by metazoans. EhLINEs/SINEs are dispersed throughout the genome, with no apparent target specificity. Here, we investigate whether pre insertion sites of EhLINEs/SINEs have any distinguishing features that favor their selection for element insertion. The two parameters studied were DNA secondary structure of pre insertion sites and sequence hotspots for nicking by the EhLINE1-encoded EN. The general validity of our results with DNA structure of target sites was tested by extending the analysis to non-LTR retrotransposons in a few selected genomes.
|
17040894_p25
|
17040894
|
DISCUSSION
| 4.173186 |
biomedical
|
Study
|
[
0.9993379712104797,
0.0003179865307174623,
0.00034407785278744996
] |
[
0.9994540810585022,
0.00017970761109609157,
0.00030854568467475474,
0.00005757065810030326
] |
en
| 0.999996 |
Different parameters that probed structural, thermodynamic or nucleosome positioning features were employed in our computational analysis of target site sequences in order to detect unique features, which may be recognized by the invading retrotransposon ( Table 2 ). This analysis showed that DNA structure is likely to be important for target site selection in many retrotransposons, although, of the features tested, none were common to insertion sites of elements in all genomes. The presence of unique DNA structure at insertion sites appears to hold both for site-specific and dispersed non-LTR elements. Similar observations with DNA transposons show that the requirement for specific DNA structure at the target site may be a common feature. The bacterial transposon Tn7 ( 35 ) and the D.melanogaster P element ( 25 ) are known to recognize optimal DNA structures, rather than specific sequences, for preferential insertion.
|
17040894_p26
|
17040894
|
DISCUSSION
| 4.205297 |
biomedical
|
Study
|
[
0.9994217157363892,
0.00031332147773355246,
0.0002649988455232233
] |
[
0.9991274476051331,
0.0001735429250402376,
0.0006252184975892305,
0.00007379725138889626
] |
en
| 0.999997 |
In our analysis of E.histolytica the most significant outcome was that in all insertion sites of EhSINE1 the region −10 to −35 bp upstream of the insertion point showed a very distinct structure. This region was also T-rich. However, the observed profiles were not attributable to T-richness alone, since shuffling the sequences in the positive dataset (while keeping base composition constant) resulted in a flat profile. In addition, a whole genome scan of E.histolytica showed several thousand T-rich sites, which scored poorly with the other structural parameters, and indeed no element was found inserted in these sites. The −10 to −35 region of a true insertion site tended to be rigid as indicated by propeller twist and bendability measures. This is due to the presence of dinucleotides, which remain rigid as shown by negative values in the profiles . Data from the various parameters used for structural analysis, put together, show that this upstream region is rigid, can melt easily and is amenable to interaction with proteins/nucleosomes in its vicinity.
|
17040894_p27
|
17040894
|
DISCUSSION
| 4.282293 |
biomedical
|
Study
|
[
0.9993483424186707,
0.0003534174175001681,
0.000298213999485597
] |
[
0.9994638562202454,
0.0002056663652183488,
0.0002556902472861111,
0.00007484802335966378
] |
en
| 0.999996 |
We have examined the sequence requirements of the EhLINE1-encoded EN and find that although the enzyme is not strictly sequence-specific (although belonging to the REL-ENDO class), it is possible to assign a consensus sequence 5′-GCATT-3′ at which the enzyme nicks most efficiently between A-T and T-T. The upstream G was not essential for activity, but its inclusion greatly improved nicking efficiency. In the context of the E.histolytica genome which is highly A + T rich ( 26 ), this enhancement of nicking activity in the vicinity of G could serve to limit the enzyme targets in vivo . The consensus nicking sequence described above was deduced from in vitro assays. Whether the same applies to in vivo nicking by EN is not clear at the moment since this sequence was not readily visible at all genomic sites where EhLINE1/SINE1 elements had inserted. It is possible that the consensus sequence is obscured after element insertion due to the addition of some non templated nucleotides by reverse transcriptase ( 36 ). This could complicate extrapolation of the pre insertion sequence from an occupied site, especially in the 3′-flank.
|
17040894_p28
|
17040894
|
DISCUSSION
| 4.334926 |
biomedical
|
Study
|
[
0.9991936087608337,
0.000442580203525722,
0.0003637844347395003
] |
[
0.9993554949760437,
0.00025443421327508986,
0.00030324445106089115,
0.00008684918429935351
] |
en
| 0.999995 |
Although, the in vitro consensus sequences preferred by EN are widely distributed in the genome, both in genic as well as intergenic regions, EhLINE1/SINE1 insertions have not been found within any gene so far. The preference of EhLINE1/SINE1 for intergenic regions would minimize direct damage to genes by insertional inactivation. It is possible that EhLINE1/SINE1 can insert in genic regions but are excluded due to selection pressure, as reported for human Alus, which can insert in A + T-rich DNA but are found more frequently in G + C-rich DNA ( 37 , 38 ).
|
17040894_p29
|
17040894
|
DISCUSSION
| 4.193258 |
biomedical
|
Study
|
[
0.9994044303894043,
0.0001616943336557597,
0.00043384230230003595
] |
[
0.9990690350532532,
0.0005950845661573112,
0.0002775678294710815,
0.00005826028063893318
] |
en
| 0.999997 |
In our earlier model of EhLINE1/SINE1 retrotransposition we had proposed a melting of the DNA duplex in the T-rich upstream region to allow positioning of the element RNA by virtue of hydrogen bonding between its T-rich 3′-tail and the A-rich bottom strand of DNA ( 22 ). In this context it is significant that the same upstream region does indeed display structural features that would enable it to interact with the element RNA in the RNP particle. From this analysis we postulate that insertion hot spots of EhLINE1/SINE1 are regions of DNA that adopt a favorable structure over a stretch of ∼25 bp (for interaction with the RNP particle), and that contain an EN-recognition sequence (upper strand 5′-AATGC-3′, or variants thereof) at a distance of ∼10 bp downstream of this structure. Similar schemes have also been proposed for selection of target sites by mammalian ( 39 , 40 ) and plant ( 41 ) retroposons based on structural features of target DNA and EN preferences. The contribution of these factors to target selection appears to be a common feature of non-LTR retrotransposons.
|
17040894_p30
|
17040894
|
DISCUSSION
| 4.544942 |
biomedical
|
Study
|
[
0.9992989301681519,
0.00036829893360845745,
0.0003327084705233574
] |
[
0.9980762004852295,
0.0005127195618115366,
0.0012699573999270797,
0.00014112656936049461
] |
en
| 0.999998 |
In summary, our combination of computational and enzymatic analysis of pre-insertion loci can lead to a more realistic understanding of why these genomic loci are preferred for retrotransposition.
|
17040894_p31
|
17040894
|
DISCUSSION
| 3.845792 |
biomedical
|
Study
|
[
0.9993996620178223,
0.0001688628108240664,
0.00043147424003109336
] |
[
0.977058470249176,
0.009947162121534348,
0.012762113474309444,
0.00023224229516927153
] |
en
| 0.999998 |
Skeletal muscle formation in vertebrates involves an interplay between the maturation of the contractile and the neural apparatus which is intricate and interdependent ( 1 – 3 ). Coordinated events in myogenesis and neuromuscular junction formation are facilitated by intercellular communication between myoprogenitors and neurons via signaling molecules on membrane surfaces as well as between neighboring myoprogenitors and myotubes via gap junctions. This communication has been repeatedly demonstrated to be essential for the formation of mature muscle and the development of a functional neural control mechanism ( 4 – 6 ).
|
17062625_p0
|
17062625
|
INTRODUCTION
| 4.327832 |
biomedical
|
Study
|
[
0.9994338154792786,
0.00030019189580343664,
0.00026590380002744496
] |
[
0.9286044836044312,
0.0017278736922889948,
0.06935823708772659,
0.00030944537138566375
] |
en
| 0.999997 |
Communication between developing muscle fibers is extensive during prenatal embryonic development. In fact, coupling is so extensive prenatally that excitation spreading laterally between myotubes gives rise to waves of excitation that propagate across the entire muscle ( 1 ). In developing intercostals muscles, for instance, electrical current applied to one muscle fiber can cause the contraction of the entire muscle; this depolarization can even spread to neighboring muscles in the next intercostal space through gap junctional communication ( 1 ). Immediately after birth, gap junctional communication in skeletal muscle sharply declines. This downregulation corresponds to the period of retraction of redundant nerve terminals from muscle fibers. During myoneural maturation, each stage requires the presence and contractile activity of functional muscle. Secondary myotube formation requires innervation, and contractile activity occurs very early after myotube formation. In utero , contraction of antagonistic muscles occurs in rhythmic alternation via reflex pathways in the spinal cord ( 7 ). Unified contraction of embryonic muscle fibers is facilitated by low-resistance electrical coupling via gap junctions composed primarily of connexin43 (Cx43). Gap junctions are hypothesized to be required in embryonic skeletal muscle to allow passage of signaling molecules and metabolites and for the coordinated maturation of contractile capabilities ( 1 , 8 , 9 ). Gap junctional coupling in hindlimb muscles disappears around birth. At this time, the only remaining mononuclear cells are the resident stem cells of skeletal muscle, the satellite cells. These cells remain quiescent until induced to proliferate and differentiate in response to injury. Upon injury, Cx43 is rapidly upregulated and functional gap junction channels couple proliferating satellite cells in vivo ( 10 ). High levels of Cx43 protein remain until after fusion of myoblasts.
|
17062625_p1
|
17062625
|
INTRODUCTION
| 4.87368 |
biomedical
|
Study
|
[
0.9986100196838379,
0.0008438715012744069,
0.0005461095133796334
] |
[
0.9703370928764343,
0.0017562257125973701,
0.027192754670977592,
0.0007138810469768941
] |
en
| 0.999995 |
In vivo observations have largely agreed with studies done on isolated primary myoblast cultures and with myoblast cell lines. Cx43 is expressed in established myoblast cell lines, and in isolated primary myoblast cultures prior to fusion ( 11 , 12 ). The fusion of myoblasts in vitro has been shown to require the presence of functional gap junctions ( 4 , 6 , 13 ). In the presence of heptanol or octanol, known gap junction channel blockers, myoblast differentiation is prevented ( 14 ). Under these conditions, the myogenic regulatory factors, myogenin and MRF4, also fail to be upregulated in response to induction ( 11 ). These results of chemical blockade suggest that functional channels are required for myogenesis in vitro . Elsewhere, it has been demonstrated that targeted gene deletion of Cx43, using a Cre-Lox system, inhibits the regenerative capability of myoblasts in vitro ( 15 ) and in vivo .
|
17062625_p2
|
17062625
|
INTRODUCTION
| 4.287226 |
biomedical
|
Study
|
[
0.9995973706245422,
0.00023378938203677535,
0.00016885530203580856
] |
[
0.9967105388641357,
0.0002727843530010432,
0.002907189540565014,
0.0001094004328479059
] |
en
| 0.999997 |
In myoblast cell lines, Cx43 is present prior to and during fusion but is rapidly downregulated after induction of differentiation despite only a slight reduction in mRNA levels ( 6 ). These studies suggested that the initial step in downregulation of Cx43 occurs at the translational level. In this study we present evidence that indicates a role for two microRNAs in the regulation of Cx43 during myogenesis in mouse tissues and from cultured myoblasts. This regulation occurs by inhibiting translation without targeting the message for degradation. MicroRNAs have been shown to regulate many other developmental processes. Despite the requirement for Cx43 during the initial phase of myogenesis, the subsequent downregulation of Cx43-dependent gap junctional communication is important in generating insulated muscle fibers that are singly innervated for fine motor control.
|
17062625_p3
|
17062625
|
INTRODUCTION
| 4.267865 |
biomedical
|
Study
|
[
0.9995575547218323,
0.00026889247237704694,
0.00017351566930301487
] |
[
0.9992380142211914,
0.0002991515211760998,
0.00037563213845714927,
0.00008727834210731089
] |
en
| 0.999998 |
Alignment of DNA sequence pairs was performed using Blast 2 Sequences available at the NCBI website ( ) ( 16 ). Multiple DNA sequence alignments were performed using ClustalX v1.8 ( 17 ). All genomic, expressed sequence tags (ESTs), and other nucleotide sequences were obtained from the NCBI online database via Genomic Blast, Blastn and Unigene, respectively.
|
17062625_p4
|
17062625
|
Bioinformatics
| 4.009953 |
biomedical
|
Study
|
[
0.9995040893554688,
0.0001392427657265216,
0.0003565812949091196
] |
[
0.997893750667572,
0.001600179122760892,
0.00043090363033115864,
0.00007523809472331777
] |
en
| 0.999996 |
Plasmids designed to express full length Cx43 mRNA sequence were subcloned from cDNA clone B0C01C08 from ATCC into the Xba I and Kpn I sites within pcDNA3.1 (Invitrogen). Reporter plasmids were constructed by PCR-amplifying the 3′-untranslated region (3′-UTR) from the cDNA clone and inserting the product into pcDNA3.1 downstream of the firefly luciferase coding region using the following primer sequences: 5′-GATCTCTAGAACAGGCTTGAACATCAAG and 5′-GATCGAATTCATTATACTAAATTAAAATTTTATTG. Mutations of miR-206 binding sites were introduced by site-directed mutagenesis using the following primers: for BS-1 5′-CCTACATCCCCGCTAAAAAACTAAGCAGTGTTTAAAAACT and 5′-AGTTTTTAAACACTGCTTAGTTTTTTAGCGGGGATGTAGG; for BS-2 5′-AACTAATTTGTTTGACTAAGCATGTTAAACTACTGTCA and 5′-TGACAGTAGTTTAACATGCTTAGTCAAACAAATTAGTT. The RNA pol III based expression plasmid, pSUPER, was constructed as described ( 18 ). pSUPER 206pri was constructed by PCR amplifying a ∼180 nt region of genomic sequence surrounding pre-miR-206 and inserting it into pSUPER using the following primers: 5′-CCTACATCCCCGCTAAAAAACTAAGCAGTGTTTAAAAACT and 5′-AGTTTTTAAACACTGCTTAGTTTTTTAGCGGGGATGTAGG. pSUPER miR-1 was constructed similarly based on the genomic sequence of the primary transcript of miR-1-2 with the following primers: 5′-CACCAAGCTTTTTGAATTCAGAGTATGGAAGTCATC and 5′-TGATGGATCCCCAAGTAATCCAAATGTCCTAC.
|
17062625_p5
|
17062625
|
Plasmid construction
| 4.280729 |
biomedical
|
Study
|
[
0.9995161294937134,
0.00024575492716394365,
0.00023801079078111798
] |
[
0.9987454414367676,
0.0007621000404469669,
0.00039643023046664894,
0.00009601177589502186
] |
en
| 0.999997 |
C2C12 cells were a generous gift from Dr Charles Murry at the University of Washington were cultured as described ( 12 ). HeLa cells were cultured and transfected as described ( 19 ). Luciferase assays were performed as described ( 19 ). Antisense inhibitors of miR-1 and miR-206 as well as negative control oligonucleotides were purchased from Ambion. For antisense inhibition of microRNAs, C2C12 cells were cultured to 50% confluence and transfected with either 100 nM negative control antisense RNA, 100 nM anti-miR-206, 100 nM anti-miR-1, or 50 nM anti-miR-206 and 50 nM anti-miR-1 for 6 h. Transfections were performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Subsequently, the cells were rescued in differentiation media [DMEM + 5% horse serum (HS) and ITS] and incubated for 48 h to induce differentiation ( 12 ).
|
17062625_p6
|
17062625
|
Cell culture
| 4.071218 |
biomedical
|
Study
|
[
0.9996740818023682,
0.00016133865574374795,
0.00016456676530651748
] |
[
0.9988304972648621,
0.0006902381428517401,
0.0004140003293287009,
0.0000652898452244699
] |
en
| 0.999997 |
Total RNA was isolated from tissues using Trizol (Invitrogen) and a Tekmar homogenizer. Total RNA was isolated from cell cultures using Trizol. Total RNA from developmental stages of neonatal mice were purchased from Zyagen. The radiolabeled antisense RNA probe targeting miR-206 was prepared by using the pSUPER 206pri plasmid linearized with EcoRV as a template with the MaxiScript-T7 in vitro Transcription Kit (Ambion). RNase protection assays (RPAs) for miR-206 were performed by using the mirVana miRNA Detection Kit (Ambion). The antisense RNA probes against β-actin and Cx43 were performed using the MaxiScript in vitro Transcription Kit (Ambion) ( 19 ). RPAs targeting β-actin and Cx43 were done by using the RPA III Kit (Ambion).
|
17062625_p7
|
17062625
|
RNase protection assay (RPA)
| 4.103028 |
biomedical
|
Study
|
[
0.9996694326400757,
0.00013902189675718546,
0.00019156518101226538
] |
[
0.9983258843421936,
0.0012377981329336762,
0.0003668112913146615,
0.000069555921072606
] |
en
| 0.999995 |
Total protein extracts from developmental stages of neonatal mice were purchased from Zyagen. Protein extracts were prepared in RIPA buffer [1% NP-40, 1% Deoxycholate, 0.1% SDS, 500 mM Tris, 150 mM NaCl, 1 mM PMSF, 1× Protease Inhibitor Cocktail (Roche)]. 10 μg of total protein was separated on precast 12% Tris–HCl PAGE gels (BioRad) and electrotransferred to PVDF membranes. Primary antibodies binding to Cx43 , myogenin , α-tubulin were detected with HRP-conjugated anti-rabbit (Sigma) or HRP-conjugated anti-mouse antibodies (Sigma) and the ECLplus Western Blotting Detection Kit (Amersham).
|
17062625_p8
|
17062625
|
Western blots
| 4.091385 |
biomedical
|
Study
|
[
0.9996299743652344,
0.00013914440933149308,
0.00023077873629517853
] |
[
0.9978121519088745,
0.0017394585302099586,
0.0003752750635612756,
0.0000730736501282081
] |
en
| 0.999996 |
Northern blots designed to detect Cx43, Luciferase and β-actin mRNA were performed on 20 μg of total RNA from transfected cells using the NorthernMax Gly Kit (Ambion). Northern blots designed to detect miR-206 and U6 RNA from transfected HeLa cells were performed on 20 μg of total RNA by separating RNA on precast 15% TBE 8M urea gels (BioRad). Northern blots designed to detect U6 RNA from tissue samples utilized 1 μg of total RNA similarly. Fractionated RNA was transferred to positively charged nylon membrane by electroblotting. The membranes were blocked in a 1:1 mixture of Ultrahyb:2× SSC, 7% SDS and probed with 5′-biotinylated DNA oligonucleotides [anti-miR-206 probe (Biotin-CCACACACTTCCTTACATTCCA) and/or an anti-U6 probe (Biotin-GCTAATCTTCTCTGTATCGTTCCAA)] at a concentration of 10 pM in the same solution. Biotinylated probe was detected with the BrightStar Detection Kit (Ambion).
|
17062625_p9
|
17062625
|
Northern blotting
| 4.161045 |
biomedical
|
Study
|
[
0.9995912909507751,
0.000216075248317793,
0.00019267183961346745
] |
[
0.9978657364845276,
0.0016064262017607689,
0.0004361695609986782,
0.0000916000353754498
] |
en
| 0.999998 |
C2C12 cells cultured in 35 mm dishes (Nunc) were induced to differentiate by switching to differentiation medium at ∼70% confluency. Cells were fixed at various time points by incubation for 5 min in −10°C methanol and air dryed. Cells were washed with PBS, further permeabilized for 5 min in 0.1% Triton in PBS and blocked with 5% HS in PBS for 1 h at room temperature. Cells were incubated with 1:100 dilution of anti-Cx43 rabbit polyclonal antibody (Sigma) and 1 μg of anti-myogenin mouse monoclonal antibody (Sigma) overnight at 4°C in 5% HS in PBS. Dishes were washed three times in PBS and incubated with 1:100 dilution of goat anti-rabbit-TRITC conjugated secondary antibody and a 1:100 dilution of goat anti-mouse-FITC conjugated secondary antibody in PBS with 5% HS. Cells were washed three times in PBS and incubated for 5 min with PBS-DAPI. Finally cells were washed three times in PBS and examined by fluorescence microscopy.
|
17062625_p10
|
17062625
|
Immunocytochemistry
| 4.157217 |
biomedical
|
Study
|
[
0.999332845211029,
0.00044166046427562833,
0.0002254614228149876
] |
[
0.9883192777633667,
0.010618488304316998,
0.0008170836954377592,
0.00024520294391550124
] |
en
| 0.999996 |
Based on evidence from previous work suggesting that downregulation of Cx43 during myogenesis occurs through a post-transcriptional mechanism, we looked for the presence of potential cis elements for translational regulation in the Cx43 mRNA sequence. Numerous investigators have published algorithms to detect possible interactions between microRNAs and target mRNA sequences. One such algorithm predicts the presence of two binding sites in the 3′-UTR of Cx43 for the microRNA, miR-206 ( 20 ). We aligned the 3′-UTR sequences of Cx43 mRNAs from the domesticated cow, ( Bos taurus ) , the common dog ( Canis familiaris ) , mouse ( Mus musculus ) , rat ( Rattus norvegicus ), chicken ( Gallus gallus ) and human ( Homo sapiens ) from Genbank and/or the EST database. The chicken and dog sequences were did not contain polyadenylation signals as reported and were extended to conserved polyadenylation signals downstream (data not shown). These sequences were confirmed by the presence of overlapping EST sequences. The ‘seed’ sequence, corresponding to the most important region for miRNA:mRNA interactions, was completely conserved in all aligned sequences. The conserved binding regions are shown in Figure 1 .
|
17062625_p11
|
17062625
|
Bioinformatics
| 4.194767 |
biomedical
|
Study
|
[
0.99950110912323,
0.000293773045996204,
0.0002050531911663711
] |
[
0.9993731379508972,
0.00018288155843038112,
0.00036666521918959916,
0.00007730381184956059
] |
en
| 0.999995 |
To determine the expression pattern of miR-206, RPAs were performed on 5 μg of total RNA from various tissues. As shown in Figure 2 , miR-206 is expressed predominantly in adult skeletal muscle; it could also be detected in skin, E11 whole embryo and lung, but not in heart muscle. The absence of miR-206 in heart is consistent with the high levels of Cx43 expression in heart tissue. Confirmation that miR-206 is indeed expressed in skeletal muscle, but is absent from cardiac muscle, was important because some published microarray data had suggested that miR-206 is expressed in heart, a tissue that expresses high levels of Cx43 ( 21 , 22 ). A northern blot directed against U6 snRNA demonstrates that small RNA molecules are represented in each sample.
|
17062625_p12
|
17062625
|
Tissue distribution of miR-206
| 4.1312 |
biomedical
|
Study
|
[
0.9995143413543701,
0.0002251557307317853,
0.00026052125031128526
] |
[
0.9995085000991821,
0.00022999623615760356,
0.00021118084259796888,
0.00005041849362896755
] |
en
| 0.999997 |
In order to test if miR-206 is indeed capable of regulating Cx43 protein expression in the context of its native mRNA sequence, we cloned the full-length cDNA corresponding to the entire Cx43 mRNA sequence from transcription start site to polyadenylation signal into pcDNA3.1 (pcDNA Cx43). Another plasmid designed to deliver miR-206 contained a ∼180 nt genomic insert under control of the H1 RNA promoter (pSUPER 206pri) ( 18 ). For expression studies, the human cervical cancer cell line HeLa was used because it expresses neither Cx43 nor miR-206. HeLa cells were grown in culture and transfected with pcDNA Cx43 and either pSUPER 206pri or pSUPER as a negative control. Forty-eight hours after transfection, protein extracts and total RNA were prepared. Western blot analysis against Cx43 shows a significant inhibition of Cx43 expression as shown in Figure 3A . A control western blot against α-tubulin showed that this protein remained unchanged. Northern blot analysis against total RNA revealed that the presence of miR-206 did not affect the level of Cx43 mRNA. As expected, miR-206 was only present when the cells were transfected with pSUPER 206pri. As a control for the efficiency of small RNA purification, the blot was stripped and rehybridized with a probe against U6 snRNA. Data from three independent experiments yielded a reproducible and considerable inhibition of Cx43 expression by miR-206. This experiment unequivocally shows that Cx43 can be regulated at the post-transcriptional level by miR-206 and further supports the hypothesis that miR-206 is responsible for the initial downregulation of Cx43 during myogenesis.
|
17062625_p13
|
17062625
|
Downregulation of Cx43 in vitro
| 4.348916 |
biomedical
|
Study
|
[
0.9994292855262756,
0.0003750818723347038,
0.00019566081755328923
] |
[
0.9991346001625061,
0.00030561009771190584,
0.00044886957039125264,
0.00011093254579463974
] |
en
| 0.999996 |
To test the specific regulation of Cx43 through the two predicted binding sites, we inserted the Cx43 3′-UTR sequence downstream of the firefly luciferase coding region. Mutants of the putative binding sites were then prepared . Known sequence similarity between miR-206 and miR-1 caused us to also consider miR-1 as a potential regulator of Cx43 despite the suggestions that miR-1 and Cx43 coexist in cardiac tissue. Therefore, a plasmid designed to deliver miR-1 utilizing the pSUPER vector was constructed for comparison. As shown in this figure, there is less complimentarity between miR-1 and binding site 1 than between miR-206 and the same sequence. As expected, the wild-type 3′-UTR (pcDNA Luc 43-3′-UTR) construct was significantly inhibited by the presence of miR-206 and to a lesser degree by miR-1 . Mutating either one of the binding sites alone had a partial effect on the ability of miR-206 to inhibit the expression of the luciferase reporter but completely abolished the ability of miR-1 to regulate luciferase expression. A double mutant completely abolished the inhibitory effect of miR-206 on the reporter expression. There was no statistically significant effect of combining miR-1 and miR-206 in this experimental model, suggesting that there is no synergistic or additive effect of the microRNAs when they are overexpressed together. In another series of experiments, two other non-related microRNAs, miR-183 and miR-137, were compared to miR-206 tested for their effect on luciferase expression. These microRNAs (also produced from pSUPER based plasmids) produced no significant difference in luciferase expression when compared to the empty vector (data not shown).
|
17062625_p14
|
17062625
|
Downregulation of Cx43 in vitro
| 4.185035 |
biomedical
|
Study
|
[
0.9994204044342041,
0.0003235973126720637,
0.0002560356224421412
] |
[
0.9993845224380493,
0.00016206008149310946,
0.0003867563500534743,
0.00006667202978860587
] |
en
| 0.999996 |
Since we determined that miR-206 and to a lesser extent miR-1 are capable of regulating Cx43 expression through direct binding to the 3′-UTR of its mRNA and that miR-206 is expressed in skeletal muscle, the next step was to look in vivo to see if miR-206 regulates Cx43 expression during myogenesis. We predicted that miR-206 RNA and Cx43 protein levels should be inversely related in a manner consistent with muscle development. To this end, we performed an RPA on total RNA isolated from skeletal muscle derived from 2 days prenatal mice (E16), and the first, third, and fifth days after birth. At 2 days prenatal, miR-206 became visible reaching a maximum level by day 3 postnatal . Western blots for Cx43 protein show that its levels decrease significantly after birth. In contrast, Cx43 mRNA levels remains unchanged during this period, as determined by RPA. The observed downregulation of Cx43 during this time period is in agreement with previous reports that have shown that myoblasts and developing myotubes express high levels of Cx43 gap junctions prior to and during fusion and growth ( 8 , 11 ). A high level of expression of miR-206 during myogenesis in vivo supports the notion that this miRNA is involved in the downregulation of connexin during perinatal development, the time when the bulk of skeletal muscle is being formed. It has been shown elsewhere that miR-1 shows a similar pattern of upregulation during in neonatal mouse muscle ( 23 ).
|
17062625_p15
|
17062625
|
Regulation of Cx43 during development
| 4.32453 |
biomedical
|
Study
|
[
0.9994710087776184,
0.00033304927637800574,
0.00019599880033638328
] |
[
0.999196469783783,
0.00023739523021504283,
0.0004712493682745844,
0.00009495379345025867
] |
en
| 0.999998 |
To confirm and extend our hypothesis that miR-206 plays an inportanat role in muscle cell development, we studied the expression of miR-206 in myoblasts in vitro , using a highly myogenic subclone of the mouse cell line C2C12. These cells are propagated as undifferentiated, mononucleated myoblasts under high serum conditions. When cultures are switched to low serum, they rapidly fuse into contractile myotubes. It has been reported that Cx43 is highly expressed in the mononucleated C2C12 myoblasts but is rapidly downregulated after induction of differentiation. C2C12 cells are often used to recapitulate the muscle differentiation pathway in vitro . To confirm the pattern of Cx43 expression in this in vitro model for muscle development, C2C12 cells were cultured to ∼70% confluency in growth media and then induced to differentiate by switching to differentiation medium. Samples were fixed at day 0 (day of induction) and every 24 h until day 3. Immunofluorescence studies were performed to detect Cx43 protein and myogenin, a marker for differentiation in skeletal muscle. As shown in Figure 5 , Cx43 was highly expressed in proliferating myoblasts at time 0. As expected, Cx43 protein is localized at the nuclear periphery, where it is synthesized on the endoplasmic reticulum and processed in the Golgi apparatus. It can also be seen at punctuate loci at cellular borders. Myogenin was undetectable at this time. After 1 day of differentiation, high levels of Cx43 were still located in the nuclear periphery and at regions of cellular apposition. Myogenin, the muscle-specific basic helix-loop-helix transcription factor, began to be expressed and was located in the nucleus as evidenced by its co-localization with the nuclear stain DAPI. At day 1 there were still no detectable multinucleated cells.
|
17062625_p16
|
17062625
|
Downregulation of Cx43 in C2C12 cells
| 4.277475 |
biomedical
|
Study
|
[
0.9994276165962219,
0.0003712760517373681,
0.0002010539174079895
] |
[
0.9993201494216919,
0.00022205838467925787,
0.00036236332380212843,
0.00009545003558741882
] |
en
| 0.999997 |
By day 2, expression of Cx43 had become undetectable in the perinuclear region and between cells, whereas myogenin expression had increased significantly. At this time, there were some multinucleated myotubes and some mononucleated myoblasts, none of which expressed Cx43. At day 3, most of the mononucleated cells had disappeared, there was no detectable Cx43 and myogenin was very highly expressed in all nuclei. These results indicate that C2C12 differentiation recapitulates the Cx43 regulatory pathway seen in developing skeletal muscle and should be a good model for dissecting the mechanism of Cx43 downregulation.
|
17062625_p17
|
17062625
|
Downregulation of Cx43 in C2C12 cells
| 4.2327 |
biomedical
|
Study
|
[
0.9996058344841003,
0.00023889953445177525,
0.00015527885989286005
] |
[
0.9988920092582703,
0.0004495815373957157,
0.0005621342570520937,
0.00009624506492400542
] |
en
| 0.999996 |
In order to further validate the in vivo correlations, we investigated the expression of Cx43 protein and mRNA as well as miR-206 during C2C12 myoblast differentiation. These cells were cultured in growth media and then induced to differentiate. Extracts were prepared at the time of induction and every 24 h thereafter. As shown in Figure 6A , Cx43 was highly expressed in proliferating myoblasts and continued its high levels of expression through day 1 of differentiation. At this time there was little expression of miR-206. By day 2 of differentiation there was a drastic reduction in the levels of Cx43 protein, and this level remained low until day 5, at which point Cx43 was undetectable. Expression of miR-206 was first clearly detectable on day 2 and came to a plateau around day 4. This is similar to the study by Chen et al . which showed that miR-1 expression was first detectible on day 3 post induction ( 23 ). The control northern blot to detect U6 showed that purification of the small RNA fraction of total RNA was consistent. The level of Cx43 mRNA was unchanged throughout the experiment, confirming that the downregulation of Cx43 protein occurred post-transcriptionally. Levels of β-actin mRNA remained unchanged throughout the experiment and served as an internal control. In fact, extended experiments have shown that Cx43 mRNA levels were largely unchanged through 12 days of differentiation in C2C12 cells (data not shown). Myogenin, the marker for myoblast differentiation, was not detectable in proliferating C2C12 cells but was rapidly upregulated in response to the medium change.
|
17062625_p18
|
17062625
|
Downregulation of Cx43 in C2C12 cells
| 4.210863 |
biomedical
|
Study
|
[
0.99946528673172,
0.0003317470254842192,
0.0002030764298979193
] |
[
0.9994149208068848,
0.00017291821131948382,
0.00033509498462080956,
0.00007700992136960849
] |
en
| 0.999998 |
Finally, to show that miR-206 and miR-1 are indeed responsible for the downregulation of Cx43 in C2C12 cells, the cells were transfected with chemically modified antisense oligonucleotide microRNA inhibitors either individually or in combination immediately prior to induction of differentiation. Extracts were harvested 48 h after induction and levels of Cx43 were measured. At 2 days of differentiation, there was significant cell fusion in all experimental conditions. There was no observable difference in cell number, morphology or gross myotube size (data not shown). Prior to differentiation, Cx43 was highly expressed as shown in Figure 6B . After 48 h, the C2C12 cells transfected with a negative control oligonucleotide showed significant downregulation of Cx43. In addition, cells transfected with either anti-miR-1 or anti-miR-206 RNA alone still downregulated Cx43 expression. However, cells transfected with both anti-miR-1 and anti-miR-206 RNA demonstrated a high level of Cx43 expression, similar to the levels seen in undifferentiated cells.
|
17062625_p19
|
17062625
|
Downregulation of Cx43 in C2C12 cells
| 4.129283 |
biomedical
|
Study
|
[
0.9994944334030151,
0.00028244280838407576,
0.00022307061590254307
] |
[
0.9994827508926392,
0.00016611095634289086,
0.00029200181597843766,
0.00005907805825700052
] |
en
| 0.999997 |
Skeletal muscle development in vertebrates is an evolutionarily conserved process that involves myoblast fusion into multinucleated myotubes. Prior to and during myoblast fusion, and during the development of the motor neuron input system, muscle precursors are electrically coupled via gap junctions. In mammals, as well as in zebrafish, frogs and chickens, Cx43 is downregulated during late embryogenesis and early post-natal life. Conservation of the Cx43 expression pattern suggests the existence of a conservation of mechanism. The work presented here demonstrates the role of two related microRNAs in the downregulation of Cx43 during differentiation of skeletal muscle.
|
17062625_p20
|
17062625
|
DISCUSSION
| 4.278356 |
biomedical
|
Study
|
[
0.9996371269226074,
0.00020602378936018795,
0.00015678966883569956
] |
[
0.9988652467727661,
0.0003601267817430198,
0.000689079228322953,
0.00008559657726436853
] |
en
| 0.999998 |
The prediction that miR-206 and to a lesser degree miR-1 would bind to the Cx43 mRNA by Lewis et al . ( 20 ) was one of many computer algorithms that predicted that a microRNA binding site was present in the Cx43 3′-UTR. Others, such as the DIANA MicroT algorithm, predict the binding of other microRNAs ( 24 ). These sites, however, are not conserved between species making them unlikely candidates to be involved in a conserved developmental mechanism. We, therefore, focused this study on the miR-206/miR-1 interaction with Cx43. An alignment of the 3′-UTR of Cx43 mRNAs from mouse, rat, human, dog, cow and chicken shows complete conservation of the ‘seed’ region of the miRNA:mRNA target interaction for both predicted targets . This region corresponds to the 5′ end of the microRNA sequence and generally consists of 6–8 consecutive Watson–Crick base pairs with the mRNA target. The seed region has been repeatedly demonstrated to be important for target recognition by an miRNA ( 20 , 25 – 28 ). The conservation of these sequences increases the likelihood that Cx43 is indeed regulated by miR-206/miR-1, particularly since the overall rate of 3′-UTR sequence identity between these species is ∼36% (data not shown).
|
17062625_p21
|
17062625
|
DISCUSSION
| 4.274966 |
biomedical
|
Study
|
[
0.9994305968284607,
0.00032618531258776784,
0.00024311061133630574
] |
[
0.9994220733642578,
0.00019590885494835675,
0.00030394826899282634,
0.00007815534627297893
] |
en
| 0.999998 |
Data presented in Figure 3 show that miR-206 expression from pSUPER 206pri is capable of inhibiting the expression of a luciferase reporter bearing the 3′-UTR of Cx43. This experiment demonstrates that even with overexpression of both components of the system, the microRNA is capable of reducing luciferase expression despite the fact that the mRNA levels are not significantly affected (data not shown). As shown in Figure 3A , miR-206 inhibits the expression of Cx43 protein significantly as compared to the empty pSUPER control. From the results shown in Figure 3B and C , one can conclude that miR-206 binds to the Cx43 3′-UTR through two independent binding sites, and that it is this binding that mediates its ability to inhibit the translation of the Cx43 message. It is also shown that miR-1 is capable of regulating Cx43, albeit to a lesser degree, by virtue of its inability to regulate expression when either binding site is mutated alone. The results provided in Figure 3 present compelling evidence that miR-206, and possibly miR-1, have the potential to regulate Cx43 expression.
|
17062625_p22
|
17062625
|
DISCUSSION
| 4.246556 |
biomedical
|
Study
|
[
0.9995427131652832,
0.00025363313034176826,
0.00020367615798022598
] |
[
0.9992974996566772,
0.0002496825181879103,
0.00037728447932749987,
0.0000754802895244211
] |
en
| 0.999994 |
The availability of myoblast cell lines that can recapitulate the myogenic process in vitro has provided a convenient method to study associated developmental processes. In C2C12 cells, myotubes begin to appear at day 2 of differentiation, concurrent with the decline in Cx43 levels. Cx43 protein levels become undetectable by day 5 of culture in differentiation medium. The downregulation of Cx43 protein is coincident with the upregulation of miR-206 which begins before day 2. The levels of Cx43 mRNA remain unchanged during the course of this experiment, consistent with the hypothesis that microRNAs are indeed responsible for the post-transcriptional inhibition of Cx43 expression. Further confirmation that miR-206 and miR-1 regulate Cx43 expression comes from the inactivation of these microRNAs by antisense RNA during C2C12 maturation. As shown in Figure 6 , both microRNAs must be inactivated for Cx43 expression to remain at its undifferentiated levels. Although there is a near total recovery of Cx43 levels in those cells transfected with anti-miR-1 and anti-miR-206 together, the magnitude of decrease in Cx43 levels during differentiation seen in Figure 6B was less than what was seen in Figure 6A . This is most likely due to the difference in experimental methods between the two experiments. In Figure 6A , the cells were induced to differentiate at 70% confluence whereas in the antisense inhibition experiment the cells were transfected and induced to differentiate at only 50% confluence. This difference in methodology was necessary because of the recommended lower optimal cell density for tranfection . It is also possible that there is a generalized effect of transfection with small RNA molecules on the downregulation of Cx43 by miR-206/1 in these cells or a generalized inhibition of differentiation by the liposome-mediated transfection process. The differentiation of C2C12 cells in culture is highly dependent on cell–cell contact and cell density; notably, these cells were subcloned to be highly myogenic ( 12 ). The finding that Cx43 protein expression during myoblast differentiation is inversely correlated with expression of miR-206 along with the finding that the inhibition of miR-206 and its family member miR-1 relieve this downregulation, suggests that miR-206 and miR-1 perform redundant or additive functions in regulating Cx43 in C2C12 cells. This correlates with the finding that miR-206 is capable of downregulating Cx43 protein expressed from full-length Cx43 mRNA expressed in HeLa cells, and luciferase expression that bears Cx43 3′-UTR sequences. It cannot, however, be entirely ruled out that the increase in Cx43 expression at day 2 in the presence of anti-miR-1 and anti-miR-206 together is due to an inhibitory effect on myoblast differentiation that was previously described but not seen by these experimental methods ( 23 ). This correlation must be further studied in vivo in developing skeletal muscle in order to further delineate the timing and quantitative levels of each microRNA during mouse development.
|
17062625_p23
|
17062625
|
DISCUSSION
| 4.560713 |
biomedical
|
Study
|
[
0.999219536781311,
0.0005244085332378745,
0.0002561099245212972
] |
[
0.9986212253570557,
0.00046350309276022017,
0.0007300368160940707,
0.00018528838700149208
] |
en
| 0.999999 |
A number of publications have implicated miR-1 in the regulation of skeletal muscle differentiation in both vertebrates and invertebrates as well as during cardiogenesis in vertebrates ( 23 , 29 , 30 ). Our observation that miR-1 downregulates Cx43 during C2C12 differentiation was somewhat surprising because of the previous reports that both Cx43 and miR-1 are present in adult cardiac tissue. Further studies are required to investigate whether miR-1 and Cx43 actually are coexpressed within cardiomyocytes in the adult and whether miR-1 plays a role in regulating Cx43 in the developing and/or adult heart.
|
17062625_p24
|
17062625
|
DISCUSSION
| 4.1435 |
biomedical
|
Study
|
[
0.9996993541717529,
0.00014535484660882503,
0.00015531116514466703
] |
[
0.9987103939056396,
0.00036357922363094985,
0.0008540931739844382,
0.0000718908486305736
] |
en
| 0.999996 |
The observation that Cx43 levels decrease during mammalian perinatal skeletal muscle development was first seen decades ago ( 1 , 2 , 9 , 11 , 14 , 31 , 32 ). Data from only one group have suggested that the downregulation may occur at the post-transcriptional level, although this possibility was not pursued in their work ( 11 ). In whole skeletal muscle extracts, isolated 2 days prior to birth (E16), Cx43 protein is highly expressed . This is in agreement with previous reports that primary and secondary myotubes, as well as undifferentiated myoblasts, are highly coupled before birth ( 9 ). In contrast, on the day of birth and thereafter, Cx43 expression is barely detectable whereas levels of miR-206 are greatly increased. Cx43 mRNA levels are not significantly altered during this period. Maximum levels of miR-206 occur around day 3 after birth and are ∼4-fold higher than adult levels (data not shown). Other reports have demonstrated that miR-1 is expressed in a similar manner during development. A quantitative comparison of miR-206 and miR-1 levels was beyond the scope of this publication.
|
17062625_p25
|
17062625
|
DISCUSSION
| 4.21367 |
biomedical
|
Study
|
[
0.9995661377906799,
0.00021476343681570143,
0.00021914260287303478
] |
[
0.9987767338752747,
0.00016176438657566905,
0.0009923068573698401,
0.0000691871318849735
] |
en
| 0.999998 |
The miR-206 gene is located between the polycystic kidney and hepatic disease 1 gene and the interleukin-17 gene in mouse (chr 1), rat (chr 9) and human (chr 6). The genomic site where the pre-miR-206 sequence is located contains another microRNA precursor sequence, miR-133b, located a mere ∼3.8 kb downstream, suggesting that miR-206 and miR-133b might be part of the same primary transcript ( 33 ). Microarray data published by other laboratories have shown that miR-133b, like miR-206, is expressed in skeletal muscle ( 21 , 22 ). Apparent tissue-specific co-expression of miR-206 and miR-133b supports the idea that they may be produced from the same primary transcript. In fact a novel noncoding RNA, 7H4, has been isolated and shown to be enriched in sub-synaptic nuclei of innervated muscle ( 34 ). This RNA corresponds to the genomic locations of both pre-miR-206 and pre-miR-133b. This suggests a possible correlation between NMJ maturation and specific microRNA expression. During the preparation of this manuscript, Rao et al . ( 35 ) published data demonstrating the upregulation of miR-1, miR-133 and miR-206 in C2C12 cells during differentiation. However, these authors identified no targets for miR-206, and there was no mention of redundancy or cooperation between these two microRNAs during muscle development. There has been one study which was able to show that a mutation in the 3′-UTR of the myostatin gene creates a cryptic microRNA binding site for miR-1/miR-206. This mutation was responsible for creating a phenotype that included hypertrophic skeletal muscle ( 36 ).
|
17062625_p26
|
17062625
|
DISCUSSION
| 4.542813 |
biomedical
|
Study
|
[
0.9993922710418701,
0.0003176173777319491,
0.0002901134721469134
] |
[
0.9956849813461304,
0.000494431471452117,
0.0036591843236237764,
0.00016143367975018919
] |
en
| 0.999999 |
Additional recent studies have demonstrated regulatory pathways responsible for the expression of miR-206 during embryonic development ( 35 , 37 ). This work and the work of others have demonstrated an early expression pattern associated with somites in chicken and mouse embryos and with developing muscle in zebrafish ( 24 , 38 , 39 ). Recently, another microRNA, miR-181 has been shown to be required for efficient myoblast differentiation by regulating Hox-A11 ( 40 ). Clearly there are multiple microRNAs that play a role in skeletal muscle development and this tissue system will provide fertile ground to study the regulatory networks involved in differentiation.
|
17062625_p27
|
17062625
|
DISCUSSION
| 4.148142 |
biomedical
|
Study
|
[
0.9995800852775574,
0.00017437557107768953,
0.00024555603158660233
] |
[
0.9779316782951355,
0.0007259193807840347,
0.021198641508817673,
0.00014380928769242018
] |
en
| 0.999999 |
Real time PCR (RT-PCR) can rapidly, reproducibly and quantitatively determine changes in gene expression ( 1 ). Although microarray analysis can measure large scale gene expression levels simultaneously, its hybridization-related variation often demands validation by other methods. Routinely, RT-PCR is used to verify the observation from microarray studies. However, several artifacts can confound the analysis including: (i) amplification of undesired template secondary to mispriming or annealing at inappropriate temperatures; and (ii) susceptibility to RNA contamination with genomic DNA, especially when collecting samples from tumor tissues ( 2 ). Though the problem of genomic contamination is partially addressed by DNase treatment this method is often incomplete and its protracted use often diminishes the sensitivity of detection. This is a costly problem particularly when the detection of rare transcripts in precious tissue samples is desired ( 3 ).
|
17068075_p0
|
17068075
|
INTRODUCTION
| 4.161116 |
biomedical
|
Study
|
[
0.9994729161262512,
0.00019992896704934537,
0.00032722949981689453
] |
[
0.9297000169754028,
0.008486329577863216,
0.06154545024037361,
0.000268177391262725
] |
en
| 0.999995 |
Low cost methods for detecting fluorescent dyes which bind to double stranded DNA, such as SYBR Green, are most widely used and suitable for high throughput screening. Since these dyes are not sequence specific, careful consideration should be given to avoid generating extraneous amplicons. One of the obstacles to high throughput RT-PCR gene expression studies in which multiple unique transcripts are simultaneously measured in 96 or 384 well formats, is the necessity to individually optimize each assay for each target ( 4 ). Currently, the criteria for successful determination by quantitative RT-PCR require that: (i) the optimal amplicon should be located in a non repetitive region without segments of low complexity, (ii) the optimal amplicon size should be ∼100 bp to ensure the efficiency of Taq polymerase processivity, (iii) if possible, primers should be designed to flank intron–exon borders or primers anneal at a splice junction to distinguish genomic DNA from cDNA template, and (iv) primers have similar melting temperatures with 20–70% GC content ( 5 , 6 ). It is a laborious and error-prone chore to design RT-PCR primers that meet these requirements. Available resources for pre-designed primers are limited. RTPrimerDB ( ), an online database, provides experimentally verified primer sets for 2699 human and 487 mouse genes ( 7 , 8 ). PrimerBank ( ) is a well known resource that covers most known human (33 741) and mouse (27 681) genes (9). However, its primer algorithm is not designed to span introns and is therefore more prone to amplify contaminating genomic sequences. Here we describe qPrimerDepot, a primer database for RT-PCR analysis of >99% of human (23 400) and mouse (18 733) RefSeq genes. These primers sets are designed to be used under uniform annealing temperatures to facilitate their application in large scale high throughput assays. Moreover, to reduce the noise from contaminating genomic DNA ( 6 ), over 90% of the primer sets are designed to produce amplicons bridging exon:exon junctions of intron-bearing genes.
|
17068075_p1
|
17068075
|
INTRODUCTION
| 4.45428 |
biomedical
|
Study
|
[
0.9994680285453796,
0.0003471296513453126,
0.0001848791871452704
] |
[
0.9981653094291687,
0.0007081531803123653,
0.0009887982159852982,
0.00013772601960226893
] |
en
| 0.999997 |
Sequences file (refMrna.zip) and intron/exon information tables (refGene.txt.gz) of 23 463 human and 18 737 mouse RefSeq genes (UCSC hg17 and mm6) were downloaded from UCSC genome browser ( ).
|
17068075_p2
|
17068075
|
Data processing
| 3.205402 |
biomedical
|
Study
|
[
0.9981479644775391,
0.00033664912916719913,
0.0015154926804825664
] |
[
0.5135436654090881,
0.48296239972114563,
0.0023166430182754993,
0.0011772643774747849
] |
en
| 0.999995 |
To assure amplicons free of repetitive elements and sequences of low complexity ( 10 ), we utilized Biowulf, a high-performance Linux cluster at the National Institutes of Health, to mask the repetitive elements using the RepeatMasker application with built-in MaskerAid ( 11 ).
|
17068075_p3
|
17068075
|
Data processing
| 3.834224 |
biomedical
|
Study
|
[
0.9990816116333008,
0.00018870987696573138,
0.0007297536940313876
] |
[
0.978884220123291,
0.020492490381002426,
0.0004160675744060427,
0.00020714954007416964
] |
en
| 0.999997 |
Primer3 ( 12 ) was used to design primers for each RefSeq entry, with the following parameters: for intronless genes (5.5% of human RefSeq genes and of 12.4% of mouse RefSeq genes), primers were set to be between 17 and 27 bp with 20 bp as optimum, and melting temperature was set to be between 57 and 63°C with 60°C as optimum, all other parameters, such as PRIMER_SELF_ANY and PRIMER_SELF_END, were set to default (8.0 and 3.0, respectively) to assure low self-complementarity. All cDNA amplions were 90–150 bp in size to ensure Taq polymerase efficiency. For 99% of intron-bearing genes (94.5% human genes and 88.6% mouse genes bear at least one intron), primers were designed to flank or cross an exon-intron border in which the intron was one of the top three largest in the gene of interest. Thus, contamination by genomic DNA would generate either a longer product, which can be detected by melting curve analysis, or no product if the contamination template length (intron > 3 Kb) is too long for Taq polymerase to traverse during the extension period.
|
17068075_p4
|
17068075
|
Data processing
| 4.231132 |
biomedical
|
Study
|
[
0.9994277358055115,
0.00034680511453188956,
0.00022545206593349576
] |
[
0.9959713816642761,
0.0034133170265704393,
0.0004653828509617597,
0.00014990277122706175
] |
en
| 0.999997 |
The BLAST algorithm was used via the NIH biowulf Linux cluster to evaluate all primers against corresponding RefSeq databases. The criteria for possible mis-priming requires that both primers have at least 15 matches in another RefSeq entry (i.e. expectation value, e < 1) ( 13 , 14 ). The BLAST result revealed that 891 of human and 420 of mouse primers may mis-prime to other RefSeq sequences. Sequence alignments of query and hit RefSeq using the BLAST2 algorithm ( 15 ) were performed and primer pairs which had <80% identities were filtered out of the database. Annotations are presented in the user interface for the individual primer sets that could ‘mis-prime’ another RefSeq gene with >80% identity. The sources of these mis-primed RefSeq will vary, but may include redundancy within the RefSeq database, transcript variants and paralogs of high sequence similarity. Each of these possibilities can be assessed by a direct link that is provided to in silico PCR ( ) for all primer pair sets. This link allows the user to rapidly identify amplicon locations in the mouse and human genomes so that primer specificity can be visually assessed and validated.
|
17068075_p5
|
17068075
|
Data processing
| 4.223305 |
biomedical
|
Study
|
[
0.9994575381278992,
0.0003564573999028653,
0.0001859796466305852
] |
[
0.9906919598579407,
0.008030429482460022,
0.0009907042840495706,
0.0002868171432055533
] |
en
| 0.999996 |
qPrimerDepot can be accessed at or by querying the database with a RefSeq ID or a gene name. Batch query service is available upon request if user provides standard gene name or accession number. Flat files and MySQL dump file which have all primer information are also available upon request.
|
17068075_p6
|
17068075
|
Data processing
| 2.053783 |
biomedical
|
Other
|
[
0.9794386625289917,
0.003232464659959078,
0.01732894778251648
] |
[
0.005339497234672308,
0.99365234375,
0.0004454561276361346,
0.000562619767151773
] |
en
| 0.999998 |
Reverse transcription was applied with Omniscript RT Kit following manufacturer's protocol (Qiagen). A 20 μl RT reaction included 2 μg Universal reference RNA (Stratagen), 1 μM Oligo-dT primer, 2 μl of 10× RT buffer, 0.5 mM each dNTP, 10 U of RNase inhibitor, 4 U of Omniscript Reverse Transcriptase, and DEPC-treated water. The reaction mix was incubated at 37°C for 60 min. After the reaction, the mix was diluted 1:5 with water for PCR analysis.
|
17068075_p7
|
17068075
|
Experimental validation
| 4.022676 |
biomedical
|
Study
|
[
0.9989219903945923,
0.0006556536536663771,
0.00042241523624397814
] |
[
0.7537045478820801,
0.24376791715621948,
0.0015039484715089202,
0.0010235405061393976
] |
en
| 0.999998 |
Primer sequences were extracted from our database and synthesized by Integrated DNA Technologies (Coralville, IA, USA). Quantitative RT-PCR was carried out in a DNA Engine Opticon-2 Real Time PCR Detection System (MJ Research). In brief, each 20 μl reaction mix comprises 0.3 μM primers (both 5′ and 3′ primers), 1 μl template from reverse transcription and 10 μl 2 × QuantiTect SYBR Green PCR Master Mix (Qiagen). Each reaction mix was incubated at 95°C for 15 min, 40 cycles of 95°C for 15 s and 60°C for 1 min. A melting curve analysis which read every 0.3°C from 65 to 95°C was followed to assess the homogeneity of a PCR product. Real-time PCR results were analyzed using the software provided by the manufacturer.
|
17068075_p8
|
17068075
|
Experimental validation
| 4.097751 |
biomedical
|
Study
|
[
0.9996026158332825,
0.000209486810490489,
0.00018793914932757616
] |
[
0.9973810315132141,
0.002161723095923662,
0.0003569548425730318,
0.00010029217082774267
] |
en
| 0.999997 |
Our database comprises pre-designed primers for 42 133 mouse and human RefSeq genes ( Table 1 ). For most genes three unique sets of primers are provided (96.3% of total). The database provides a simple user interface where the user may enter either the HUGO approved gene symbol or the RefSeq gene identifier . The database graphic output provides information on the primary transcript location, number of introns, primer sequence, primer length, GC%, amplicon size, and genomic amplicon size. Also a direct link is provided for location of the genomic amplicon by in silico PCR .
|
17068075_p9
|
17068075
|
RESULTS AND DISCUSSION
| 3.997545 |
biomedical
|
Study
|
[
0.9991638660430908,
0.0005168126081116498,
0.00031938706524670124
] |
[
0.6752943992614746,
0.319161057472229,
0.004138320684432983,
0.0014061912661418319
] |
en
| 0.999997 |
To experimentally evaluate the primer quality, 288 genes were arbitrarily selected from a list of genes known to function in the immune response. 288 primer sets were retrieved from the database and synthesized in 96-well plates. Universal human reference RNA was reverse transcribed and used as template in PCR to examine the quality of the primers.
|
17068075_p10
|
17068075
|
RESULTS AND DISCUSSION
| 3.996752 |
biomedical
|
Study
|
[
0.9995673298835754,
0.00018120255845133215,
0.0002513851213734597
] |
[
0.9991880059242249,
0.0005297598545439541,
0.00022376146807800978,
0.00005842507744091563
] |
en
| 0.999995 |
Given the variation of transcript abundance, melting curve analysis followed by gel electrophoresis has been suggested to verify RT-PCR products ( 6 ). Melting curve analysis revealed that 94.1% generate unique product. Visualization by the less sensitive ethidium bromide stain shows that >70% of the primer sets produce an amplicon of the correct molecular weight that will amplify and be detected as a single species by quantitative PCR . Several of the failures detected by gel electrophoresis are likely due to very low abundance transcripts in the universal RNA, imperfect primer design, unanticipated high secondary mRNA structure, or erroneous exon annotation in UCSC Genome Browser. Approximately 88.5% of primer sets produced no product in the absence of reverse transcriptase and the remaining sets produced detectable product only beyond 34 cycles of amplification possibly, due to primer dimers.
|
17068075_p11
|
17068075
|
RESULTS AND DISCUSSION
| 4.170248 |
biomedical
|
Study
|
[
0.9995465874671936,
0.00024520099395886064,
0.00020816567121073604
] |
[
0.9992971420288086,
0.00034499677713029087,
0.00029121042462065816,
0.00006658874190179631
] |
en
| 0.999997 |
The resistance of most qPrimerDepot primer sets to contaminating input genomic DNA is illustrated in Figure 4 . Here the real-time amplification profiles of three intron-bearing genes (VEFG,VEGFB and VEGFC) was compared to that of three non intron-bearing genes (XCR1, SSTR4 and MC1R) after challenge with increasing concentrations of contaminating genomic DNA (0.5–500 pg/μl). As demonstrated in Figure 4 , all three intron-bearing genes show robust resistance to >500 pg/μl of input genomic DNA. This is in stark contrast to the three non intron-bearing genes where as little as 5 pg/μl produces a significant false signal.
|
17068075_p12
|
17068075
|
RESULTS AND DISCUSSION
| 4.099805 |
biomedical
|
Study
|
[
0.9994485974311829,
0.0002788244455587119,
0.00027261467766948044
] |
[
0.9995101690292358,
0.00020783120999112725,
0.00022771717340219766,
0.00005427550422609784
] |
en
| 0.999997 |
Taking advantage of the intron/exon inventory of RefSeq genes and Primer3, a paradigm primer design tool, we designed primers which are contamination resistant for 99% of human and mouse RefSeq genes ( Table 1 ). Since the majority of the primer sets will amplify desired templates under unified annealing temperatures, high throughput multiplex analysis is achievable at a reasonable cost. Empirical screening and validation of primer set performance conservatively suggests that 70–90% of the primer set designs are likely to perform effectively ‘right out of the box’ with no need to adjust the conditions of amplification. Therefore, qPrimerDepot is a valuable resource for qRT applications, especially in those circumstances requiring high throughput detection of rare transcripts in curated and/or patient-derived samples that often contain unavoidable contamination with genomic DNA.
|
17068075_p13
|
17068075
|
CONCLUSION
| 4.148656 |
biomedical
|
Study
|
[
0.9996429681777954,
0.00022202912077773362,
0.0001350377278868109
] |
[
0.9977682828903198,
0.0015154170105233788,
0.0006061280146241188,
0.00011012324830517173
] |
en
| 0.999996 |
DNA polymerase δ (pol δ) has a major and essential role in eukaryotic nuclear DNA replication ( 1 ). Pol δ also performs DNA synthesis during homologous recombination and fills DNA gaps during mismatch repair, long patch base excision repair of damaged bases and nucleotide excision repair of bulky DNA lesions [reviewed in ( 2 )]. Because all these transactions influence eukaryotic genome stability, it is of interest to understand the fidelity of DNA synthesis conducted by pol δ. Previous studies ( 3 – 5 ) have shown that pol δ is a highly accurate enzyme whose fidelity derives from high nucleotide selectivity at the polymerase active site and from proofreading by its intrinsic 3′ exonuclease activity.
|
16936322_p0
|
16936322
|
INTRODUCTION
| 4.354514 |
biomedical
|
Study
|
[
0.9992204904556274,
0.00038552944897674024,
0.0003938662412110716
] |
[
0.7913095355033875,
0.0015327547444030643,
0.20670540630817413,
0.0004523451207205653
] |
en
| 0.999997 |
In performing its roles in replication and repair, pol δ is assisted by accessory proteins. The three-subunit replication protein A (RPA) complex ( 6 ) binds single-stranded DNA and coordinates the exchange of pol δ and other proteins at template–primer termini ( 7 ). In addition, the processivity of pol δ is enhanced by proliferating cell nuclear antigen (PCNA) ( 8 ), the sliding clamp that is loaded onto template–primers by the five-subunit RFC complex ( 9 ). There are several reasons to consider whether RPA or PCNA modulate the fidelity of DNA synthesis by pol δ. Genetic studies have identified mutations in the genes encoding the large subunit of RPA ( 10 , 11 ), RFC subunits ( 12 , 13 ) and PCNA ( 14 , 15 ) that elevate mutation rates. Among several possible explanations for these mutator effects, one is that they may result from reduced DNA synthesis fidelity by pol δ during replication ( 15 ), repair or recombination. Single-stranded DNA-binding proteins have been shown previously to affect the fidelity of other DNA polymerases [e.g. see ( 16 , 17 ) and references therein]. Proteins that enhance polymerase processivity promote the extension of mismatches ( 18 – 20 ), which could reduce base substitution fidelity by preventing partitioning of mismatches to the active sites of proofreading exonucleases ( 21 ). On the other hand, several studies [reviewed in ( 22 )] have shown that the processivity of DNA polymerases correlates with their insertion/deletion (indel) fidelity in mononucleotide repeat sequences, such that proteins that increase processivity may improve indel fidelity.
|
16936322_p1
|
16936322
|
INTRODUCTION
| 4.660524 |
biomedical
|
Study
|
[
0.997572124004364,
0.0012702889507636428,
0.0011576216202229261
] |
[
0.5802931785583496,
0.0019176877103745937,
0.41673606634140015,
0.0010530196595937014
] |
en
| 0.999998 |
Here we test these ideas by examining the fidelity of DNA synthesis by three-subunit yeast DNA polymerase δ alone and its fidelity in the presence of RPA alone, PCNA (plus its loader RFC) and all three accessory protein complexes. To evaluate the effects of these accessory proteins on both nucleotide selectivity and proofreading, we compare error rates of proofreading-proficient (wild-type) pol δ to those observed with two different proofreading-deficient derivatives. To obtain a comprehensive view of the effects of accessory proteins on pol δ fidelity, we use an assay ( 23 ) that scores a variety of base substitution and indel errors. The results indicate that fidelity for errors involving single base pairs is largely determined by pol δ itself. However, the accessory proteins strongly modulate the ability of pol δ to delete large numbers of nucleotides between directly repeated sequences. The results are discussed in relation to earlier studies (cited below) on the effects of accessory proteins, and in light of models for how accessory proteins may modulate fidelity.
|
16936322_p2
|
16936322
|
INTRODUCTION
| 4.33088 |
biomedical
|
Study
|
[
0.9992882609367371,
0.0004586867871694267,
0.0002530812635086477
] |
[
0.9983721375465393,
0.000262034242041409,
0.0012530818348750472,
0.00011279070895398036
] |
en
| 0.999998 |
Yeast RPA, PCNA and RFC were purified from Escherichia coli overproducing strains as described elsewhere ( 24 , 25 ). All materials for the fidelity assay were from previously described sources ( 23 , 26 ).
|
16936322_p3
|
16936322
|
Materials
| 3.551251 |
biomedical
|
Study
|
[
0.9984921216964722,
0.00023108458844944835,
0.001276839291676879
] |
[
0.9798737168312073,
0.01843363791704178,
0.0014449912123382092,
0.0002477301750332117
] |
en
| 0.999995 |
Plasmid pBL335 (bluescript, 2 µM ori, TRP1 , M13 ori, GAL1-10 GST-POL3 ) contains the Schistosoma japanicum glutathione S -transferase gene (GST) fused to the N-terminus of the POL3 gene in vector pRS424-GALGSTPKA. The GST tag is separated from the POL3 gene by a recognition sequence for the human rhinoviral protease (LEVLFQ/GP), followed by a recognition site for the catalytic subunit of cAMP dependent protein kinase ( 27 ). After cleavage by the protease the N-terminal sequence of the Pol3 polypeptide is altered from MSEKRSLP M to GPEFRRASVGS M . Plasmid pBL341 (bluescript, 2 µM ori, URA3 , M13 ori, GAL1-10, POL31, POL32 ) has the POL31 and POL32 genes placed divergently under control of the bidirectional GAL1-10 promoter into vector pRS426-GAL. Plasmids and sequences are available upon request from P.M.B. Plasmid pBL335-DV (as pBL335, but pol3-5DV = pol3D520V ) was made by gap repair. pBL335 was cut with BglII and NdeI, releasing that portion of the POL3 gene containing the intended mutation, and the isolated large fragment was transformed into strain YH712 ( MAT α ade5-1 his7-2 leu2-3,112::lys2D5'-LEU2 lys2::InsHS-D trp1-289 ura3-52 pep4::KanMX pol3-5DV ) ( 28 ). After plasmid recovery, the mutation was confirmed by sequencing.
|
16936322_p4
|
16936322
|
Overexpression and purification of Pol δ
| 4.345788 |
biomedical
|
Study
|
[
0.9990509152412415,
0.0005788258276879787,
0.0003702749090734869
] |
[
0.9963139891624451,
0.003142179688438773,
0.00031859276350587606,
0.00022535726020578295
] |
en
| 0.999996 |
Overexpression was in Saccharomyces cerevisiae strain BJ2168 transformed with pBL341 and with either pBL335 or pBL335-DV. Growth and induction was as described elsewhere, and so was the preparation of cell lysates by blending with dry ice ( 29 ). Cells (60 g of packed cells resuspended in 20 ml of water) frozen previously in liquid nitrogen in the form of popcorn were blended with 40 ml of buffer 3A (buffer A, 30 mM HEPES–NaOH, pH 7.8, 10% glycerol, 2 mM EDTA, 1 mM EGTA, 0.02% Nonidet P-40, 2 mM DTT, 10 mM sodium bisulfite, 10 µM pepstatin A and 10 µM leupeptin). All further operations were carried out at 0–4°C. After thawing of the lysate, 1 mM phenylmethanesulfonyl fluoride (from a 100 mM stock in isopropanol) and 150 mM ammonium sulfate (from a 4 M stock) were stirred in, followed by 0.45% polymin P (from a 10% stock at pH 7.3). After stirring for 15 min, the lysate was cleared at 40 000 g for 40 min, and the supernatant precipitated with 0.28 g/ml of solid ammonium sulfate. The precipitate was collected at 40 000 g for 45 min, and redissolved in ∼75–125 ml of buffer A until the conductivity equals that of A 250 (subscript denotes NaCl concentration). Batch binding to 2 ml of glutathione–Sepharose 4B beads (GE Healthcare), equilibrated previously in buffer A 250 , was accomplished by gentle rotation in the cold room for 2 h. The beads were collected at 1000 r.p.m. in a swinging bucket rotor, batch washed, by resuspension and spinning, with 3× 30 ml of buffer A 250 , transferred to a 10 ml column, and washed at 2 ml/min with 100 ml of A 250 . Bound chaperones, particularly Ssa1, were removed by a 30 ml wash with A 250 containing 1 mM ATP and 5 mM Mg-acetate. After another 10 ml wash with A 150 to remove residual nucleotide and decrease salt, the beads were resuspended in 2 ml A 150 containing 20 mM glutathione (pH adjusted to 8.0). The capped column was incubated on ice for 10 min, and the eluant collected. This procedure was repeated four times. Most of the protein eluted in fractions 1–4. These fractions (∼0.5–1 mg protein) were incubated overnight at 4°C with 30 U of PreScission protease (GE Healthcare), and then directly loaded onto a 1 ml MonoS column as described elsewhere ( 30 ). Concentrated pure enzyme eluted at ∼350–400 mM NaCl.
|
16936322_p5
|
16936322
|
Overexpression and purification of Pol δ
| 4.37264 |
biomedical
|
Study
|
[
0.9989776611328125,
0.0006788953905925155,
0.0003434818936511874
] |
[
0.9978042244911194,
0.0015380593249574304,
0.0004629317845683545,
0.0001948211865965277
] |
en
| 0.999996 |
Reactions (25 µl) contained 20 mM Tris–HCl (pH 7.7), 8 mM MgAc 2 , 75 mM NaCl, 0.5 mM ATP, 100 µM of each dNTP, 1 mM DTT, 100 mg/ml BSA and 40 fmol (1.6 nM) gapped M13mp2 DNA. When included, the amounts of the accessory proteins used were 500 fmol PCNA, 200 fmol RFC and 10 pmol RPA, an amount more than sufficient to coat the single-stranded DNA within the gap. Polymerization reactions were performed at 30°C. The amount of pol δ and reaction times were: pol δ alone, 2.0 pmol for naked DNA or 1.5 pmol with RPA-coated DNA, both 30 min; pol δ plus accessory proteins, 150 fmol pol δ for 5 min for naked DNA and for 2 min with RPA-coated DNA. These quantities of pol δ and incubation times were chosen such that, when DNA products were analyzed by agarose gel electrophoresis as described elsewhere ( 23 ), all reactions filled the 407 nt gap without obvious strand displacement [data not shown, but for typical result see Figure 3 in Ref. ( 23 )]. Note that synthesis by pol δ alone is only moderately processive, such that complete gap filling likely involves multiple cycles of binding and dissociation. Importantly, reactions containing 150 fmol of pol δ alone failed to fill the gap, indicating that synthesis catalyzed by pol δ was indeed stimulated by the presence of the accessory proteins.
|
16936322_p6
|
16936322
|
Gap-filling DNA synthesis reactions and product analysis
| 4.226756 |
biomedical
|
Study
|
[
0.999423623085022,
0.0003474555560387671,
0.0002289632539032027
] |
[
0.9992350339889526,
0.0003723388654179871,
0.00030839364626444876,
0.00008420141239184886
] |
en
| 0.999998 |
DNA products of gap-filling reactions were introduced into E.coli cells and plated as described elsewhere ( 23 ) to score blue M13 plaques (correct synthesis) and light blue and colorless plaques (containing errors). The types of errors were determined by sequencing the lacZ α-complementation gene in single-stranded DNA isolated from independent mutant M13 plaques, allowing calculation of error rates as described previously ( 5 ). The statistical significance of differences in error rates with and without accessory proteins was calculated using the Fisher's exact test ( 31 ). Because several such comparisons were made, the multiple comparisons method of Benjamini and Hochberg ( 32 ) was used to control the false discovery rate to no more than 0.05.
|
16936322_p7
|
16936322
|
Gap-filling DNA synthesis reactions and product analysis
| 4.108135 |
biomedical
|
Study
|
[
0.9994716048240662,
0.0002644987544044852,
0.00026389784761704504
] |
[
0.9993495345115662,
0.0002585139009170234,
0.0003370384802110493,
0.000054967393225524575
] |
en
| 0.999996 |
Pol δ fidelity with and without accessory proteins was determined for synthesis to fill a single-stranded gap in a circular duplex M13mp2 DNA substrate. This gap contains the lacZ α-complementation template sequence that when copied correctly results in a blue M13 plaque phenotype. Polymerization errors are detected as light blue and colorless plaques. A total of 12 gap-filling reactions were conducted and the products analyzed for lacZ mutant frequencies. Four reactions contained wild-type pol δ ( Table 1 , Experiment 1), either alone, with RPA, with PCNA and RFC or with all three accessory protein complexes. The lacZ mutant frequencies for all reactions with wild-type pol δ were several-fold lower than for parallel reactions performed with exonuclease-deficient pol3-5DV pol δ (Experiment 2) or exonuclease-deficient pol3-01 pol δ (Experiment 3), reflecting the contribution of proofreading to the overall fidelity of the wild-type enzyme (see more below). DNA samples were prepared from independent lacZ mutants collected from each of the 12 reactions, and were sequenced to identify the types ( Table 1 ) and locations of sequence changes responsible for reduced plaque colors. As observed previously for three-subunit yeast pol δ alone ( 5 ), four main classes of sequence changes were observed: single base substitutions, single nucleotide deletions, single nucleotide insertions and deletions of larger numbers of nucleotides between direct repeat sequences ( Table 1 ). The mutant frequency and sequence specificity information ( Table1 ) was then used to calculate average rates (errors per detectable nucleotide polymerized) for single base errors ( Table 2 ) and mutant frequencies for large deletions ( Table 3 ).
|
16936322_p8
|
16936322
|
Fidelity measurements and calculation of error rates
| 4.246671 |
biomedical
|
Study
|
[
0.9993785619735718,
0.0003755320212803781,
0.00024588964879512787
] |
[
0.9993340373039246,
0.0002266761293867603,
0.00035289369407109916,
0.00008638112194603309
] |
en
| 0.999997 |
In order to separate the effects of the accessory factors on polymerase insertion fidelity from those on proofreading efficiency, we will first discuss our results with the exonuclease-deficient pol δ, followed by those with the wild-type enzyme. Because similar results were obtained for each of the two exonuclease-deficient polymerases, those data were combined.
|
16936322_p9
|
16936322
|
Fidelity measurements and calculation of error rates
| 3.954427 |
biomedical
|
Study
|
[
0.9994125366210938,
0.0002293591242050752,
0.00035815907176584005
] |
[
0.9984173774719238,
0.0007181827677413821,
0.0007872851565480232,
0.00007708404882578179
] |
en
| 0.999996 |
The most common errors generated by pol δ were single base substitutions. The calculated average single base substitution error rate of exonuclease-deficient pol δ alone is 6.3 × 10 −5 ( Table 2 ), confirming an earlier report that yeast pol δ has high base substitution fidelity ( 5 ). Interestingly, similar base substitution error rates were obtained for pol δ reactions that contained RPA, PCNA plus RFC or all three accessory proteins. Detecting errors in this type of fidelity assay requires both nucleotide misinsertion and extension of the resulting mismatch. Thus, the similarities in average base substitution error rates suggest that the accessory proteins do not strongly influence the nucleotide selectivity of the pol δ active site or strongly alter discrimination for extension of matched versus mismatched primer termini.
|
16936322_p10
|
16936322
|
Effects on selectivity against base–base mismatches
| 4.197137 |
biomedical
|
Study
|
[
0.99945467710495,
0.00030549027724191546,
0.00023984794097486883
] |
[
0.9993897676467896,
0.0002459139795973897,
0.00029660225845873356,
0.00006767040758859366
] |
en
| 0.999997 |
The base substitution values in Table 2 are average error rates for numerous different mismatches in a variety of sequence contexts. From these average rates, it cannot be excluded that the accessory proteins have modest effects on nucleotide selectivity and/or mismatch extension for specific mismatches and/or in specific sequence contexts. For example, a previous kinetic study demonstrated that PCNA decreases the selectivity of exonuclease-deficient yeast pol δ for misinsertions opposite a specific template guanine by 2- to 4-fold ( 4 ). Any such effects here would be small compared to the >10 000-fold contribution to selectivity conferred by exonuclease-deficient pol δ alone. This conclusion is reinforced by studies of several other replicative polymerases ( 17 – 20 , 33 – 38 ) indicating that accessory proteins have only small effects that sometimes enhance and sometimes reduce discrimination against base substitution errors. Thus we conclude that pol δ itself is by far the primary determinant of selectivity against base substitution errors resulting from copying an undamaged DNA template.
|
16936322_p11
|
16936322
|
Effects on selectivity against base–base mismatches
| 4.304262 |
biomedical
|
Study
|
[
0.9994376301765442,
0.0003007578488904983,
0.000261655542999506
] |
[
0.99919193983078,
0.00027821792173199356,
0.0004539591900538653,
0.00007588072185171768
] |
en
| 0.999997 |
An estimate of the contribution of proofreading to base substitution fidelity in the absence and presence of the accessory proteins can be obtained by comparing error rates for exonuclease-deficient pol δ to those for wild-type pol δ ( Table 2 ). We believe that these are minimal estimates because the base substitutions recovered from reactions by wild-type pol δ are thought to largely reflect background noise in the assay due to very low levels of spontaneous substitutions and cryptic damage (e.g. cytosine deamination) in the gapped DNA substrate. With this caveat in mind, the ratios of base substitution error rates are similar for all four reactions. This is interesting in light of studies indicating that proteins that increase processivity promote extension of base–base mismatches by T7 DNA polymerase ( 18 ), T4 DNA polymerase ( 19 ) and human DNA polymerase γ ( 20 ). The present results suggest that the accessory proteins neither strongly enhance nor strongly suppress proofreading by pol δ when copying an undamaged DNA template, as expected in order to maintain both high processivity and high fidelity during yeast chromosomal DNA replication.
|
16936322_p12
|
16936322
|
Effects on proofreading of base–base mismatches
| 4.30914 |
biomedical
|
Study
|
[
0.9993759989738464,
0.0003786291053984314,
0.0002454398781992495
] |
[
0.9992375373840332,
0.00026505341520532966,
0.00040180422365665436,
0.00009564169158693403
] |
en
| 0.999997 |
The second most common single base error made by pol δ is deletion of 1 nt ( Table 2 ). The rates at which these errors are generated by pol δ in the absence or presence of RFC/PCNA and/or RPA either do not differ in a statistically significant manner, or they differ in a statistically significant manner but by <2-fold. Such effects are small relative to the 100 000-fold discrimination imposed by the polymerase alone, indicating that PCNA, RFC and RPA contribute very little to preventing single base deletion errors by pol δ. However, in all four reactions, single base deletion error rates are higher for exonuclease-deficient pol δ than for wild-type pol δ, suggesting that misaligned intermediates are proofread with or without the accessory proteins present.
|
16936322_p13
|
16936322
|
Effects on single nucleotide deletion and addition errors
| 4.228887 |
biomedical
|
Study
|
[
0.999363124370575,
0.00035503454273566604,
0.0002819089568220079
] |
[
0.9993101358413696,
0.0003247549175284803,
0.0002893579949159175,
0.00007569317676825449
] |
en
| 0.999998 |
Exonuclease-deficient pol δ also generates single base additions at a readily detectable overall average rate of 0.66 × 10 −5 ( Table 2 ). This rate is not appreciably influenced by RPA (0.80 × 10 −5 ), but is reduced ∼7-fold by PCNA plus RFC . Wild-type pol δ alone (error rate ≥0.026 × 10 −5 ) is at least 25-fold more accurate for one base additions than exonuclease-deficient pol δ. This difference is larger than observed for single base deletions, supporting our earlier interpretation ( 5 ) that pol δ proofreads addition intermediates more efficiently than deletion intermediates. Just as for deletions, single base addition error rates in all four reactions are higher for exonuclease-deficient pol δ than for wild-type pol δ, suggesting that addition intermediates are proofread with or without accessory proteins present.
|
16936322_p14
|
16936322
|
Effects on single nucleotide deletion and addition errors
| 4.260118 |
biomedical
|
Study
|
[
0.9993782043457031,
0.0003368646721355617,
0.0002848147414624691
] |
[
0.9993047714233398,
0.0003400136192794889,
0.000274633668595925,
0.00008061242260737345
] |
en
| 0.999997 |
The effects of accessory proteins on single base error rates are modest in comparison with the dominant role of the polymerase itself in discriminating against single base errors. However, the situation is different for deletions of larger numbers of nucleotides located between direct repeat sequences. We found previously that pol δ alone is particularly prone to generating these types of deletions ( 5 ), and does so at frequencies that are similar for the wild-type and exonuclease-deficient enzymes, indicating that the misaligned intermediates are not efficiently proofread. The present study confirms those observations ( Table 3 , line 1). More importantly, the results show that the ability of pol δ to generate large deletions between direct repeats is strongly suppressed by RPA alone (line 2) or by PCNA plus RFC (line 3). Moreover, no deletions between direct repeats were generated by wild-type pol δ in the presence of all three accessory proteins, representing a ≥90-fold increase in fidelity for this class of errors.
|
16936322_p15
|
16936322
|
Effects on deletions between direct repeats
| 4.227528 |
biomedical
|
Study
|
[
0.9994602799415588,
0.0003055566630791873,
0.0002341776853427291
] |
[
0.9993498921394348,
0.00020924358977936208,
0.000367062195437029,
0.00007378601731033996
] |
en
| 0.999998 |
How RPA and PCNA may suppress formation of large deletions by pol δ can be considered in light of a model for deletions between direct repeats [ Figure 2 ( 39 )]. In this model, after the first repeat sequence encountered by the polymerase is copied, the primer frays and then relocates to the second repeat sequence. Hybridization would provide a duplex primer–template for continued polymerization, but in a misaligned intermediate that contains a loop of unpaired, template strand bases that are eventually deleted. In certain sequence environments, when an inverted repeat is present at the deletion junction, the misaligned intermediate may be stabilized through formation of a stem at the base of the single-stranded loop . Within the context of this model, PCNA may suppress deletion formation by preventing fraying of the primer terminus at the first repeat, and/or by preventing relocation of the primer to the downstream repeat. RPA may suppress deletion formation by coating the single-stranded DNA to reduce the probability of DNA rearrangement and/or prevent the frayed primer terminus from annealing to the downstream repeat. The observation that all three accessory proteins together yield lower large deletion frequencies than either RPA alone or PCNA plus RFC ( Table 3 ) is consistent with the idea that RPA and PCNA affect large deletion fidelity at least partly by different mechanisms. This possibility is further supported by the observation that RPA suppresses deletions generated by exonuclease-deficient pol δ that can be modeled by both pathways depicted in Figure 2 to about the same extent (∼10-fold, Table 3 ). However, while PCNA also strongly suppresses deletions between repeats that are potentially stabilized by a stem containing two or more correct base pairs , it has little effect on deletions that lack an obvious inverted repeat . By virtue of their ability to prevent wild-type pol δ from generating these types of replication errors, PCNA and RPA may have important roles in protecting eukaryotic genomes against the biological consequences of large deletions, and perhaps also in modulating the stability of large tracts of repetitive sequences whose instabilities are associated with hereditary degenerative diseases.
|
16936322_p16
|
16936322
|
Effects on deletions between direct repeats
| 4.737188 |
biomedical
|
Study
|
[
0.9987497329711914,
0.0008398336940445006,
0.0004103759420104325
] |
[
0.9962372779846191,
0.0010342964669689536,
0.002378112869337201,
0.00035032755113206804
] |
en
| 0.999997 |
The accuracy of intron excision from pre-mRNAs relies on the precise recognition of rather degenerate splicing signals. Splice sites are defined by consensus sequences where only the two terminal nucleotides [GT at the acceptor (5′) and AG at the donor (3′) splice sites for the majority of introns] are highly conserved. The fidelity of the splicing process is attained through various exonic and intronic sequences acting as splicing enhancers or silencers thereby influencing splice site selection. The precise recognition of splice sites is achieved by the spliceosome, a large RNP particle consisting of 5 snRNPs and ∼200 different proteins ( 1 ). Out of these, Ser/Arg-rich (SR) proteins play an important role in the regulation of splice site selection. SR proteins are a family of evolutionary conserved, structurally related, splicing factors which possess one or two RNA-recognition motifs (RRM) at the N-terminus and a C-terminal arginine/serine-rich (RS) domain ( 2 , 3 ). They are important for recognizing splice sites or exonic splicing enhancers and facilitate spliceosome assembly. It has been demonstrated that some SR proteins influence alternative splice site selection in quantitative manner ( 4 ). Consequently, SR proteins are one of the determinants of splicing pattern present in different cells and at specific developmental stages or conditions.
|
16936312_p0
|
16936312
|
INTRODUCTION
| 4.917306 |
biomedical
|
Study
|
[
0.9972122311592102,
0.001499249367043376,
0.0012885017786175013
] |
[
0.7512384057044983,
0.003608601400628686,
0.24382120370864868,
0.0013318239944055676
] |
en
| 0.999996 |
Alternative splicing is an important mechanism for the generation of proteome complexity and fine tuning of gene expression at the post-transcriptional level both in the metazoan and plant species. Differential intron removal allows production of splice variants which may code for distinct protein isoforms affecting their post-translational modification, subcellular localization and the ability to interact with their binding partners. In addition, alternative splicing can also influence translational control and stability of mRNA transcripts.
|
16936312_p1
|
16936312
|
INTRODUCTION
| 4.274549 |
biomedical
|
Study
|
[
0.9984182119369507,
0.0007500601350329816,
0.0008317132014781237
] |
[
0.452223002910614,
0.09580313414335251,
0.4504108130931854,
0.0015631492715328932
] |
en
| 0.999996 |
Our analysis of the Arabidopsis genome has revealed 19 SR proteins which is almost twice as much as in humans ( 5 , 6 ). They fall into seven subfamilies ( 6 ) some of which have orthologues in metazoan (SF2/ASF, 9G8, SC35) but interestingly three of them seem to be plant-specific (RS, RS2Z and SCL). Many of the SR protein genes are alternatively spliced both in metazoa and in Arabidopsis ( 7 – 16 ). It has been noted that alternative splicing occurs mainly in and around the long introns of the Arabidopsis SR genes ranging in size from ∼400 to 1100 nt ( 6 ), while the typical size of plant introns is <150 nt ( 17 ). In atSRp30 and atSRp34/SR1 , two Arabidopsis homologues of human SF2/ASF, such a long intron is situated near the 3′ end of the gene, and alternative splicing in this intron results in protein isoforms with shortened RS domains ( 12 , 15 ). Interestingly, all three plant-specific gene subfamilies possess a long intron at the beginning of the gene dividing the first RRM. Alternative splicing of this intron occurs in all three subfamilies and in a way that the potential protein is extremely truncated containing only a part of the RRM ( 6 , 13 , 14 ). Alternative splicing in the long intron of atRSZ33 , a member of the RS2Z subfamily is autoregulated, as demonstrated by overexpression experiments ( 16 ). This autoregulation is crucial for correct gene expression levels, because overexpression of an intronless version of atRSZ33 is lethal.
|
16936312_p2
|
16936312
|
INTRODUCTION
| 4.487518 |
biomedical
|
Study
|
[
0.9991469383239746,
0.0004139212251175195,
0.00043918672599829733
] |
[
0.9989853501319885,
0.0003690442827064544,
0.0005295180599205196,
0.0001161774416686967
] |
en
| 0.999996 |
In this study, we extend the analysis of alternative splicing and its regulation to the plant-specific RS subfamily, focusing on atRSp31 . We also show that atRSZ33 , a member of the RS2Z subfamily, is involved in this regulation. In addition, we show that the position of the long intron and its capacity for alternative splicing is a characteristic feature of the plant-specific families of SR genes and is conserved from green algae to flowering plants. Moreover, sequences around the alternative splice sites in the long introns are much more conserved over large evolutionary distances than those in the region of the constitutive splice sites in the respective genes, indicating that they are under strong selective pressure.
|
16936312_p3
|
16936312
|
INTRODUCTION
| 4.204835 |
biomedical
|
Study
|
[
0.9991374015808105,
0.00033018813701346517,
0.000532439153175801
] |
[
0.9995300769805908,
0.00017079136159736663,
0.00024647609097883105,
0.000052569354011211544
] |
en
| 0.999997 |
Arabidopsis and rice sequences can be found at and under following numbers: atRSp31 , atRSp31a , atRSp40 , atRSp41 , osRSp29 , osRSp33 , atRSZ32 , atRSZ33 , osRSZ36 , osRSZ37a , osRSZ37b and osRSZ39 . Accession numbers of Arabidopsis , rice, maize, Pinus taeda , Physcomitrella patens and Chlamydomonas reinhardtii transcripts are in Supplementary Data.
|
16936312_p4
|
16936312
|
Accession numbers
| 1.761809 |
biomedical
|
Other
|
[
0.9825687408447266,
0.0009179117623716593,
0.01651330105960369
] |
[
0.09299678355455399,
0.9045491814613342,
0.0013974595349282026,
0.0010566443670541048
] |
en
| 0.999997 |
To identify splice variants of Arabidopsis SR proteins, corresponding genomic sequences were used in BLAST search against EST database limited to Arabidopsis thaliana at . ESTs with matches to corresponding gene were selected and then aligned using Geneseqer ( 18 ) at .
|
16936312_p5
|
16936312
|
Sequence retrieval and analysis
| 3.892373 |
biomedical
|
Study
|
[
0.9991328120231628,
0.00017631259106565267,
0.0006909661460667849
] |
[
0.9978062510490417,
0.0018866690807044506,
0.0002415578783256933,
0.00006550041143782437
] |
en
| 0.999996 |
Subsets and Splits
Clinical Cases Sample
Retrieves 100 samples of clinical cases, providing a basic overview of this specific document type.
High-Score Clinical Cases
The query retrieves a limited set of clinical case documents with a high educational score, providing a basic filtered view of the dataset.