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These probes would also be phylogenetically scalable, providing nested sets of informative markers from the population level up to major clades. This type of data would overcome many of the shortcomings currently apparent in the field of phylogenomics ( 23 , 24 ). If the standard set of probe sequences could not provide enough detail at finer scales of populations and recent evolutionary events, the informative sequences could be used as ‘seeds’ from which to produce a large degenerate sets of anonymous oligonucleotide sequences. These custom designed degenerate sets would then provide a more focused and detailed result for the target genomes. By applying a more detailed set of sequence selection criteria, certain regions of known genomes or genomic compartments within cells could be targeted and screened in a much wider range of organisms. These informative sets of sequences could address a wide range of ecological questions, including the rates of gene flow across landscapes. The development of commercial applications, particularly in the natural resources trade, using genomic signatures to identify species and geographic origin certainly seem possible and could provide an objective means for determining legality of the harvest. It would also allow evolutionary biologists to pinpoint the relevant genomic differences among sets of target genomes and generate sequence tags to explore these regions more in detail.
|
17000641_p25
|
17000641
|
DISCUSSION
| 4.291623 |
biomedical
|
Study
|
[
0.9992144107818604,
0.0003025720070581883,
0.00048305970267392695
] |
[
0.8884519934654236,
0.010202236473560333,
0.10090956836938858,
0.00043626787373796105
] |
en
| 0.999997 |
The primary motivation for this study and the development of the SHyP approach is the speed with which human activity is significantly modifying the distribution and composition of genomic diversity in natural communities around the globe ( 25 ). To properly interpret the present ecological and evolutionary situation and to plan for the future management and conservation of these natural resources, the historical past holds the key. This fundamental baseline of data must be collected prior to wholescale modification. The SHyP genomic signature database, once constructed, could provide great acceleration in the effort to recognize and catalog life's diversity using a DNA fingerprint. The Bar Code of Life approach hangs the identification of all taxa on the DNA sequence variation at one or a few genomic loci. While this approach appears promising in some situations ( 26 – 29 ), the possibility of obtaining positively misleading results, particularly if the Bar Code is based upon a cytoplasmic locus ( 30 – 33 ), could easily skew the original objectives of the initiative. Further perspective on the BCoL approach could easily be gained by following our example here and exploring its application in the best known groups, like mice and men. While the current Bar Code has been lauded as a way to identify ‘cryptic’ species ( 34 ), the objective definition of ‘species’ may become particularly problematic when applied to human races ( 35 , 36 ). For the analogy to the commercial version of barcode or ‘automated identification and data capture’ technologies to be true, the code must involve more than a single or even a handful of bars. The SHyP-CGH database would fulfill this analogy by examining the hybridization intensities at tens of thousands of loci and with a quick query of the database, a biologist could obtain potentially thousands of informative markers directly pertinent to the question without performing any microarray work. These markers would also not be limited to simply identifying the organism but could be used for a wide variety of other purposes.
|
17000641_p26
|
17000641
|
DISCUSSION
| 4.284527 |
biomedical
|
Study
|
[
0.9993135929107666,
0.00027266755932942033,
0.0004137340874876827
] |
[
0.9982203841209412,
0.0004262768488842994,
0.0012789495522156358,
0.00007436375017277896
] |
en
| 0.999997 |
RNA interference is a process which leads to degradation of endogenous mRNA via formation of duplexes with small non-coding RNAs. The application of RNA interference has revolutionized the functional analysis of genes and may also facilitate gene therapeutic approaches ( 1 – 5 ). Several different strategies have been devised to achieve RNA interference in cells. For example, synthetic short interfering RNAs (siRNA) of 21 nt can be efficiently transfected into cells. Alternatively, transfection of plasmids encoding shRNAs (short hairpin), which are processed to siRNAs by the enzyme DROSHA, is commonly used. It is possible to achieve up to 90% reduction in expression of the respective protein by RNA interference. Transiently introduced siRNAs are effective for up to 5 days as they are degraded with time and diluted during cell divisions. For permanent gene inactivation it is necessary to stably integrate plasmids driving the expression of shRNAs into the genome of the respective target cell. Initially, RNA polymerase III (Pol III) promoters were used to mediate permanent shRNA expression ( 6 ). Recently, Pol II promoters have been employed to drive the expression of precursor microRNAs ( 7 ). The constitutive expression of siRNAs may however compete with endogenous microRNAs, leading to toxic side effects.
|
16998186_p0
|
16998186
|
INTRODUCTION
| 4.71657 |
biomedical
|
Study
|
[
0.9982341527938843,
0.0008702241466380656,
0.0008955924422480166
] |
[
0.647683322429657,
0.0028180787339806557,
0.3486301600933075,
0.0008683750638738275
] |
en
| 0.999996 |
For the biochemical and functional analysis of essential genes conditional approaches are necessary. Furthermore, the analysis of isogenic cell populations which only differ in the activation state of a conditional allele has advantages when compared to the analysis of cells permanently expressing a knock-down construct. In the latter case multiple clones obtained by tedious single cell cloning and expansion have to be analyzed to avoid clonal variations. In addition, the long-term down-regulation of proteins by stable knock-down strategies may lead to compensatory activation of parallel pathways, obscuring the initial effect of the gene-specific inactivation. Therefore, the conditional activation of microRNA expression provides significant advantages.
|
16998186_p1
|
16998186
|
INTRODUCTION
| 4.137682 |
biomedical
|
Study
|
[
0.9995611310005188,
0.00017499097157269716,
0.0002638069854583591
] |
[
0.9743826389312744,
0.012160475365817547,
0.013254926539957523,
0.00020190233772154897
] |
en
| 0.999999 |
Although a number of systems for conditional gene expression have been developed, the Tet-repressor system was most widely applied in recent years ( 8 ). In this system elements of the bacterial tet operon were transferred into mammalian cells: the tet-repressor was fused to the VP16 transactivation domain (tTA). In the presence of tetracycline or its derivative doxycycline (DOX) this fusion protein will not bind a promoter which harbors a tet-repressor binding site. Later a mutant tet-VP16 fusion was developed which only binds to DNA in the presence of DOX (rTA, reverse tetracycline controlled transactivator) ( 9 ). In many cases the leakiness of the regulated promoters has presented a problem. This can be avoided by the simultaneous expression of a tet-repressor fused to the KRAB protein (tTS KRAB ), which keeps the inducible gene inactive in the absence of doxycycline ( 10 ). Bornkamm et al. ( 11 ) combined recently the rTA and tTS KRAB in the episomal pRTS-1 vector, which allows conditional expression of cDNAs in mammalian cells. Here we demonstrate that the pRTS-1 vector can be used to conditionally regulate the expression of microRNAs mediating RNA-interference, thereby providing a convenient tool to determine gene functions.
|
16998186_p2
|
16998186
|
INTRODUCTION
| 4.421978 |
biomedical
|
Study
|
[
0.99954754114151,
0.00028300302801653743,
0.00016946571122389287
] |
[
0.9980511665344238,
0.0006183635559864342,
0.0012075906852260232,
0.0001229017652804032
] |
en
| 0.999996 |
U2OS osteosarcoma cells were maintained in DMEM containing 10% fetal bovine serum and penicillin (100IE)/streptomycin (100 μg/ml). Doxycycline (Sigma) was resolved in water (1 mg/ml). Etoposide was resolved in DMSO (40 mg/ml) and used at a final concentration of 20 μg/ml. Poly(I:C) (Sigma) was resolved in water (10 mg/ml) and used at a final concentration of 10 μg/ml.
|
16998186_p3
|
16998186
|
Cell lines/culture and reagents
| 3.947846 |
biomedical
|
Study
|
[
0.9991796612739563,
0.000370372406905517,
0.0004499954520724714
] |
[
0.7924782633781433,
0.20506015419960022,
0.001613493892364204,
0.0008481579716317356
] |
en
| 0.999996 |
For generation of the shuttle vector pSHUMI the oligos pUC19linker-fw 5′-AATTGGGCCTCACTGGCCACCGGAGATCTGTCGACGGACGCGTACCGGTG-3′ and pUC19linker-rv 5′-TCGACACCGGTACGCGTCCGTCGACAGATCTCCGGTGGCCAGTGAGGCCC-3′ were annealed and inserted into the EcoRI/XhoI sites of pUC19 resulting in pUC19m. A BglII/AgeI fragment containing miR30 sequences from the LMP plasmid ( 12 ) was inserted into the BglII/AgeI sites of pUC19m. The resulting pSHUMI plasmid can be used to subclone short hairpin sequences using the XhoI and EcoRI restriction sites.
|
16998186_p4
|
16998186
|
Generation of plasmids
| 4.16721 |
biomedical
|
Study
|
[
0.999297022819519,
0.0003019866708200425,
0.0004009674012195319
] |
[
0.9810982942581177,
0.0180300772190094,
0.0005874946364201605,
0.0002841320529114455
] |
en
| 0.999997 |
For generation of the pEMI vector, regions containing 5′miR30 and homology region 2 (HR2) were amplified from pSM2c ( 7 ) using the primers 5′miR30-frw 5′-CGAGATCTTGTTTGAATGAGGCTTCAGTAC-3′ and 5′miR30-rev 5′-GCACCGGTGCGGCCGCCTCGAGCCTTCTGTTGGGTTAACC-3′ and HR2-frw 5′-CGCTCGAGATCCATGGCATATGGGATCCAAGGCAGTTATTGGTGCCCTTAAAC-3′HR2-rev 5′-GCACCGGTTCAGATCCTCTTCGGAGATCAG-3′and inserted into the pTOPO vector (Invitrogen). The 5′miR30 part was subcloned into pSHUMI using BglII/AgeI restriction sites and the HR2 region was introduced using XhoI/AgeI restriction sites. The PheS expressing cassette was released from pBSPheS ( 13 ) using NcoI/BglII and ligated between the 5′miR30 and HR2 sequences into the NcoI/BamHI sites. From the resulting vector a fragment containing the 5′miR30/PheS/HR2 region was released by SfiI and inserted into the SfiI sites of pRTS-1 resulting in pEMI (plasmid for episomal microRNA expression).
|
16998186_p5
|
16998186
|
Generation of plasmids
| 4.236403 |
biomedical
|
Study
|
[
0.9993446469306946,
0.0003126001392956823,
0.00034276925725862384
] |
[
0.9970183372497559,
0.002541683614253998,
0.0003092436818405986,
0.00013071842840872705
] |
en
| 0.999997 |
A p53-specific hairpin was released from pSM2c ( 7 ) using XhoI/EcoRI and inserted into pSHUMI. A MAD2 -specific microRNA was generated in a two-step PCR: the primers forward 5′-tgctgttgacagtgagcgCTGGGAAGAGTCGGGACCACAGtagtgaagccacagatg-3′ and reverse 5′-tccgaggcagtaggcaATGGGAAGAGTCGGGACCACAGtacatctgtggcttcac-3′were annealed, extended by PCR and amplified using universal miR30XhoI/EcoRI primers (miR30Xho I Fw, 5′-CAGAAGGCTCGAGAAGGTATATTGCTGTTGACAGTGAGCG-3′; miR30EcoR I Rv, 5′-CTAAAGTAGCCCCTTGAATTCCGAGGCAGTAGGCA-3′. The resulting fragment was cut with XhoI and EcoRI and inserted into pSHUMI. The SfiI fragments from pSHUMI containing the microRNA cassettes were inserted into pRTS-1.
|
16998186_p6
|
16998186
|
Restriction mediated microRNA transfer
| 4.212659 |
biomedical
|
Study
|
[
0.9994320273399353,
0.00027145753847435117,
0.000296577753033489
] |
[
0.9966579675674438,
0.0029194410890340805,
0.00027821780531667173,
0.00014434616605285555
] |
en
| 0.999998 |
Ligation-free gene transfer using the MAGIC system was done essentially as described ( 13 ). In brief, the donor bacterial strain (DH10βF′DOT sbcC, PIR1 positive) was transformed with a p53-specific pSM2c vector and grown on kanamycin (30 μg/ml) containing Luria–Bertani (LB) plates at 37°C. The recipient strain was transformed with the recipient vector pEMI and grown in the presence of 100 μg/ml carbenicilin, 50 μg/ml spectinomycin and 0.2% (w/v) glucose on LB plates at 30°C. Donor and recipient colonies were used to inoculate overnight liquid cultures. The recipient bacteria were washed twice with LB. Both donor and recipient bacteria were diluted 1:50 with LB/0.2% (w/v) rhamnose and grown at 30°C until an OD 600 of 0.15–0.25. The bacteria were mixed for conjugation in the presence of 0.2% (w/v) l -arabinose and incubated at 37°C for 2 h without and for 2 h with agitation. Recombinant bacteria were plated on a selection plate containing of chloramphenicol (30 μg/ml), carbenicilin (100 μg/ml) and 10 mM DL-p-chlorophenylalanine and incubated at 42°C overnight. Recombination events were detected by colony PCR using the primers [CmR-frw, 5′-CCGTTTGTGATGGCTTCCATGTC-3′ (corresponding to the chloramphenicol resistance) and pEMI-rev 5′-AATCAAGGGTCCCCAAACTC-3′ (matching to pEMI)].
|
16998186_p7
|
16998186
|
Mating-assisted genetically integrated cloning (MAGIC)
| 4.20002 |
biomedical
|
Study
|
[
0.9994208812713623,
0.00039037843816913664,
0.00018876424292102456
] |
[
0.9987542629241943,
0.0007035377784632146,
0.0004297562991268933,
0.00011233749683015049
] |
en
| 0.999996 |
U2OS osteosarcoma cells were transfected by lipofection with pEMI-plasmids using FuGene reagents (Roche) according to the manufacturer's instructions. After 48 h, cells were selected in media containing 150 μg/ml hygromycin for 7 days. Homogenicity of the selected cell pools was tested by addition of 100 ng/ml doxycycline for 24 h and monomeric red fluorescent protein (mRFP)-fluorescence detection.
|
16998186_p8
|
16998186
|
Generation of cell pools
| 4.150452 |
biomedical
|
Study
|
[
0.9994537234306335,
0.0003581817145459354,
0.00018817390082404017
] |
[
0.9943168759346008,
0.004997221753001213,
0.0005082395509816706,
0.00017763857613317668
] |
en
| 0.999998 |
U2OS (5 × 10 4 ) were plated into T25 cell culture flasks. Floating cells and trypsinized cells were collected by centrifugation at 1700 r.p.m. (600 g ) for 7 min, cells were fixed with ice-cold 70% ethanol and stored over night on ice. After washing with phosphate-buffered saline (PBS), 1 ml FACS solution [PBS, 0.1% Triton X-100, 60 μg/ml propidium iodide (PI) and 0.5 mg/ml DNase free RNase] was added per sample and incubated at room temperature for 30 min. DNA content was determined by propidium iodide staining (FACSCalibur, Becton-Dickinson).
|
16998186_p9
|
16998186
|
DNA content analysis by FACS
| 4.150245 |
biomedical
|
Study
|
[
0.999376118183136,
0.0004030982090625912,
0.00022079450718592852
] |
[
0.9923323392868042,
0.006934768054634333,
0.0005379607900977135,
0.00019484569202177227
] |
en
| 0.999994 |
Cells were lysed in RIPA lysis buffer [50 mM Tris–HCl, pH 8.0, 250 mM NaCl, 1% NP40, 0.5% (w/v) sodium deoxycholate, 0.1% SDS and complete mini protease inhibitors (Roche)]. Lysates were sonicated and centrifuged at 13 000 r.p.m. (16 060 g ) for 15 min at 4°C. Per lane 30 μg of whole cell lysate was loaded on 12% SDS–acrylamide gels, separated and transfered on Immobilon PVDF membranes (Millipore corporation, MA). For immunodetection membranes were incubated with antibodies specific for p53 (DO-1; Santa Cruz), p21 (Ab-11; NeoMarkers), Mad2 (Clone 48, BD Biosciences) and β-actin . HRP (horse-radish peroxidase)-coupled secondary antibodies were detected by enhanced chemiluminescence (Perkin Elmer Life Sciences, Boston).
|
16998186_p10
|
16998186
|
Western blot analysis
| 4.129846 |
biomedical
|
Study
|
[
0.9995338916778564,
0.0002132873487425968,
0.00025280186673626304
] |
[
0.9959171414375305,
0.0033931778743863106,
0.000574059784412384,
0.00011569795606192201
] |
en
| 0.999996 |
After treatment of U2OS with 100 ng/ml doxycycline or transfection with poly(I:C) (Sigma), total RNA was isolated (Total RNA Isolation System; Promega, Madison, USA). cDNA was generated from 1 μg total RNA per sample using anchored oligo-dT primers (Reverse-iT First Strand Synthesis; ABgene). qPCR was performed by using the LightCycler (Roche) and the FastStart DNA Master SYBR Green 1 kit (Roche Applied Science). The following primer sets were used: IFIT1 , 5′-GCCATTTTCTTTGCTTCCCCTA-3′and 5′-TGCCCTTTTGTAGCCTCCTTG-3′; β- actin , 5′-tgacattaaggagaagctgtgctac-3′ and 5′-gagttgaaggtagtttcgtggatg-3′; Mad2-specific-microRNA, 5′-GATGTACTGTGGTCCCGACTCT-3′ and 5′-TCAAAGAGATAGCAAGGTATTCAGT-3′.
|
16998186_p11
|
16998186
|
Quantitative real-time PCR (qPCR)
| 4.113687 |
biomedical
|
Study
|
[
0.9995889067649841,
0.00020918468362651765,
0.0002019870007643476
] |
[
0.9984933137893677,
0.0011147498153150082,
0.0003042583412025124,
0.0000877135171322152
] |
en
| 0.999996 |
The generation of specific PCR products was confirmed by melting curve analysis and gel electrophoresis (data not shown). Fold inductions were calculated as described previously ( 14 ) with standardization using β-actin.
|
16998186_p12
|
16998186
|
Quantitative real-time PCR (qPCR)
| 3.803547 |
biomedical
|
Study
|
[
0.9995075464248657,
0.00020332279382273555,
0.00028919693431816995
] |
[
0.9977790713310242,
0.0017564635490998626,
0.0003632022126112133,
0.0001011847925838083
] |
en
| 0.999997 |
mRFP and phase contrast images of living cells were obtained on an inverted Axiovert 200M microscope (Zeiss, Oberkochem, Germany) using Metamorph software (Universal Imaging).
|
16998186_p13
|
16998186
|
Microscopy
| 3.053557 |
biomedical
|
Study
|
[
0.998627781867981,
0.0005295926239341497,
0.0008425769046880305
] |
[
0.5519657731056213,
0.44360774755477905,
0.0028542932122945786,
0.0015721700619906187
] |
en
| 0.999996 |
Recently, comprehensive libraries of microRNAs which were designed to facilitate the RNA interference mediated down-regulation of all human or mouse genes have been described ( 7 ). These microRNAs are publicly available and are provided in the pSHAG-MAGIC 2c (pSM2c) retroviral vector ( ), which provides constitutive expression driven by a long-terminal repeat (LTR). Here, we have chosen several microRNA cassettes from the human library for transfer from pSM2c vectors to the pRTS-1 vector. Since pRTS is a relatively large vector (∼18 kb) the intermediate pUC19-based shuttle vector pSHUMI (plasmid for shuttling microRNAs) is necessary for the transfer procedure . For a faster transfer of microRNAs we also adapted the pRTS vector to the ligation-free MAGIC technique (mating-assisted genetically integrated cloning) ( 13 ). The resulting pEMI harbors a pheS Gly294 allele encoding a tRNA synthase for phenylalanine with relaxed specificity, which incorporates toxic chloro-phenylalanine and thereby facilitates selection against non-recombinant clones. Bacteria containing the pEMI-recipient vector were conjugated with bacteria containing a pSM2c vector encoding a p53-specific microRNA. Of 80 resulting bacterial colonies 79 (98.7%) harbored pEMI vectors containing the p53-microRNA as determined by colony PCR (data not shown). Successful recombination was also confirmed by restriction and sequence analysis (data not shown).
|
16998186_p14
|
16998186
|
Construction of episomal vectors for RNAi
| 4.219982 |
biomedical
|
Study
|
[
0.9994814991950989,
0.0002729529805947095,
0.0002455758221913129
] |
[
0.9993945360183716,
0.00024122932518366724,
0.00030106341000646353,
0.0000631782240816392
] |
en
| 0.999998 |
The human osteosarcoma cell line U2OS was transfected with pEMI vectors encoding either p53- or MAD2-specific or non-silencing microRNAs, which do not recognize any human mRNA. Selection for cells containing the pEMI vectors with hygromycin B was completed within 7 days. The resulting pools of resistant cells were analyzed for RT–PCR analysis to determine the expression of the ectopic microRNA after addition of doxycycline (DOX) . In the absence of DOX no MAD2-specific microRNA was detected after 30 PCR cycles. However, 24 h after addition of DOX the microRNA was expressed. By 48 h the expression increased further as determined by quantitative PCR (data not shown). As no microRNA expression was detected in the absence of DOX, these results show that the pEMI vectors mediate an extremely stringent control over microRNA expression. In line with this observation the cell pools were consistently devoid of mRFP expression in the absence of DOX as determined by live cell fluorescence microscopy . Within 24 h after addition of DOX approximately half of the cells were positive for mRFP at 3.2 ng/ml DOX and virtually all cells were positive at 25 ng/ml . Even at only 0.8 ng/ml DOX a decrease in p53 protein levels was observed within 24 h . At a DOX concentration of 25 ng/ml the degree of p53 down-regulation and frequency of mRFP-positive cells reached its maximum and p53 protein was hardly detectable. Similar results were obtained with microRNAs specific for MAD2 , although the maximal down-regulation of MAD2 was reached at higher DOX-concentrations, presumably due to the different half-lives of p53 and MAD2 or varying efficiencies of the respective microRNAs. Induction of a non-silencing microRNA did not affect the levels of p53 protein. In a time course analysis the level of p53 protein began to decrease one day after induction of the p53 microRNA . At 5 ng/ml DOX the maximum knock-down was reached after 2–3 days. After a 48 h exposure to DOX, its removal led to complete restoration of p53 expression within 6 days which was paralleled by the disappearance of mRFP as determined by fluorescence microscopy .
|
16998186_p15
|
16998186
|
Functional evaluation of pEMI vectors
| 4.452823 |
biomedical
|
Study
|
[
0.9992071986198425,
0.0005454130005091429,
0.0002473622444085777
] |
[
0.9988501071929932,
0.0003910122613888234,
0.0005984892486594617,
0.00016046561358962208
] |
en
| 0.999997 |
When p53 was activated by treatment with the DNA damaging agent etoposide, an increase in the levels of p53 and its target p21 was observed within 3.5 h . Induction of the p53-specific microRNA 2 days before etoposide addition resulted in strong suppression of p53 and prevented any significant increase in p53 and p21 protein after DNA damage. A control cell population expressing a non-silencing microRNA showed normal stabilization of p53 and p21 induction after addition of etoposide . Furthermore, a flow cytometric analysis revealed that suppression of p53 inhibited arrest in the G 1 phase upon etoposide treatment, whereas in the absence of p53-specific microRNAs, or upon expression of the non-silencing microRNA cells arrested normally .
|
16998186_p16
|
16998186
|
Functional evaluation of pEMI vectors
| 4.291458 |
biomedical
|
Study
|
[
0.9994943141937256,
0.0003137407766189426,
0.0001919569622259587
] |
[
0.9990973472595215,
0.0003139980253763497,
0.0004978134529665112,
0.00009083792974706739
] |
en
| 0.999998 |
As an example for the inactivation of an essential gene by the system introduced here we conditionally down-regulated the expression of the MAD2 protein, which was shown to result in mitotic failure and extensive cell death when permanently inactivated ( 15 , 16 ). After introduction of the pEMI-plasmid encoding a MAD2-specific microRNA we observed no effect on the viability and cell cycle distribution. Only when MAD2 was down-regulated by addition of DOX an increased fraction of apoptotic cells and altered cell cycle distribution was observed (data not shown). These results show that the expression of microRNAs can be tightly controlled using pEMI vectors, which are therefore useful for studying essential genes.
|
16998186_p17
|
16998186
|
Functional evaluation of pEMI vectors
| 4.122629 |
biomedical
|
Study
|
[
0.9995874762535095,
0.0002170320658478886,
0.00019548641284927726
] |
[
0.9993661046028137,
0.0002959724806714803,
0.0002774081949610263,
0.00006050752926967107
] |
en
| 0.999998 |
To rule out toxic side effects mediated by the pEMI vector-driven microRNA expression, we analyzed the expression of the IFIT1 (interferon-induced protein with tetratricopeptide repeats 1) gene by qPCR. Others have shown that expression of IFIT1 mRNA is rapidly induced upon IFN treatment ( 17 ). Besides interferon, double-stranded RNA (dsRNA) and viral infection have been shown to increase the expression of IFIT1 ( 18 ). pEMI-driven expression of a non-silencing microRNA for 2 and 4 days did not provoke an increased IFIT1 expression . Also expression of a p53-specific microRNA did not lead to any increase in IFIT1 expression, whereas transfection of U2OS cells with a synthetic dsRNA [poly(I:C)] led to ∼40-fold increase in IFIT1 mRNA expression within 18 h after transfection. Incubation of U2OS cells in media containing poly(I:C) also increased the level of IFIT1 up to ∼7-fold (data not shown) indicating this cell line is in principle very sensitive towards the presence of dsRNA. Furthermore, proliferation assays showed no significant anti-proliferative effect of pEMI-driven microRNA over-expression and cells were viable for several weeks when expressing microRNAs not targeting essential genes, whereas treatment with poly(I:C) led to an apoptotic response 2 days post transfection presumably by activating the dsRNA response (data not shown). Taken together, expression of ectopic microRNA by pEMI vectors does not lead to a dsRNA response or other toxic side effects.
|
16998186_p18
|
16998186
|
Functional evaluation of pEMI vectors
| 4.275587 |
biomedical
|
Study
|
[
0.9994550347328186,
0.00034256838262081146,
0.00020238182332832366
] |
[
0.9991825222969055,
0.0002828157739713788,
0.0004464886733330786,
0.00008821598021313548
] |
en
| 0.999996 |
A number of different conditional systems for RNA interference have been described previously ( 12 , 19 – 26 ). They utilize tetracycline regulation or CRE-recombinase mediated activation of shRNAs or microRNAs). However, they rely on several separate plasmids providing the necessary regulators in trans . One major advantage of the system described in this manuscript lies in the possibility to establish cells exhibiting inducible knock-down of the gene/protein of interest in one step as all components have been integrated on a single vector. This feature facilitates the analysis of cells which are characterized by a low transfection efficiency or a limited life span which presumably applies to most primary cells. With this vector system we could generate cell pools which homogenously express the microRNA of interest in an inducible manner in only one week. Furthermore, the analysis is not restricted to a cell clone expressing the trans-regulator (tTA or CRE) at the appropriate level. Instead, a pool of different cells expressing the pEMI vector can be analyzed. Obviously, this provides significant advantages and protects against misleading clonal effects.
|
16998186_p19
|
16998186
|
DISCUSSION
| 4.174123 |
biomedical
|
Study
|
[
0.9996275901794434,
0.00018576276488602161,
0.00018661613285075873
] |
[
0.998737633228302,
0.00037011856329627335,
0.0008274903520941734,
0.00006483185279648751
] |
en
| 0.999998 |
The vector system employed here is episomal and therefore microRNA expression levels are not dependent on the site of integration. Furthermore, the combination of a repressor and an activator acting through the tet-operon prevents expression in the off-state and allows precise titration of the expression level of the respective microRNA. Our determination of ectopic microRNA expression validate this point, as we were unable to detect expression in the uninduced state. The pEMI vector allows the conditional knockdown of proteins within a short period without the need for viral infection procedures or repeated transfections of several plasmids encoding the different components of the system. The expression of a fluorescent protein from the bidirectional promoter allows transfected cells to be sorted by flow cytometry, circumventing single cell cloning. Furthermore, cells exhibiting RNA interference can be traced in a heterogeneous population of cells in cell culture or in animals. In addition, the fluorescent protein marker facilitates the detection of any potential loss of microRNA expression in a cell population. Therefore, laborious, parallel analyses to confirm microRNA expression or its effects on the targeted gene are not necessary for every experiment. A further useful feature of this system is the possibility to reverse the knockdown and hence the induced phenotype. Thereby the causal link between the knockdown of any gene and the observed phenotype can be strengthened as non-reversible phenotypes may represent secondary effects.
|
16998186_p20
|
16998186
|
DISCUSSION
| 4.465582 |
biomedical
|
Study
|
[
0.9994365572929382,
0.0003772575000766665,
0.0001862075150711462
] |
[
0.9975879192352295,
0.0010668783215805888,
0.0011912687914445996,
0.0001539816294098273
] |
en
| 0.999998 |
We observed different degrees of inducibility over time in different cell lines. Whereas U2OS and H1299 cells showed a decrease of inducibility after 4 weeks of passaging, MCF7 cells did not show any decrease in the frequency of inducible (mRFP-positive) cells after several month of propagation. Cells which show loss of expression after continued passaging should therefore be conserved by freezing at early passages.
|
16998186_p21
|
16998186
|
DISCUSSION
| 4.042316 |
biomedical
|
Study
|
[
0.9994152784347534,
0.0002076349628623575,
0.0003771201299969107
] |
[
0.9985103011131287,
0.001081550377421081,
0.00033987394999712706,
0.00006833780207671225
] |
en
| 0.999995 |
Mammalian cells discriminate between microRNAs that are endogenous DICER products and non-self dsRNAs, e.g. by-products of viral replication ( 27 ). The microRNAs which were used here are embedded in an authentic human miRNA miR30 ( 28 ). This human microRNA has been shown to be efficiently recognized and processed by the Drosha–DGCR8 complex and DICER in mammalian cells ( 28 ) and therefore should not induce non-specific effects that are triggered by viral dsRNAs, as, for example, activation of the interferon pathway. A commonly used and sensitive marker for the activation of these dsRNA signaling pathways leading to IRF-3 and IFN induction is the increased expression of the dsRNA induced protein 56K (p56) encoded by the IFIT1 (interferon-induced protein with tetratricopeptide repeats 1) gene ( 18 , 29 – 31 ). Expression of p56 has furthermore been closely linked to the general toxicity of distinct siRNA duplexes ( 27 ). We found that neither non-silencing microRNA nor a p53-specific microRNA expressed by the episomal pEMI vector triggered a dsRNA pathway response.
|
16998186_p22
|
16998186
|
DISCUSSION
| 4.322606 |
biomedical
|
Study
|
[
0.999454915523529,
0.0002860381209757179,
0.00025902854395098984
] |
[
0.9992011189460754,
0.00032221307628788054,
0.00039657874731346965,
0.00008010182500584051
] |
en
| 0.999998 |
The controlled inactivation of genes by the system introduced here may be useful in certain therapeutic regimes and prevent the potential toxicity or immunogenicity which has been discussed for therapeutic applications of ectopic RNA interference. Using the bi-directional promoter it may be possible to express an RNA interference resistant wild-type protein to substitute for a mutant protein, which is simultaneously down-regulated by the ectopic microRNA. A similar approach has been recently described for the treatment of sickle cell anemia ( 32 ). As described above the pEMI vector is compatible with recently generated microRNA libraries and will therefore presumably become a widely used tool for conditional RNA interference ( 7 ).
|
16998186_p23
|
16998186
|
DISCUSSION
| 4.153084 |
biomedical
|
Study
|
[
0.9997146725654602,
0.00013259713887237012,
0.0001526345149613917
] |
[
0.991168737411499,
0.0033273783046752214,
0.005342939402908087,
0.00016097232582978904
] |
en
| 0.999997 |
Before transport to the cytoplasm for translation, RNA transcripts in the form of pre-mRNA assemble into a macromolecular structure termed the spliceosome. During subsequent remodeling events of spliceosomes, introns are removed and exons are ligated to form mature mRNA. In vitro cell-free assays using simple splicing substrates and nuclear extracts (NE) have established the basic sequence of biochemical events and ordered series of complexes in the pathway ( 1 – 4 ). In this dynamic, multi-step process, over 300 proteins and five ribonucleoprotein particles (snRNPs) orchestrate two trans -esterification reactions that result in the formation of mRNA ( 5 – 9 ). We previously identified two polypeptides in NE from HeLa cells (galectin-1 and galectin-3) required for splicing ( 10 , 11 ). Depletion of both galectins from NE, either by lactose–agarose affinity or double antibody adsorption chromatography, abolished splicing activity and halted spliceosome assembly at an early step. Addition of either galectin restored both splicing activity and spliceosome formation. These data suggested that galectins are, indeed, splicing factors and that they are functionally redundant.
|
16998182_p0
|
16998182
|
INTRODUCTION
| 4.460386 |
biomedical
|
Study
|
[
0.9993762373924255,
0.0004053847515024245,
0.0002183412725571543
] |
[
0.9987187385559082,
0.0004012457502540201,
0.0007289906498044729,
0.00015104741032700986
] |
en
| 0.999997 |
Galectins are a family of carbohydrate-binding proteins with specificity for galactose or galactose-containing glycoconjugates that localize both intracellularly and extracellularly ( 12 ). Two of the galectins (galectins-1 and -3) are ubiquitous in mammalian tissues and have been documented in the nucleus and cytosol. Galectin-1 (gal-1) is composed of a single carbohydrate recognition domain (CRD) of ∼130 amino acids. Galectin-3 (gal-3) has an N-terminal domain (ND) of ∼130 amino acids containing multiple repeats of theamino acid sequence PGAYPGXXX of unknown function fused to a CRD whose sequence is conserved when compared to the CRD of gal-1 and the other galectins. As we investigated the role of gal-1 and gal-3 in splicing, we noted several key differences. First, recombinant gal-3 is ∼5–10 times more efficient at reconstitution of splicing than gal-1 or the isolated CRD of gal-3 ( 11 ). Second, although both galectins co-localize with known splicing factors (i.e. the Sm core polypeptides of snRNPs and SC35) and each other in nuclear speckles, there are regions of non-overlap ( 13 ). Lastly, the isolated ND of gal-3 inhibits splicing chemistry and spliceosome formation in a dominant negative manner when added to splicing competent NE ( 14 ).
|
16998182_p1
|
16998182
|
INTRODUCTION
| 4.394701 |
biomedical
|
Study
|
[
0.9994694590568542,
0.00026305526262149215,
0.0002675323048606515
] |
[
0.9990492463111877,
0.0002509090700186789,
0.0006239509675651789,
0.00007584355626022443
] |
en
| 0.999997 |
Although these data are consistent with galectins being splicing factors, they do not address whether their role in splicing is direct or indirect. In this study, we provide evidence for a direct role of galectins in the splicing pathway by showing that galectin-specific antibodies co-immunoprecipitate pre-mRNA, splicing intermediates and products throughout the entire splicing pathway. We have investigated various characteristics of galectin incorporation into spliceosomes and conclude they interact via weak protein–protein interactions with another spliceosomal component.
|
16998182_p2
|
16998182
|
INTRODUCTION
| 4.139836 |
biomedical
|
Study
|
[
0.9995356798171997,
0.0002671056427061558,
0.0001972581521840766
] |
[
0.999387264251709,
0.0002418202202534303,
0.0002982009027618915,
0.00007273054507095367
] |
en
| 0.999998 |
Polyclonal rabbit antiserum against recombinant rat gal-1 was a gift from Doug Cooper (University of California, San Francisco, CA, USA) ( 14 ). Additionally, we raised a second polyclonal rabbit antiserum against recombinant human gal-1 expressed as a fusion protein with glutathione S -transferase (GST). A human gal-1 cDNA clone was kindly provided by Jun Hirabayashi ( 15 ). The gal-1 cDNA insert was isolated after BamH1 digestion and inserted into pGEX-2T. Following expression in DH5α, the fusion protein was purified by glutathione–agarose affinity chromatography and used to immunize rabbits. Several polyclonal rabbit antisera to murine gal-3 were produced as described in Agrwal et al . ( 16 ). When used in immunoprecipitation experiments , each galectin antiserum was mono-specific for a galectin (i.e. anti-gal-1 precipitated only gal-1 from splicing reactions and anti-gal-3 precipitated only gal-3). Human autoimmune serum (ENA anti-Sm) was purchased from The Binding Site. Anti-Slu7 antibodies were purchased from Santa Cruz Biotechnology and anti-Ran antibodies from BD Biosciences. A monoclonal antibody against hnRNP C1/C2 (4F4) was provided by Gideon Dreyfuss (University of Pennsylvania). For immunoprecipitation experiments, all antibodies were covalently cross-linked to protein G-Sepharose fast flow 4B beads (Sigma) as previously described ( 11 , 14 ) generally using a 2:1 ratio of antiserum to protein G beads.
|
16998182_p3
|
16998182
|
Antibodies
| 4.127363 |
biomedical
|
Study
|
[
0.9995844960212708,
0.00017192881205119193,
0.00024358995142392814
] |
[
0.9991531372070312,
0.0004290317592676729,
0.00035860060597769916,
0.00005917189264437184
] |
en
| 0.999998 |
Splicing reactions containing 60% by vol. HeLa NE were assembled with 32 P-MINX RNA without or with ATP (1.5 mM) and creatine phosphate (20 mM) and incubated at 30°C for the times indicated as previously described ( 10 , 11 , 14 ). HeLa cells were obtained from the National Cell Culture Center. Typically 60–100 μl of the splicing reaction mixture was diluted to 0.5 ml with 60% buffer D (D60) and incubated with 30–50 μl antibody cross-linked protein G-Sepharose beads at 4°C for 1–2 h. After washing with D60 containing 0.05% Triton X-100 (three washes, each with 1 ml buffer), the bound material was eluted with 20 μl of SDS–PAGE sample buffer. The eluted sample was divided into two aliquots. RNA was purified from one by incubating at 37°C for 20 min with proteinase K (4 mg/ml final concentration) and diluting to 100 μl with 125 mM Tris (pH 8), 1 mM EDTA, 300 mM sodium acetate. RNA was extracted with 200 μl of phenol–chloroform (50:50 [vol/vol]), followed by 200 μl of chloroform. RNA was precipitated with 300 μl of ethanol at −80°C. The extracted RNA was dissolved in 10 μl of sample buffer (9:1/formamide:bromophenol blue) and subjected to electrophoresis through 13% polyacrylamide (bisacrylamide–acrylamide 1.9:50 [wt/wt])-8.3 M urea gels. The radioactive RNA species were revealed by autoradiography and quantitated by phosphorimage analysis (Molecular Dynamics). The other aliquot was subjected to electrophoresis in 12.5% or 15% SDS–PAGE (bisacrylamide–acrylamide 0.9:30 [wt/wt]) and analyzed by western blotting.
|
16998182_p4
|
16998182
|
Co-immunoprecipitation of 32 P-labeled-MINX RNAs
| 4.266192 |
biomedical
|
Study
|
[
0.9994556307792664,
0.00033294159220531583,
0.0002115096867782995
] |
[
0.9985137581825256,
0.0008973926305770874,
0.00047662961878813803,
0.00011226119386265054
] |
en
| 0.999997 |
The salt sensitivity of the association of galectins and snRNPs with spliceosomes was determined as follows. Active splicing complexes (incubated for 60 min with ATP at 30°C) were incubated with antibody-coupled beads. Following the initial binding, the beads were washed either with 60, 130 or 250 mM KCl-containing buffer and the bound material was eluted with SDS–PAGE sample buffer. The RNA was extracted and analyzed by denaturing gel electrophoresis as described above.
|
16998182_p5
|
16998182
|
Co-immunoprecipitation of 32 P-labeled-MINX RNAs
| 4.105859 |
biomedical
|
Study
|
[
0.9995620846748352,
0.00023878156207501888,
0.00019908620743080974
] |
[
0.9991422891616821,
0.0004605861031450331,
0.0003320892574265599,
0.00006500414019683376
] |
en
| 0.999998 |
To evaluate binding of the galectins to the splicing substrate, recombinant gal-1 or recombinant gal-3 (expressed in DH5α and purified by lactose–agarose affinity chromatography), or NE was incubated with 32 P-MINX under splicing conditions. Following incubation, the reactions were subjected to native gel electrophoresis (4% polyacrylamide gels [bisacrylamide:acrylamide 1:80 wt/wt]) ( 17 ) and complex formation revealed by autoradiography. Agarose native gel electrophoresis to identify H or E complexes was performed as described by Das et al . ( 18 ) followed by autoradiography.
|
16998182_p6
|
16998182
|
Native gel electrophoresis
| 4.104616 |
biomedical
|
Study
|
[
0.9995490908622742,
0.00021374753850977868,
0.00023717890144325793
] |
[
0.9993059635162354,
0.0003533635172061622,
0.0002861359971575439,
0.00005453553603729233
] |
en
| 0.999997 |
Previously we showed that depletion of gal-1 and gal-3 from HeLa NE abolished pre-mRNA splicing and arrested spliceosomes before formation of active complexes (i.e. only early complexes formed) ( 10 ). Addition of either recombinant gal-1 or gal-3 to the galectin-depleted NE resulted in reconstitution of splicing activity and active spliceosome formation ( 10 , 11 ). We concluded that these two nuclear galectins are redundant splicing factors. However, there was little information to suggest whether their involvement in the splicing pathway was direct or indirect.
|
16998182_p7
|
16998182
|
Galectins-1 and -3 are associated with spliceosomes
| 4.126079 |
biomedical
|
Study
|
[
0.999568521976471,
0.0002020493702730164,
0.00022932908905204386
] |
[
0.9992520213127136,
0.00025498305330984294,
0.00042764790123328567,
0.00006534581916639581
] |
en
| 0.999998 |
To test for a direct association of galectins with spliceosomes, we determined whether radiolabeled splicing substrate in splicing complexes could be precipitated by galectin-specific antisera. As seen in Figure 1 (lane 1), after incubation in HeLa NE with ATP for 60 min, the 32 P-MINX splicing substrate is converted to processing intermediates and products. Typically, 20–35% of the RNA detected following 60 min is ligated product. In the absence of ATP, no processing of the MINX substrate occurs (see below). When splicing reactions that had been incubated for 60 min are immunoprecipitated by galectin-specific antisera, all species of RNA, the pre-mRNA substrate, the intermediates and the mature products, are found in the precipitates (lanes 4 and 5). In contrast, pre-immune serum did not precipitate significant quantities of labeled RNA (lane 2). Human autoimmune serum reactive against the Sm polypeptides of the snRNPs precipitated all species of radiolabeled spliceosomal-associated MINX as expected (lane 3). Thus, gal-1 and gal-3, like Sm proteins, are associated with active spliceosomes.
|
16998182_p8
|
16998182
|
Galectins-1 and -3 are associated with spliceosomes
| 4.247985 |
biomedical
|
Study
|
[
0.9994158744812012,
0.0003451957309152931,
0.0002389976871199906
] |
[
0.9992637038230896,
0.00033266469836235046,
0.0003243829123675823,
0.00007920891948742792
] |
en
| 0.999998 |
To characterize in greater detail galectin-containing splicing complexes, splicing reactions were subjected to antiserum selection during a time course experiment and RNAs and some of the proteins in the immunoprecipitates were characterized. Each galectin antisera co-immunoprecipitated the splicing substrate throughout the time course of the splicing reaction. At the earliest times sampled, both antisera precipitated MINX pre-mRNA, most probably in the form of H/E complexes (see below). Splicing intermediates and ligated exons were precipitated as they appeared during the kinetic analysis . Less spliceosomal RNAs were precipitated in the gal-3 time course compared to the gal-1 time course in this experiment due to use of a lower quantity of anti-gal-3 serum for precipitation. An internal control for the specificity of RNA precipitation is apparent in these analyses. Degraded RNAs of the gal-1 time course observed at 0, 5 and 10 min are not detected in the immunoprecipitated complexes at these times. Both galectin antisera appeared to immunoprecipitate the excised lariat RNA species preferentially . Both observations (i.e. no precipitation of degraded RNAs and preferential precipitation of free lariat) argue against non-specific adsorption of radioactive RNA species to beads since the precipitated RNAs do not reflect the same relative amounts of the different RNA species in the sample used for immunoselection (input).
|
16998182_p9
|
16998182
|
Galectins-1 and -3 are associated with spliceosomes
| 4.281387 |
biomedical
|
Study
|
[
0.9993543028831482,
0.00036874247598461807,
0.0002769692800939083
] |
[
0.999215841293335,
0.0002581668959464878,
0.00044143444392830133,
0.0000845745817059651
] |
en
| 0.999996 |
Detecting MINX pre-mRNA in galectin immunoprecipitates at early times in the splicing reaction prompted us to investigate the association of galectins with complexes characterized as H or E splicing complexes. H complexes form upon addition of a substrate to a splicing extract even when incubated on ice. Following incubation at elevated temperatures, H complexes are converted to E (early) complexes that are the immediate precursors of active spliceosomes. Neither H nor E complex assembly requires ATP ( 18 ). To test for galectin association with these two complexes, MINX pre-mRNA was incubated without ATP in NE at 30°C for 0 min to assemble H complexes or 20 and 40 min to chase H complexes into E complexes. Figure 3A shows MINX pre-mRNA formed H complexes upon addition to NE (lane 1). Nearly all of the MINX RNA was chased into E complexes following incubation for 20 and 40 min at 30°C (lanes 2 and 3). In a time course experiment, anti-gal-1 antiserum immunoprecipitated splicing competent pre-mRNA assembled in H complexes at 0 time and E complexes after 20 through 60 min of incubation at 30°C . Thus, complexes formed in the absence of ATP with mobilities characteristic of H and E pre-splicing complexes contain gal-1. Similar results for gal-3 were obtained (data not shown).
|
16998182_p10
|
16998182
|
Galectins-1 and -3 are associated with spliceosomes
| 4.231816 |
biomedical
|
Study
|
[
0.9994080066680908,
0.0003204061067663133,
0.00027164287166669965
] |
[
0.9994342923164368,
0.00023480894742533565,
0.00025866503710858524,
0.00007223466673167422
] |
en
| 0.999997 |
To confirm further that the immunoprecipitated complexes represented spliceosomes, antisera specific for several splicing factors were used to probe the precipitated fractions . Antiserum specific for gal-1 co-precipitated the Sm B/B′ core polypeptides of snRNPs, hnRNP C1/C2 and a factor required for in the second trans -esterification reaction Slu7. In contrast, gal-3 was not detected in the anti-gal-1 precipitate. We have also tested for and failed to find other nuclear proteins such as Ran co-immunoprecipitated by anti-gal-1 (data not shown). As expected, gal-1 antiserum precipitated gal-1. Similar results were obtained when the immunoprecipitates of anti-gal-3 antiserum were analyzed by western analysis (data not shown).
|
16998182_p11
|
16998182
|
Galectins-1 and -3 are associated with spliceosomes
| 4.087194 |
biomedical
|
Study
|
[
0.9994868040084839,
0.0002317214966751635,
0.00028138808556832373
] |
[
0.9994428753852844,
0.00024101999588310719,
0.00025999260833486915,
0.00005617551869363524
] |
en
| 0.999994 |
Galectins should be detected in spliceosomes isolated by precipitation with antiserum directed against another splicing factor. A reciprocal co-immunoprecipitation experiment was performed using anti-Sm antisera . As expected, spliceosomes selected by the Sm antiserum contained the splicing substrate, intermediates and products. Immunoprecipitation with human IgG revealed only background levels of splicing RNAs. Most importantly, gal-1 was detected in the Sm selected complexes, but not detected in the material precipitated by the control human IgG. These data strongly support our contention that galectins are splicing factors associated with the splicing machinery.
|
16998182_p12
|
16998182
|
Galectins-1 and -3 are associated with spliceosomes
| 4.166385 |
biomedical
|
Study
|
[
0.9995285272598267,
0.00020734542340505868,
0.00026418911875225604
] |
[
0.999278724193573,
0.0004015849845018238,
0.0002607466303743422,
0.00005895536014577374
] |
en
| 0.999998 |
Spliceosomes selected by galectin-specific antisera are a heterogeneous mixture of complexes (e.g. spliceosomes containing pre-mRNA substrate would be expected to contain different RNA and protein components than spliceosomes containing ligated exon product). While the stoichiometry of most of the proteins in spliceosomes has not been determined, it is assumed that under standard splicing conditions using a single splicing substrate each distinct processing pathway intermediate will contain the same complement of proteins. Thus, it is reasonable to predict that spliceosomes containing gal-1 would also contain gal-3. However, in our previous depletion–reconstitution studies, we showed that either gal-1 or gal-3 could restore splicing activity to a galectin-depleted NE. Obviously, under these reconstitution conditions, spliceosomes containing a galectin would contain only that particular member of the galectin family.
|
16998182_p13
|
16998182
|
Galectin-1 and galectin-3 reside on distinct splicing complexes
| 4.213806 |
biomedical
|
Study
|
[
0.9995381832122803,
0.0001877260801848024,
0.00027413616771809757
] |
[
0.9989350438117981,
0.000638549099676311,
0.000355553871486336,
0.00007084597018547356
] |
en
| 0.999997 |
To distinguish between these two formal possibilities (spliceosomes precipitated by one galectin antibody contain one or both nuclear galectins), we performed sequential immunoprecipitations as outlined in Figure 6A . Standard splicing reactions were incubated for 60 min. and divided into two equal portions. One aliquot was immunoprecipitated with anti-gal-1 and the other with anti-gal-3. The unbound fractions were then subjected to a second immunoprecipitation using the other galectin antiserum. Radiolabeled RNA in the bound fractions from each immunoprecipitation was analyzed . Roughly the same quantity of spliceosomes was precipitated by the anti-gal-1 antiserum in the two sequential selections (compare lanes 3 and 10). Similar results were obtained following the two anti-gal-3 immunoprecipitations (compares lanes 4 and 8). In order to interpret these results, the efficiency of each galectin antiserum to quantitatively immunoprecipitate its cognate antigen was determined. We analyzed the bound and unbound fractions from the first immunoprecipitation for gal-1 and gal-3 . The bound fraction from the first anti-gal-1 precipitate showed only gal-1 with no detectable gal-3. Further, the unbound fraction of this precipitation showed nearly quantitative depletion of gal-1 (lane 6; the amount of gal-1 in this fraction represents <10% of the total gal-1 in the reaction used for immunoprecipitation). Similar results were obtained with gal-3. Analysis of the bound fraction of the first anti-gal-3 precipitation showed only gal-3 (lane 4) and nearly all of gal-3 was removed by this immunoprecipitation (lane 5; <15% of the total gal-3 in the reaction remained in the unbound fraction of the first precipitation). We interpret these data to indicate that gal-1 and gal-3 were quantitatively removed during the initial immunoselection and that the two galectins reside on different splicing complexes. Finally, spliceosomal RNAs could be immunoprecipitated by anti-Sm serum from the material remaining after the two sequential galectin adsorptions (data not shown), indicating that some spliceosomal complexes contained neither gal-1 nor gal-3.
|
16998182_p14
|
16998182
|
Galectin-1 and galectin-3 reside on distinct splicing complexes
| 4.268844 |
biomedical
|
Study
|
[
0.9993509650230408,
0.0003847746120300144,
0.0002641752071212977
] |
[
0.9993106126785278,
0.0002674270363058895,
0.0003377665707375854,
0.00008419717778451741
] |
en
| 0.999995 |
As an additional evaluation of the nearly quantitative and specific removal of each galectin by its respective antiserum, this experiment was repeated with the following modification. The unbound fraction of the first immunoprecipitation was incubated with the same antiserum as that used in the first antibody selection (i.e. unbound material of anti-gal-1 precipitation was rebound to anti-gal-1 coated beads). Less than 5% of the initially precipitated RNA was bound to the antiserum in the second round of precipitation (data not shown). This low level of binding to the same antiserum matched the low levels of galectin found in the unbound material after the first precipitation.
|
16998182_p15
|
16998182
|
Galectin-1 and galectin-3 reside on distinct splicing complexes
| 4.067185 |
biomedical
|
Study
|
[
0.9994674324989319,
0.00021600375475827605,
0.0003165571251884103
] |
[
0.9994215965270996,
0.00035837842733599246,
0.00017015296907629818,
0.000049940270400838926
] |
en
| 0.999997 |
It is important to note that these results provide another control for the specificity of the spliceosomes precipitated by the anti-galectin antisera. The fact that each galectin antiserum precipitates only its respective antigen indicates that these antibodies do not precipitate nuclear proteins/complexes non-specifically and further suggests that galectins do not bind to splicing complexes non-specifically.
|
16998182_p16
|
16998182
|
Galectin-1 and galectin-3 reside on distinct splicing complexes
| 3.986313 |
biomedical
|
Study
|
[
0.9994388222694397,
0.00016078251064755023,
0.0004004057846032083
] |
[
0.9961997866630554,
0.0033316139597445726,
0.0003796567616518587,
0.00008891431207302958
] |
en
| 0.999995 |
The findings described above could be explained by a direct interaction of the galectins with the MINX pre-mRNA. If this interaction was with a unique site in the pre-mRNA, then only one galectin would bind per pre-mRNA and spliceosomes containing this pre-mRNA would have either gal-1 or gal-3 associated. To test for direct galectin–MINX interactions, we used an electrophoretic mobility shift assay . 32 P-MINX RNA migrated in native polyacrylamide gels as shown in lane 1 of Figure 7A and B. Incubation of MINX with recombinant human gal-1 or recombinant gal-3 did not alter the mobility of MINX when compared to the mobility of the substrate alone. Various incubation conditions (i.e. changing temperature, time of incubation and incubation with or without ATP) yielded the same results. As a positive control for altered mobility, MINX was incubated with NE for 15 min on ice. As expected, MINX was assembled into an H complex that had a slower mobility in the native gel . We conclude that the association of galectins with spliceosomes occurs through interaction with another splicing component rather than through direct binding to the splicing substrate.
|
16998182_p17
|
16998182
|
Galectin-1 and galectin-3 do not bind MINX RNA directly
| 4.35893 |
biomedical
|
Study
|
[
0.9994359612464905,
0.0003662374510895461,
0.00019778491696342826
] |
[
0.9991074204444885,
0.0003788285539485514,
0.00038875589962117374,
0.00012495907139964402
] |
en
| 0.999996 |
Splicing activity in a complete HeLa NE is inhibited in a dose dependent manner by the addition of the ND of gal-3. At the highest concentration of ND, neither splicing activity nor spliceosome complex formation could be detected even though gal-1 was available ( 14 ). Our finding that each galectin resides on separate splicing complexes suggests a mechanism for this dominant negative effect of the ND. Splicing requires binding of galectins to another splicing factor. Likely this partner is shared by the galectins in a mutually exclusive manner. We suggest that excess ND binds to this partner and blocks the interaction of this partner with both nuclear galectins. To test this prediction, we quantitated spliceosomes immunoprecipitated by anti-gal-1 antiserum in reactions inhibited by the ND of gal-3 (added as a GST fusion protein). The results are shown in Figure 8 . As previously reported ( 14 ), the GST-ND polypeptide inhibited product formation nearly 100% (lane 5) compared to reactions incubated with (lane 3) or without (lane 1) GST. The addition of GST to the reaction did not inhibit the precipitation of spliceosomes by anti-gal-1 (compare lane 4 to lane 2). In contrast, the addition of GST-ND nearly completely inhibited the association of gal-1 with the splicing machinery (compare lane 6 to lanes 2 and 4). Thus, the ND of gal-3 exerts its dominant negative effect by regulating the incorporation of gal-1 (and gal-3) into splicing complexes.
|
16998182_p18
|
16998182
|
ND of gal-3 blocks gal-1 association with spliceosomes
| 4.34898 |
biomedical
|
Study
|
[
0.999448835849762,
0.00031665843562223017,
0.00023445073748007417
] |
[
0.9992073178291321,
0.0003013919049408287,
0.0004052393196616322,
0.00008611461817054078
] |
en
| 0.999997 |
The strength of the association of galectins with spliceosomes was evaluated in relation to the stable association of the snRNPs with splicing complexes. Splicing complexes formed after a 60 min. splicing reaction were incubated with each galectin antiserum or pre-immune serum in 60 mM KCl buffer (the buffer used for optimal splicing efficiency). Aliquots of the antibody-bound spliceosomes were then washed with 60 mM (lanes 2–5), 130 mM (lanes 6–9) or 250 mM (lanes 10–13) KCl buffers. The bound fractions were eluted and analyzed for radiolabeled RNA. Salt concentrations of 130 or 250 mM released most of the splicing substrate from the galectin-selected columns (>90% of the spliceosomes were released as determined by quantitation from phosphorimage analysis) whereas 130 mM KCl had no effect on spliceosomes selected by anti-Sm antiserum. Even when the salt was increased to 250 mM KCl, ∼20% of the snRNPs remained stably associated with spliceosomes. The loss of spliceosomal RNAs from the antibody columns was due to dissociation of the complexes from each galectin and not due to release of the galectins from their respective antibody. At 130 mM KCl, virtually no gal-1 or gal-3 was released from the immobilized antibodies compared to the galectins bound at 60 mM KCl. At 250 mM KCl, more than 70% of the galectins remained bound to the antibodies (data not shown). We conclude that the association of galectins with the splicing machinery is sensitive to perturbation of ionic strength.
|
16998182_p19
|
16998182
|
Galectin association with spliceosomes is salt labile
| 4.33463 |
biomedical
|
Study
|
[
0.9994567036628723,
0.0003400883579161018,
0.0002033050695899874
] |
[
0.9991126656532288,
0.0003018172283191234,
0.0004912393051199615,
0.00009430493810214102
] |
en
| 0.999996 |
We previously have shown by depletion–reconstitution experiments that galectins-1 and -3 are required splicing factors ( 10 , 11 ). Further, the splicing activity of the galectins appears to be redundant in that either galectin can reconstitute splicing in a galectin-depleted NE. Now we document a direct association of these galectins with spliceosomal complexes. The key findings are (i) mono-specific galectin antisera immunoprecipitate splicing substrate RNAs in H, E and active spliceosomes along with Sm proteins of snRNPs, hnRNP C1/C2 and Slu7 (all known splicing factors), (ii) spliceosomal complexes contain either gal-1, gal-3 or no galectin (iii) neither galectin interacts directly with the pre-mRNA substrate, (iv) the amino terminus of gal-3 exerts a dominant negative effect on splicing by regulating the entry of gal-1 (and presumably gal-3) into splicing complexes and (v) the association of galectins with spliceosomes is salt-sensitive compared to the stable association of the snRNPs.
|
16998182_p20
|
16998182
|
DISCUSSION
| 4.364563 |
biomedical
|
Study
|
[
0.9994193315505981,
0.00035379675682634115,
0.00022687215823680162
] |
[
0.9990919828414917,
0.000279938627500087,
0.0005272348644211888,
0.00010079510684590787
] |
en
| 0.999997 |
The data presented are significant in evaluating the role of galectins as splicing factors. First, these data support our hypothesis that nuclear galectins are indeed splicing factors that can be incorporated into the canonical model for pre-mRNA splicing ( 1 ). The fact that both galectin antibodies co-immunoprecipitate pre-mRNA associated with early (i.e. H and E) complexes illustrates the initial entry of galectins during spliceosome assembly. While a precise molecular function of galectins during spliceosome assembly is not known, it appears to be in the recruitment or supplying of snRNPs to pre-mRNA based on two observations: (i) depletion of galectins from splicing extracts inhibits transition of early (e.g. H and/or E) complexes to active spliceosomes ( 1 ) and (ii) galectins are associated with gemin4 in SMN complexes ( 14 ) which are implicated in recycling snRNPs to pre-mRNA in the early commitment complex ( 19 ).
|
16998182_p21
|
16998182
|
DISCUSSION
| 4.239635 |
biomedical
|
Study
|
[
0.9995325803756714,
0.0002646375505719334,
0.00020275820861570537
] |
[
0.9989075660705566,
0.0002814353792928159,
0.0007401534239761531,
0.00007082254160195589
] |
en
| 0.999996 |
Since the anti-galectin antibodies precipitated not only the pre-mRNA substrate but also the intermediates and products of the splicing reaction generated on the spliceosome, our results suggest that gal-1 and gal-3 are associated with the assembled machinery throughout the reaction cycle. It is clear that these galectin-containing complexes are spliceosomes as the hnRNP C1/C2 polypeptides [known splicing factors ( 5 – 9 , 20 )], the snRNP-specific Sm proteins and Slu7 ( 2 , 5 – 9 ) co-precipitate with the substrate intermediates and products. Results of three controls strengthen our interpretation of the data. First, the nuclear shuttling protein Ran, which has not been found associated with spliceosomes, is not co-precipitated by the galectin antisera. Second, gal-1-containing spliceosomes are not precipitated by antibodies specific for gal-3 and vice versa. These results indicate (i) the galectin antisera are mono-specific and (ii) galectins do not adhere non-specifically to splicing complexes. Third, spliceosomes isolated by precipitation with antibodies directed against the Sm polypeptides of snRNPs co-precipitate galectins.
|
16998182_p22
|
16998182
|
DISCUSSION
| 4.298268 |
biomedical
|
Study
|
[
0.999396800994873,
0.00031390407821163535,
0.00028924515936523676
] |
[
0.999204695224762,
0.0003256174677517265,
0.0003911984385922551,
0.00007851789268897846
] |
en
| 0.999997 |
An obvious question of our contention that galectins are splicing factors is that they have not been identified in any of the proteomic analyses of spliceosomes ( 5 – 9 ). Several reasons can be offered to explain this discrepancy. First, most of the spliceosome isolation procedures use 150–250 mM salt during binding or washing to select stable complexes. The rationale has been to correctly identify components with a stable association in the spliceosome, rather than catalogue all associations. As we have shown , ionic conditions >60 mM release the galectins from spliceosomal complexes. We contend the buffer conditions we use for immunoprecipitation (which are optimal for in vitro splicing activity) allow a more complete cataloguing of spliceosomal proteins. Could the transient/loose association of galectins hint to a regulatory role? Second, the galectins may be in low abundance in spliceosomes. Our observation that not all spliceosomes contain galectins speaks to this point. Third, it is possible that galectins only assist in initiating spliceosome assembly (i.e. only associate with a complex containing the pre-mRNA substrate) and are not stable components of active splicing complexes. Only a thorough and careful evaluation of these early complexes would reveal this association. Finally, the stringency set for the identification of peptides and subsequent database searches results in missing members of a complex. For example, of the four massive proteomic analyses published, none have detected the U6-associated polypeptide LSm5 and there are several instances where one of the four studies detected a core spliceosomal component and the other three did not.
|
16998182_p23
|
16998182
|
DISCUSSION
| 4.371016 |
biomedical
|
Study
|
[
0.999427855014801,
0.00029707077192142606,
0.00027511786902323365
] |
[
0.9967576861381531,
0.00036287750117480755,
0.0027670697309076786,
0.00011241732136113569
] |
en
| 0.999998 |
Other significant aspects of our findings include providing an explanation for functional redundancy of the galectins and hinting at the nature of the spliceosome-associated binding partner for the galectins. Reconstitution of a galectin-depleted NE can be achieved by either gal-1 or gal-3 ( 10 , 11 ). The sequential immunoprecipitation data provide experimental proof for the exclusive incorporation of only a single galectin into a splicing complex in a complete (i.e. non-depleted) splicing extract. Thus, functional redundancy means spliceosomes contain only one galectin. We show that the ND of gal-3 regulates the entry of gal-1 into spliceosomes. In aggregate, these data suggest the galectins share a common binding partner. This partner is probably a polypeptide splicing factor that weakly interacts with the galectins. In a splicing extract, gal-3 interacts with this partner via its two domains (the ND which contains the PGAYPGXXX repeats of unknown function and the C-terminus which is the CRD) whereas gal-1 only binds via its single CRD. The observation that gal-3 is 8–10 times more efficient in reconstituting splicing activity in galectin-depleted extracts compared to gal-1 supports this contention ( 11 ). Addition of excess ND binds to this common partner and blocks binding of gal-1 or gal-3. Abrogation of gal-1 and gal-3 binding results in inhibition of splicing. It remains to be determined whether the galectin-binding partner is assembled into splicing complexes when bound to the isolated ND.
|
16998182_p24
|
16998182
|
DISCUSSION
| 4.404207 |
biomedical
|
Study
|
[
0.9992938041687012,
0.00044689667993225157,
0.00025938847102224827
] |
[
0.9990013241767883,
0.00032407170510850847,
0.0005608077626675367,
0.00011382162483641878
] |
en
| 0.999998 |
Crucial to providing a mechanistic interpretation of these data regarding the association of galectins with spliceosomal complexes is the identification of the splicing partner for the galectins. While we have identified gemin4 as a galectin binding protein ( 14 ), neither gemin4 nor other members of the SMN complex ( 19 ) have been identified as spliceosomal components in proteomic analyses ( 5 – 9 ). Is this due to the fact that gemin4 and interacting proteins are not spliceosomal proteins or, as with the galectins, that they are weakly associated with spliceosome complexes and released under the conditions used for spliceosome isolation? From a different perspective, gal-1 has been shown to be a component of the nuclear matrix ( 21 ) that has been proposed to serve as a scaffold for the splicing process. The nuclear matrix partner with which gal-1 interacts is unknown. Also, proteomic analysis of interchromatin granule clusters (IGC) has identified a galectin as a member of this nuclear organelle. Over 80% of the proteins identified as IGC components play a role in RNA biogenesis with >50% having a splicing function ( 22 ). Both of these findings lend additional, albeit indirect, support to our assertion that the galectins are splicing factors.
|
16998182_p25
|
16998182
|
DISCUSSION
| 4.383926 |
biomedical
|
Study
|
[
0.9993841648101807,
0.0003399974957574159,
0.0002757972397375852
] |
[
0.9987192153930664,
0.0003281364042777568,
0.0008580087451264262,
0.00009461175068281591
] |
en
| 0.999997 |
Galectin-containing spliceosomes have been precipitated with several different galectin-specific antisera using three conditions to assemble various splicing complexes. The association of galectins with spliceosomes is through a salt-sensitive protein–protein interaction rather than a galectin-splicing substrate interaction. The commonly shared binding partner/splicing factor explains the mutually exclusive incorporation of gal-1 or gal-3 into splicing complexes and the dominant negative inhibition of splicing demonstrated by the ND of gal-3.
|
16998182_p26
|
16998182
|
DISCUSSION
| 4.303033 |
biomedical
|
Study
|
[
0.9995482563972473,
0.00022134995379019529,
0.00023031377349980175
] |
[
0.9988365769386292,
0.0005444318521767855,
0.000538364693056792,
0.00008071909542195499
] |
en
| 0.999997 |
The TATA-binding protein (TBP) plays an integral role in transcription by all three nuclear RNA polymerases, including transcription from promoters without a TATA box ( 1 ). In the Saccharomyces cerevisiae RNA polymerase (pol) III apparatus, TBP is found in the recruitment factor TFIIIB, along with the TFIIB-related factor, Brf1 and the pol III-specific Bdp1. In vivo , TFIIIB is assembled onto the DNA via TFIIIC ( 2 – 5 ), but this requirement can be bypassed in vitro if a TATA box is present ( 6 , 7 ), allowing TBP to bind the DNA and nucleate a stepwise TFIIIB assembly requiring Brf1 to bind the TBP–DNA complex before Bdp1 can associate. These TFIIIB complexes are indistinguishable in vitro from those assembled by TFIIIC ( 8 ). Even when TFIIIB is assembled by TFIIIC, direct interaction of TBP with its cognate site contributes to accurate transcriptional initiation ( 9 ).
|
17028095_p0
|
17028095
|
INTRODUCTION
| 4.610941 |
biomedical
|
Study
|
[
0.9992431402206421,
0.0004567215219140053,
0.00030013377545401454
] |
[
0.9965522289276123,
0.0010005789808928967,
0.0022430315148085356,
0.00020414992468431592
] |
en
| 0.999996 |
TBP binds DNA in the minor groove and induces a bend of the order of ∼80° ( 10 – 15 ). TBP binds an 8 bp site with the consensus sequence TATAa/tAa/tN, where N is any base ( 16 – 18 ), and an iterative in vitro selection performed using Acanthamoeba TBP on 84 bp DNA containing 40 randomized positions yielded only four classes of TATA box and a preference for TATATAAG [35 of 54 clones; ( 19 )]. Complex stability measurements using DNase I footprinting and the frequency of a sequence appearing in the clones indicate that TBP is able to differentiate between A:T and T:A base pairs, and that the frequency with which a sequence is selected correlates with complex stability ( 19 ).
|
17028095_p1
|
17028095
|
INTRODUCTION
| 4.286775 |
biomedical
|
Study
|
[
0.9995624423027039,
0.0002162205200875178,
0.00022125348914414644
] |
[
0.9991993308067322,
0.0003539208264555782,
0.0003732166369445622,
0.00007362076576100662
] |
en
| 0.999996 |
While an A→G substitution at the second base pair of the TATA box abolishes specific DNA binding by wild-type TBP, a mutant TBP known as TBPm3 was previously isolated from yeast and found to bind to the sequence TGTA as well as to the wild-type TATA box ( 20 ). The three mutations (I194F, L205V, and V203T) that confer this altered specificity, are in close proximity in the folded protein and are in a position to interact with the second base pair of the TATA box ( 20 ). TBPm3 assembles a stable TFIIIB-DNA complex that is functional for pol III transcription ( 21 ).
|
17028095_p2
|
17028095
|
INTRODUCTION
| 4.323566 |
biomedical
|
Study
|
[
0.9995013475418091,
0.00023522097035311162,
0.0002634716802276671
] |
[
0.9990407824516296,
0.0005767120746895671,
0.0002975121606141329,
0.00008503032586304471
] |
en
| 0.999997 |
We are exploiting the specificity of binding of TBPm3 to orient the protein unidirectionally on the DNA and investigate TBP–DNA contacts within the downstream half of the TATA box as a function of Brf1 and Bdp1 association. It has been previously suggested that addition of Brf1 and Bdp1 to the TBP–DNA complex alter its conformation or dynamics: (i) While a missing nucleoside at the downstream end of the TATA box, coinciding with the site of TBP-mediated DNA kinking (base pair −23), significantly enhances complex formation, the TFIIIB complex abrogates this preference, instead preferring missing nucleosides within an extended region downstream of the TATA box ( 22 ), (ii) examination of TFIIIB interacting with a region upstream of the SUP4 tRNA Tyr gene by photochemical crosslinking showed TBP in proximity to the DNA minor groove, except for contacts to the DNA major groove at base pair −23 of the transcribed strand which were enhanced upon TFIIIB-DNA complex formation ( 23 ) and (iii) the structure of a ternary complex composed of TBP, DNA and the primary TBP-binding domain of Brf1 revealed an exceptionally large number of interactions that bury 3230 Å 2 of TBP surface area ( 24 ).
|
17028095_p3
|
17028095
|
INTRODUCTION
| 4.491686 |
biomedical
|
Study
|
[
0.9990942478179932,
0.0005705594667233527,
0.0003351172199472785
] |
[
0.9988849759101868,
0.0003522861225064844,
0.0006077766884118319,
0.0001549696025904268
] |
en
| 0.999998 |
Here, we use an iterative in vitro selection to compare the sequence preference exhibited by TBPm3 and TFIIIB assembled with TBPm3. We show that the sequence preference of TBPm3 is less stringent than that reported for wild-type TBP ( 19 ). Notably, entry of Brf1 and Bdp1 into the complex imposes a strict sequence preference for the downstream half of the TATA box that matches the TATA box of the pol III-transcribed U6 small nuclear RNA ( SNR6 ) gene.
|
17028095_p4
|
17028095
|
INTRODUCTION
| 4.181899 |
biomedical
|
Study
|
[
0.9994148015975952,
0.0003238949866499752,
0.00026132812490686774
] |
[
0.9994152784347534,
0.0002723072830121964,
0.00024159715394489467,
0.00007085083780111745
] |
en
| 0.999996 |
Plasmids expressing TBPm3, Brf1 and Bdp1 were generous gifts from E. P. Geiduschek and G. A. Kassavetis, University of California San Diego, CA. Purification of TBPm3 was modified from ( 25 ), briefly, plasmid containing the gene encoding TBPm3 was transformed into Escherichia coli BL21(DE3)pLysS and grown to OD 600 = 0.4 in LB broth containing 100 μg/ml ampicillin. Protein overexpression was induced with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 2 h and the pelleted cells frozen at −80°C. Forty-five ml lysis buffer A [50 mM Tris–HCl (pH 8.0), 0.1 mM EDTA, 5% glycerol, 10 mM β-mercaptoethanol, 300 μg/ml lysozyme and 0.5 mM phenylmethylsulfonyl fluoride (PMSF)] were added to ∼5 g thawed cells and incubated for 1 h on ice. All steps after lysis are carried out at 0–4°C. The lysate was diluted ∼1:1 with 60 ml lysis buffer B [50 mM Tris–HCl (pH 8.0), 1 M NaCl, 5% glycerol, 10 mM β-mercaptoethanol, and 0.5 mM PMSF], CaCl 2 added to 0.5 mM, and incubated for 1 h with 10 μl DNase I (10 U/μl). The mixture was dialyzed overnight against 3 l buffer A [50 mM Tris–HCl (pH 8.0), 100 mM KCl, 20% glycerol, 1 mM EDTA, 10 mM β-mercaptoethanol and 0.5 mM PMSF] and loaded on tandem DEAE-heparin columns. TBPm3 was eluted from the heparin column using a linear gradient (100–500 mM KCl) and fractions containing TBPm3 identified by SDS–PAGE. Active fractions were pooled and dialyzed against 2 l buffer A for 2 h prior to loading on a CM-Sepharose column. The protein was eluted as described above. TBPm3 concentration was determined by Coomassie blue-staining of SDS–PAGE gels using BSA as a standard. The activity of TBPm3 was determined by electrophoretic mobility shift assays (EMSA), and the preparation found to be essentially 100% active.
|
17028095_p5
|
17028095
|
Protein purification
| 4.329089 |
biomedical
|
Study
|
[
0.9993589520454407,
0.00040346625610254705,
0.0002375158219365403
] |
[
0.9986696243286133,
0.0007532319868914783,
0.0004278082924429327,
0.00014938884123694152
] |
en
| 0.999997 |
The plasmid containing the gene encoding N- and C-terminally-His-tagged Brf1 was transformed into E.coli BL21(DE3)pLysS and grown to OD 600 = 0.4 in LB broth containing 60 μg/ml ampicillin. Protein overexpression was induced with 0.4 mM IPTG for 2 h and the pelleted cells frozen at −80°C. Approximately 5 g of thawed cells were resuspended in 15 mL lysis buffer A supplemented with 1 μg/ml pepstatin and 1 μg/ml leupeptin, lysozyme was added to a final concentration of 300 μg/ml and the suspension was allowed to incubate on ice for 30 min. Tween-20 was added to a final concentration of 0.1%, and the cells sonicated on ice five times for 30 sec. The lysate was diluted ∼1:1 with 20 ml lysis buffer B supplemented with 1 μg/ml pepstatin and 1 μg/ml leupeptin, sonicated on ice five times for 30 seconds, then centrifuged at 20 000× g for 1 h at 4°C. The pellet was resuspended in 10 ml Buffer G [50 mM Tris–HCl (pH 8.0), 6 M guanidinium-HCl, 10% glycerol, 7 mM β-mercaptoethanol, 0.5 mM PMSF, 1 μg/ml pepstatin and 1 μg/ml leupeptin], then centrifuged at 20 000× g to pellet insoluble material. The supernatant fraction was added to 5 ml nickel-NTA agarose beads equilibrated in buffer G and incubated for 1 h at 4°C. The beads were washed three times for 15 min at 4°C with 10 ml buffer G. After harvesting the beads by centrifugation, the protein was eluted by a pH gradient (6.7, 6.5, 5.9, 5.7, 5.5, 5.1 and 4.7), accomplished via successive 15 min washes at 4°C with 10 ml buffer B (7 M urea, 7 mM β-mercaptoethanol, 0.5 mM PMSF, 1 μg/ml pepstatin, and 1 μg/ml leupeptin and 100 mM sodium phosphate at appropriate pH), and fractions containing the double His-tagged Brf1 determined via electrophoresis on a 12% SDS–PAGE gel. The active fractions were pooled, the pH adjusted to 7.9 with 1.5 M Tris–HCl (pH 8.7), and urea removed by sequential dialysis for 2 h against 500 ml buffer C-500 [20 mM HEPES (pH 7.8), 10% glycerol, 7 mM MgCl 2 , 500 mM NaCl, 10 mM β-mercaptoethanol, 0.01% Tween-20, 0.5 mM PMSF and 10 μM ZnSO 4 ] containing 3.0, 1.5, 0.75 and 0 M urea, respectively ( 8 ). The preparation was judged by Coomassie blue-staining of SDS–PAGE gels to be without contamination by truncated variants. Protein concentration was determined by Coomassie blue-staining of SDS–PAGE gels using BSA as a standard. EMSA measuring assembly of TFIIIB suggests that the preparation is ∼15% active.
|
17028095_p6
|
17028095
|
Protein purification
| 4.400983 |
biomedical
|
Study
|
[
0.9991445541381836,
0.0005793277523480356,
0.00027613769634626806
] |
[
0.9975984692573547,
0.0015611822018399835,
0.0006344371940940619,
0.00020585300808306783
] |
en
| 0.999994 |
Bdp1 was overexpressed as described for Brf1 and purification procedures carried out at 0–4°C. Cells were resuspended in buffer W (50 mM potassium phosphate buffer pH 7.0, 350 mM KCl, 5% glycerol, 4 mM β-mercaptoethanol and 0.2 mM PMSF). Lysozyme was added to 0.5 mg/ml followed by incubation for 1 h on ice. Following addition of 10% Triton X-100 to a final concentration of 0.5% (v/v), polymin P was added dropwise from a 13% solution to a final concentration of 0.5%. Cell debris and precipitates were removed by centrifugation for 10 min at 6000× g . The supernatant was incubated with Talon beads (BD Biosciences) for 1 h, washed twice with buffer W, and the N-terminally His 6 -tagged Bdp1 eluted batchwise by successive 15 min incubations with buffer W supplemented with increasing concentrations of imidazole (10, 25, 50, 75, 100 and 150 mM). Fractions containing Bdp1 were pooled, diluted 1:2 with buffer W and loaded on CM-Sepharose equilibrated in buffer W. Bdp1 was eluted with a linear gradient from 350 mM to 1 M KCl in buffer W. Protein concentration was determined by Coomassie blue-staining of SDS–PAGE gels using BSA as a standard. EMSA indicates that the preparation is at least 25% active. TFIIIB assembled with the proteins used for selection studies is transcriptionally active (data not shown).
|
17028095_p7
|
17028095
|
Protein purification
| 4.214655 |
biomedical
|
Study
|
[
0.9993674159049988,
0.00040339346742257476,
0.0002292278513778001
] |
[
0.9988605976104736,
0.0007593526388518512,
0.0002706544182728976,
0.00010943856614176184
] |
en
| 0.999996 |
Oligonucleotides used to generate duplex DNA containing 8 bp TATA- (5′-CGT GAC TAC TAT AAA TA G ATG ATC CG-3′) or TGTA-boxes (5′-CGT GAC TAC TGT AAA TA G ATG ATC CG-3′) were purified on denaturing polyacrylamide gels. For EMSA, the top strand was 5′ end-labeled using T4 polynucleotide kinase and [γ- 32 P]ATP, and annealed to the bottom strand by heating to 90°C, followed by slow cooling to room temperature.
|
17028095_p8
|
17028095
|
TBPm3–DNA complex formation using EMSA
| 4.137538 |
biomedical
|
Study
|
[
0.9995371103286743,
0.00024536316050216556,
0.00021758023649454117
] |
[
0.9939824938774109,
0.0052969492971897125,
0.0005659469752572477,
0.00015466030163224787
] |
en
| 0.999996 |
Reactions for kinetics assays contained 44 mM Tris (pH 8.0), 8.4 mM NaHEPES (pH 7.8), 50.5 mM NaCl, 7 mM MgCl 2 , 1 mM EDTA, 8% (v/v) glycerol, 3 mM DTT, 4 mM β-mercaptoethanol and 84 μg/ml BSA. Samples were subjected to electrophoresis on native 10% polyacrylamide gels and in buffer containing 0.5× TBE (45 mM Tris-borate, pH 8.0 and 1 mM EDTA) and 2.5 mM MgCl 2 .
|
17028095_p9
|
17028095
|
TBPm3–DNA complex formation using EMSA
| 4.116044 |
biomedical
|
Study
|
[
0.9993888139724731,
0.000358265038812533,
0.0002529402554500848
] |
[
0.9942458271980286,
0.005251104943454266,
0.00032589054899290204,
0.00017711755936034024
] |
en
| 0.999997 |
To determine the rate of complex dissociation during electrophoresis ( k diss ), 200 fmol TBPm3 and 50 fmol DNA were incubated for 55 min, 400 ng competitor DNA added [poly(dA–dT):poly(dA–dT)], and subjected to electrophoresis for time t . The gels were dried, exposed to a phosphorimaging screen, and the data quantitated using ImageQuant 1.1. Data were fitted to F obs = F * exp(− k diss t ), where F obs is the observed fractional complex, F is the fractional complex present at t = 0, k diss is the rate of dissociation on the gel, and t is the time of electrophoresis ( 26 ).
|
17028095_p10
|
17028095
|
TBPm3–DNA complex formation using EMSA
| 4.168571 |
biomedical
|
Study
|
[
0.9995052814483643,
0.000255470018601045,
0.00023919784871395677
] |
[
0.9990917444229126,
0.0005679429741576314,
0.00027455834788270295,
0.0000657409691484645
] |
en
| 0.999996 |
For determination of the off-rate in solution, 750 fmol of DNA and 3000 fmol TBPm3 was incubated at room temperature for 1 h, and aliquots loaded on the gel at time t after addition of 6000 ng poly(dA–dT):poly(dA–dT). Data were corrected by F corr = F /exp(− k diss t ), where F corr is the fractional complex corrected for dissociation during electrophoresis, F is the observed fractional complex and t is the time of electrophoresis. The corrected data were fitted to F corr = F 0 * exp(− k off t ), where F 0 is the fractional complex present before addition of competitor, k off is the off-rate in solution, and t is time after addition of poly(dA–dT):poly(dA–dT).
|
17028095_p11
|
17028095
|
TBPm3–DNA complex formation using EMSA
| 4.182946 |
biomedical
|
Study
|
[
0.99947589635849,
0.0002613433462101966,
0.0002628756337799132
] |
[
0.999103307723999,
0.0005726016242988408,
0.00026167978649027646,
0.00006250201113289222
] |
en
| 0.999998 |
The on-rate in solution was determined for a protein concentration range of 20–80 nM. Protein and 50 fmol DNA were incubated for time t and loaded on the gel immediately after addition of poly(dA–dT):poly(dA–dT). The observed fractional complex was corrected for dissociation during electrophoresis as described above and fitted to F corr = F final [1 − exp(− k obs t )], where F final is the calculated fractional complex present at completion of the reaction and k obs the apparent first-order rate constant. The reciprocal of the slope of a plot of 1/ k obs versus 1/[protein] yielded the second-order rate constant, k on ( 26 ). All rate constants represent the mean of at least three experiments.
|
17028095_p12
|
17028095
|
TBPm3–DNA complex formation using EMSA
| 4.180088 |
biomedical
|
Study
|
[
0.9995074272155762,
0.0002722321660257876,
0.0002203678450314328
] |
[
0.9993506073951721,
0.0002920996630564332,
0.00029180871206335723,
0.00006551994010806084
] |
en
| 0.999997 |
The oligonucleotide (5′-CGC TGC AAT CTC TTT TTC AAT TGC TCC GGA C TG TAA NNN NGC GGT CCC TTA CTC TTT CCT CAA CAA TTA ACG GCC C-3′, mutant TATA box underlined and bold) was purified on a denaturing 5% polyacrylamide gel, and amplified by PCR using Taq polymerase and 40 pmol of primers PSXB (5′-GGG CCG TTA ATT GTT GAG-3′) and PSXT (5′-CGC TGC AAT CTC TTT TTC-3′). Reaction conditions included buffer supplied by the manufacturer containing 2.0 mM MgCl 2 and 250 μM dNTP. The starting pool of oligonucleotides easily contains every possible sequence (4 4 or 256 sequences). The double-stranded product was 5′ end-labeled using T4 polynucleotide kinase and [γ- 32 P]ATP and at least 40 ng was incubated with 400 fmol TBPm3 to yield no more than ∼10% complex in early rounds (this fraction should yield a consensus sequence from the 256 possible sequences within 4–5 rounds of selection) for 1 h in the buffer listed above, except with 150 mM NaCl. After addition of 800 ng poly(dA–dT):poly(dA–dT), the reaction was loaded onto a native 10% polyacrylamide gel with the power on and subjected to electrophoresis at 175 V for 1 h. The gel was exposed to a phosphorimaging screen, the TBPm3–DNA complex excised from the gel, and the DNA passively eluted overnight in 1 ml elution buffer [20 mM Tris–HCl (pH 8.0), 1 M LiCl, 0.2 mM EDTA, 0.2% SDS] with rotation. The recovered DNA was amplified by PCR as described above, the PCR product purified on a native 7% polyacrylamide gel, radioactively labeled, and used as template for the next round of selection. After 10 rounds of selection, the DNA was cloned into the pCR T7/NT-TOPO vector (Invitrogen) and transformed into E.coli TOP10. Sequences containing the TGTAA box were aligned with ClustalX ( 27 ).
|
17028095_p13
|
17028095
|
Determination of TBPm3 and TFIIIBm3 sequence preference by iterative in vitro selection
| 4.35904 |
biomedical
|
Study
|
[
0.999090313911438,
0.0006383402505889535,
0.0002713307912927121
] |
[
0.9983291029930115,
0.001070351805537939,
0.0004119484219700098,
0.00018863628793042153
] |
en
| 0.999997 |
For the TFIIIBm3 selection, the selection was performed using the same conditions as for TBPm3, except that 40 ng labeled DNA was incubated with 120 fmol TBPm3, 520 fmol of total Brf1 and 1200 fmol of total Bdp1 for 1 h, with 100 mM NaCl. Active Brf1 was chosen as the limiting component to avoid trapping all existing TBPm3–DNA complex ( 28 ). Poly(dA–dT):poly(dA–dT) (240 ng) was added, and the reaction was loaded onto a native 4% polyacrylamide gel with the power on and subjected to electrophoresis at 175 V for 3 h. The recovered DNA was amplified by PCR, the PCR product purified on a native 10% polyacrylamide gel, radioactively labeled, and used as template for the next round of selection. After 10 rounds of selection, the DNA was cloned into the pCR4-TOPO vector (Invitrogen) and transformed into E.coli TOP10. Sequenced DNA was aligned using ClustalX ( 27 ).
|
17028095_p14
|
17028095
|
Determination of TBPm3 and TFIIIBm3 sequence preference by iterative in vitro selection
| 4.168137 |
biomedical
|
Study
|
[
0.9994121789932251,
0.0003381970163900405,
0.0002495709341019392
] |
[
0.9991338849067688,
0.0005165507318452001,
0.00027028293698094785,
0.0000793821454863064
] |
en
| 0.999998 |
To confirm complex formation by TBPm3, EMSA was performed as described above using 26 bp constructs, except that the sequence was modified to represent sequences selected by TBPm3.
|
17028095_p15
|
17028095
|
Determination of TBPm3 and TFIIIBm3 sequence preference by iterative in vitro selection
| 3.951577 |
biomedical
|
Study
|
[
0.9993694424629211,
0.00018241233192384243,
0.0004481323412619531
] |
[
0.9984246492385864,
0.001283730030991137,
0.00022775314573664218,
0.00006382777792168781
] |
en
| 0.999995 |
Oligonucleotides were purchased and purified on 5% denaturing polyacrylamide gels. The top strand of the 76 bp DNA probe (5′-CGC TGC AAT CTC TTT TTC AAT TGC TCC GGA CTG TAA ATT G GC GGT CCC TTA CTC TTT CCT CAA CAA TTA ACG GCC C-3′) was 5′ end-labeled using T4 polynucleotide kinase and [γ 32 -P]ATP, and annealed to the unlabeled complementary strand. One hundred twenty-five fmol of 76 bp DNA was incubated with or without 500 fmol TBPm3 at room temperature for 1 h using the buffer conditions described above, except with 3 mM MgCl 2 . After addition of 1 μg poly(dA–dT):poly(dA–dT), 1 μl of 10 mM sodium ascorbate and 4 μl freshly prepared 25 μM Fe-MPE were added, incubated for 1 min and the reaction was stopped by loading onto a native 10% polyacrylamide gel with the power on and subjected to electrophoresis at 175 V for 1 h. Free DNA and TBPm3–DNA complex were excised from the gel, and the DNA eluted and purified, as described above.
|
17028095_p16
|
17028095
|
Two-dimensional methidiumpropyl-EDTA (MPE)-Fe(II) footprinting
| 4.205553 |
biomedical
|
Study
|
[
0.9994750618934631,
0.0003158376202918589,
0.00020910122839268297
] |
[
0.9984643459320068,
0.0010913970181718469,
0.00034064645296894014,
0.00010361513704992831
] |
en
| 0.999997 |
Samples were resuspended in formamide loading buffer and heated at 95°C for 2 min prior to loading on a 10% polyacrylamide sequencing gel. The gel was run at 35 W in 1× TBE for ∼4 h, and dried. The gel was exposed to a phosphorimaging screen, and the gel image quantitated in ImageQuant 1.1.
|
17028095_p17
|
17028095
|
Two-dimensional methidiumpropyl-EDTA (MPE)-Fe(II) footprinting
| 3.975178 |
biomedical
|
Study
|
[
0.9988704323768616,
0.0005682575283572078,
0.0005612998502328992
] |
[
0.7175125479698181,
0.2796879708766937,
0.0018374407663941383,
0.000962038291618228
] |
en
| 0.999998 |
TBPm3 does not fully substitute for wild-type TBP in vivo , as evidenced for example by the slower growth phenotype of yeast strains carrying TBPm3 as the only TBP variant ( 20 ). As a basis for comparison of sequence preferences exhibited by TBPm3 and TFIIIB assembled with TBPm3 to those reported for wild-type TBP, we therefore first determined the affinity of TBPm3 for DNA constructs carrying either an 8 bp TATA box (the U6 TATA box) or the corresponding TGTA box. To calculate the equilibrium binding constant, rates of association and dissociation in solution were determined using EMSA ( 26 , 29 ) ( Table 1 ). As shown in Figure 1 , the TBPm3–DNA complex is quite stable in solution and decays with first-order kinetics with t 1/2 of 72 and 61 min, respectively, for DNA containing either the TATA or TGTA box. The observed rate of dissociation is comparable with that observed for wild-type TBP ( 26 , 29 ). Association of TBPm3 with DNA is detectable after 10 s, and a gradual increase in complex formation is observed after longer incubation times. The second-order rate constants for association of TBPm3 with DNA containing either the TATA or TGTA box are comparable and well within the range of values reported for association of wild-type TBP with various DNA substrates, using a variety of techniques ( 26 , 29 – 35 ). The calculated equilibrium dissociation constant for TBPm3 binding to the TATA or TGTA probe is 0.3 and 0.6 nM, respectively.
|
17028095_p18
|
17028095
|
Characterization of TBPm3
| 4.339205 |
biomedical
|
Study
|
[
0.9993976354598999,
0.00038031034637242556,
0.00022201526735443622
] |
[
0.9992491602897644,
0.00023041400709189475,
0.00041893470915965736,
0.00010143840336240828
] |
en
| 0.999997 |
The ability of TBPm3 to bind the TGTA box unidirectionally was exploited to perform an iterative in vitro selection on a 76 bp DNA construct in which four bases at the downstream end of the TGTA box were randomized; this DNA construct derives from a modified tRNA Tyr gene in which a 6 bp TATA box was embedded in a G+C-rich surrounding sequence ( 9 ). The randomized region was selected to coincide with sites at which modulation by Brf1 and Bdp1 may be expected and includes positions 6, 7 and 8 of the TBP site and one base pair downstream ( 22 , 23 ). Positions 1–5 of the TBP site were retained to ensure unidirectional binding, and inclusion of 1 bp downstream of the 8 bp TBP site in the randomized segment was chosen as TBP has been seen also to exhibit a sequence preference at this position ( 19 ). For stringency of selection, the concentration of NaCl was raised to 150 mM during incubation of TBPm3 with DNA. With only 256 possible sequences, a consensus should be reached within 4–5 rounds of selection. However, the sequence preference exhibited by TBPm3 was only modest after five rounds of selection (data not shown), so we elected to continue the selection for another five rounds. After 10 rounds of selection, the selected pool of DNA was again sequenced and a favored sequence determined for the four randomized bases ( Table 2 ). Of the sequenced clones, 66 contained the original TGTAA sequence and were used to determine the consensus for this selection. A total of 29 clones contained a TATAA box, suggesting its generation as a result of errors introduced during PCR; these sequences were not included in the alignment to avoid potential introduction of sequences arising from TBPm3 binding in the reverse orientation. A total of 17 clones contained a sequence comprised of a series of GTG repeats, with only the regions complementary to the primer sequences constant. The remainder of the clones contained sequence with no match to either of the above categories, such as other alterations to original TGTAA sequence. Alignment of the 66 TGTAA-containing sequences still showed a surprisingly modest sequence preference for each of the randomized positions. Whereas a C is generally disfavored at every position, position N1, corresponding to the sixth base pair of the TBP site, shows essentially only selection against C. Positions 7 and 8 of the TBP site (N2 and N3) reveal a modest preference for T, while a G is preferred at position N4. This is in contrast to bases selected by wild-type TBP, for which a G at positions equivalent to N1 and N2 was not observed . Apparently, TBPm3 exhibits a less stringent sequence preference compared with wild-type TBP.
|
17028095_p19
|
17028095
|
Determination of TBPm3 sequence preference for the downstream half of the TGTA box
| 4.418171 |
biomedical
|
Study
|
[
0.999103844165802,
0.0005924690049141645,
0.00030365915154106915
] |
[
0.999178946018219,
0.0002905815199483186,
0.00040031922981143,
0.00013010724796913564
] |
en
| 0.999998 |
The results of the TBPm3 selection were verified by EMSA and MPE-Fe(II) footprinting on a DNA probe representing the most frequently selected bases at each position, TGTAA ATTG (note that this sequence represents the most frequently occurring bases at each randomized position, but was not found among the selected clones). TBPm3 was seen to bind to 26 bp DNA containing this sequence, while disfavored sequences, containing for example a C in position N1 (TGTAA CTGG ) yield barely detectable complex formation . MPE-Fe(II) footprinting on the 76 bp DNA containing the favored sequence indicates that TBPm3 is binding at the TGTA box, despite the fact that this sequence was not found among the selected clones, as seen by the partial protection from cleavage at positions −28 to −23 , where the first T of the TGTA box is designated −30. Enhanced cleavage was observed at base pair −19, −18 and −31 consistent with that observed for wild-type TBP at these positions ( 22 , 29 ).
|
17028095_p20
|
17028095
|
Determination of TBPm3 sequence preference for the downstream half of the TGTA box
| 4.239038 |
biomedical
|
Study
|
[
0.9994217157363892,
0.0003135169972665608,
0.0002648447407409549
] |
[
0.9994245767593384,
0.0002592467935755849,
0.00024273151939269155,
0.00007348373037530109
] |
en
| 0.999993 |
When the selection was performed using TFIIIB assembled with TBPm3, a distinct sequence preference emerged. A total of 25 clones containing the TGTAA sequence were aligned to determine the consensus for this selection. As for the TBPm3 selection, clones containing the TATAA sequence (25 clones) were excluded from the alignment, as were 13 clones containing the series of GTG repeats. While the occurrence of the GTG-repeat sequences in both selections is curious, we did not pursue this observation further. The remainder of the clones contained sequence with no match to the above categories, including other alterations to the 5′ half of the TGTA box. Alignment of the 25 TGTAA-containing sequences ( Table 3 ) showed a much stronger sequence preference compared with TBPm3 alone, despite reaction conditions that should have allowed less stringent binding . Notably, the selected consensus sequence (TGTAA ATAG ) is a perfect match to the 8 bp U6 TATA box (TATAAATA).
|
17028095_p21
|
17028095
|
Determination of TBPm3 sequence preference for the downstream half of the TGTA box
| 4.161219 |
biomedical
|
Study
|
[
0.9993261098861694,
0.0003306673897895962,
0.0003431715886108577
] |
[
0.9995586276054382,
0.00018956315761897713,
0.00019125445396639407,
0.00006050926094758324
] |
en
| 0.999995 |
TBPm3 dissociates from its DNA site with first-order kinetics and exhibits second-order kinetics of association, as reported for wild-type TBP ( 26 , 29 – 35 ). As also seen for wild-type TBP under comparable experimental conditions, rate determinations do not indicate any contribution from a competing TBPm3 monomer–dimer equilibrium ( 29 , 36 ). Rates of association with either DNA probe are within the range reported for wild-type TBP, while the rate of dissociation is slower ( t 1/2 of 61 and 72 min) compared with ∼10 min for wild-type TBP using DNA containing the 8 bp U6 TATA box ( 29 ). This difference may be owing to the lower [NaCl] used here, as shown by the enhanced rate of dissociation of wild-type TBP that accompanies an increase in [KCl] from 60 to 80 mM [ t 1/2 100 versus 65 min using the AdML promoter TATA sequence; ( 26 )]. In addition, more stable complex formation may be the consequence of sequence flanking the 8 bp TATA box [the A immediately downstream of the U6 TATA box used in previous assays ( 29 ) was replaced with a G in the constructs used here]; while TBPm3 dissociates from TATAAATA G with t 1/2 = 72 min ( Table 1 ), t 1/2 for dissociation from TATAAATA A is 53 min (data not shown). Sequence flanking the TATA box has been previously shown to modulate kinetics of TBP dissociation, in particular for TBP sites characterized by alternating A-T base pairs ( 35 , 37 ).
|
17028095_p22
|
17028095
|
TBPm3–DNA complex formation
| 4.4484 |
biomedical
|
Study
|
[
0.9993423819541931,
0.00038489719736389816,
0.0002727396204136312
] |
[
0.9990174770355225,
0.00029052680474705994,
0.000580689636990428,
0.00011126864410471171
] |
en
| 0.999997 |
TBPm3 binds to the TGTA and TATA probes with comparable affinity, but we note that the modestly higher affinity for the TATA-containing DNA ( K d = 0.3 nM versus 0.6 nM for TGTA-containing DNA) is consistent with the identification of numerous TATA-containing sequences in the in vitro selections . The basis for this difference in affinity may be the increased flexibility of the T•A step relative to the T•G step ( 38 , 39 ). As for wild-type TBP, the rate of association of TBPm3 with DNA is orders of magnitude slower than the diffusion limit; for wild-type TBP, the rate of association appears not to be affected by flexure at the sites of DNA kinking, whereas complex stability is ( 29 ). Consistent with this observation, rates of association of TBPm3 with either TATA- or TGTA-containing DNA are equivalent.
|
17028095_p23
|
17028095
|
TBPm3–DNA complex formation
| 4.309201 |
biomedical
|
Study
|
[
0.9994032382965088,
0.0003115176223218441,
0.00028522987850010395
] |
[
0.9993304014205933,
0.0003046397469006479,
0.0002908165333792567,
0.00007419064786517993
] |
en
| 0.999997 |
The orientation of TBP on the TATA box is such that the C-proximal TBP domain interacts with the 5′ half of the TATA box, while the N-proximal domain contacts the less-conserved 3′ half-site ( 11 – 13 , 40 ). Sequence specificity at the upstream half of the TATA box has been suggested to be in part imposed by the presence of a proline (Pro191) that would disallow any base other than a T at the 5′ end of the TATA box owing to steric clashes with other bases ( 13 ). The equivalent residue in the N-proximal TBP repeat is alanine (Ala100) which imposes no such steric constraints. The modest orientational preference of TBP observed in vitro has been suggested to derive also from differential DNA flexure at the two sites of kinking ( 29 ). In the preinitiation complex (PIC), however, the orientation of TBP is largely determined by interaction with other transcription factors ( 21 , 29 , 41 ).
|
17028095_p24
|
17028095
|
Sequence preference of TBPm3
| 4.51771 |
biomedical
|
Study
|
[
0.999258816242218,
0.00038665771717205644,
0.0003545518557075411
] |
[
0.9983643889427185,
0.0004581001994665712,
0.0010602179681882262,
0.00011736241867765784
] |
en
| 0.999995 |
For TBPm3, three substitutions create a binding pocket that can accommodate G at position two of the TATA box. TBPm3 exhibits an only modest sequence preference for the last four bases of the TGTA box, with C generally disfavored at every position. While A→T and T→A transversions cause little change to the chemical environment of the DNA minor groove, the introduction of GC or CG base pairs results in the exocyclic amino group of G protruding into the minor groove. For wild-type TBP, cavities in the interface between TBP and the DNA minor groove can be seen to accommodate a G in positions 3 and 6 of the TATA box ( 40 ). The frequent occurrence of a G at position N1 (position 6 of the TBP site) was somewhat unexpected, but this portion of the helix is flattened and unwound in the wild-type TBP–DNA co-crystal structure, and there may likewise not be steric clashes between TBPm3 and the DNA. The widening of the minor groove that accompanies bending into the major groove is more difficult with GC base pairs, hence the more easily deformable TA sequence is preferred by wild-type TBP. Perhaps TBPm3 features additional contacts that may support bending of more rigid sequences.
|
17028095_p25
|
17028095
|
Sequence preference of TBPm3
| 4.611961 |
biomedical
|
Study
|
[
0.9987744688987732,
0.0007368001970462501,
0.0004888122202828526
] |
[
0.9978528022766113,
0.0012118951417505741,
0.0006764847203157842,
0.00025878899032250047
] |
en
| 0.999998 |
We note also that the bases most frequently selected at each position do not occur together. In its association with DNA, TBP introduces a significant bend at both ends of the TATA box. The energetically most favorable bending of B -DNA occurs by compression of the major groove with concomitant opening of the opposing minor groove; consequently, TBP generally targets A+T-rich regions that feature a greater range of minor groove widths. An exception is poly(dA) runs that exhibit local structural rigidity, as seen by the interlocking of major groove methyl groups of consecutive thymines ( 13 , 38 , 39 , 42 ). In an A•T base pair step, the stacking of the methyl group of thymine against the adjacent adenine is likewise extensive. Consequently, TBPm3 may select against the sequence TGTAA ATTG owing to its inherent stiffness, even though each base is the most frequently selected at its respective position.
|
17028095_p26
|
17028095
|
Sequence preference of TBPm3
| 4.35667 |
biomedical
|
Study
|
[
0.9993804693222046,
0.00030834792414680123,
0.0003111975674983114
] |
[
0.9989473223686218,
0.0005692641134373844,
0.0003936977300327271,
0.00008968896872829646
] |
en
| 0.999996 |
The sequence most frequently selected by wild-type TBP, TATATAA is followed by a G to complete the 8 bp TBP site, with a G or C found at the position immediately downstream ( 19 ). This sequence is selected against in our assay as the first five bases of the TGTA box (TGTA A ) were held constant, thus a T in position five of the TATA box could only have arisen as a result of errors during PCR. Notably, of all the selected sequences, only one featured the sequence TGTA T , suggesting that it is not favored by TBPm3 (unlike the sequence T A TAA, which occurred in 29 of the clones, despite position two of the TBP site also not corresponding to a randomized position). Selection by wild-type TBP for a sequence that includes an A at position five of the TATA box was followed by the sequence A-T-A, generating the U6 TATA box TATAA ATA , with a C preferred at the position immediately downstream of this 8 bp TBP site (4 of 54 clones; ( 19 )). Eight base pair alternating TA sequences were generally followed by a G or C (12 of 54 clones). Accordingly, the presence of a G following the 8 bp TBP site preferred by TBPm3 is consistent with the preferred base following an 8 bp A+T-containing sequences selected by wild-type TBP ( 19 , 37 ). Since sequence flanking the 8 bp TBP site affects complex stability but not the rate at which TBP associates with the TATA box, flanking the A+T-rich TBP site with G+C-rich sequence may create border effects that stabilize bound TBP ( 35 , 37 ).
|
17028095_p27
|
17028095
|
Sequence preference of TBPm3
| 4.465019 |
biomedical
|
Study
|
[
0.9992561936378479,
0.0004078946076333523,
0.00033591940882615745
] |
[
0.9990585446357727,
0.00043053567060269415,
0.00039999966975301504,
0.00011091194028267637
] |
en
| 0.999996 |
The sequence preference of TFIIIB assembled with TBPm3 for the downstream half of the TGTA box differs significantly from that exhibited by TBPm3 alone. While TBPm3 mainly discriminates against C in positions N1–N3, entry of Brf1 and Bdp1 into the complex imposes a strict preference for the sequence A-T-A. In both selections, a G at position N4 is preferred, although only modestly so for TBPm3. Comparable with the TBPm3 selection, no sequences occur in the TFIIIB selection with the sequence TGTA T . We also discount the possibility that TFIIIB may reverse orientation, as a C is strongly disfavored at position N2. Accordingly, the sequence selected by TFIIIB matches that of the native U6 TATA box, except that a G immediately downstream of the 8 bp TATA box is seen in preference to the naturally occurring A.
|
17028095_p28
|
17028095
|
Brf1 and Bdp1 impose a strict sequence preference for the downstream half of the TATA box
| 4.402875 |
biomedical
|
Study
|
[
0.9992030262947083,
0.0003895019181072712,
0.00040748194442130625
] |
[
0.9989973902702332,
0.0005032626795582473,
0.00040580303175374866,
0.00009343773854197934
] |
en
| 0.999996 |
It was previously shown that stability of TBP on a 6 bp TATA box, which is suboptimal for TFIIIB assembly, is comparable with that of an 8 bp TATA box, which efficiently supports assembly of TFIIIB ( 29 ). The significant difference between TATA box sequences must therefore be structural or dynamic adaptations to interaction with Brf1 and Bdp1. In general, the DNA bending that occurs upon association with TBP brings flanking DNA segments closer together to facilitate contacts with other transcription factors that make up the PIC ( 24 , 43 – 45 ), and sequences that promote a disposition of DNA flanking the TBP-mediated DNA bends in a direction consistent with association of Brf1 and Bdp1 may be preferred. Indeed, analysis of TBP in complex with several divergent TATA sequences reveals comparable structures, yet only some are permissible for PIC formation; base pair changes may well be tolerated in terms of binding to TBP, but may negatively affect recruitment of other transcription factors ( 40 ).
|
17028095_p29
|
17028095
|
Brf1 and Bdp1 impose a strict sequence preference for the downstream half of the TATA box
| 4.360122 |
biomedical
|
Study
|
[
0.9994025230407715,
0.0003082627081312239,
0.00028929588734172285
] |
[
0.9990550875663757,
0.0002914557117037475,
0.0005769084673374891,
0.00007654506771359593
] |
en
| 0.999997 |
The efficiency with which the TBP–TATA complex promotes transcriptional activity depends on the sequence of the TATA box, including A-T transversions that do not alter functional groups present in the DNA minor groove. Presumably, TBP depends significantly on recognition of inherent flexibility of the TATA box, and such differences may also affect PIC assembly ( 40 , 46 ). For example, molecular dynamics simulations of different TATA variants suggest that DNA flexibility is correlated with transcriptional activity by RNA pol II ( 47 ). Correlating molecular dynamics simulations of TBP–TATA complexes involving different TATA sequences with reported transcriptional activity by pol II further suggests that optimal pol II activity occurs on DNA that allows the two domains of TBP to rotate relative to each other and that allows the H2 helix of TBP to assume an optimal disposition to interact with factors that bind both TBP domains (such as Brf1). In contrast, low activity DNA sequences appear to promote movement of the H1 helix of TBP and to involve conformational changes in the DNA ( 48 ).
|
17028095_p30
|
17028095
|
Brf1 and Bdp1 impose a strict sequence preference for the downstream half of the TATA box
| 4.404297 |
biomedical
|
Study
|
[
0.999438464641571,
0.00030440263799391687,
0.0002571286750026047
] |
[
0.9987871050834656,
0.0002954488736577332,
0.0008263669442385435,
0.00009117287845583633
] |
en
| 0.999998 |
TBP introduces roll deformations at either end of the TATA box ( 11 – 13 ). The T•A base pair step is easily deformable owing to its large range of allowable roll angles and is often found in DNA sequences requiring a sharp bend ( 38 , 39 , 49 ). Indeed, roll deformations at the downstream kink of TATA DNA in complex with TBP vary from ∼30° for A•G steps to >45° for T•A steps ( 49 ). A unique feature of the U6 TATA box sequence identified in our selections with TFIIIB is the presence of a T•A step at the downstream end of the 8 bp TBP site. While this sequence is not strongly favored by either wild-type TBP or TBPm3 alone, it clearly promotes formation of the TFIIIB-DNA complex. Consistent with this interpretation, in vitro transcription with Drosophila nuclear extract indicated that while pol II utilizes the TATA box TATAAAAA in the forward direction, pol III reverses orientation ( 50 ). We suggest that the unique feature of the selected sequence is a flexibility at the downstream end of the 8 bp TATA box that promotes Brf1 and Bdp1 binding and the associated DNA deformation downstream of the TATA box ( 22 ).
|
17028095_p31
|
17028095
|
Brf1 and Bdp1 impose a strict sequence preference for the downstream half of the TATA box
| 4.482472 |
biomedical
|
Study
|
[
0.9991756081581116,
0.0004999364027753472,
0.0003245019179303199
] |
[
0.9989540576934814,
0.000470149825559929,
0.0004199576796963811,
0.00015575259749311954
] |
en
| 0.999999 |
In Saccharomyces cerevisiae , the dosage and expression level of the SIR2 gene has been implicated in the regulation of important processes, such as transcriptional silencing, genome stability, DNA repair, chromatin structure and cellular aging ( 1 ). Sir2p is a NAD-dependent histone deacetylase ( 2 – 4 ), suggesting that its role in transcriptional silencing might be enzymatic. In addition, Sir2p can perform a weak ADP-ribosylation reaction using NAD as the donor of an ADP-ribose moiety ( 5 , 6 ). Further evidence for the importance of this protein comes from the discovery of the ‘sirtuin’ protein family, which is conserved from bacteria to humans. Sirtuins are also involved in cell cycle progression, chromosome stability and aging ( 7 , 8 ). Four proteins, designated homologues of sir two (HST), have also been found in S.cerevisiae ( 9 ).
|
17012273_p0
|
17012273
|
INTRODUCTION
| 4.49376 |
biomedical
|
Study
|
[
0.9993900060653687,
0.00028214274789206684,
0.0003278367512393743
] |
[
0.9741570949554443,
0.0008968815091066062,
0.024751044809818268,
0.00019493408035486937
] |
en
| 0.999997 |
To carry out its role in transcriptional silencing, Sir2p acts together with three additional silent information proteins, Sir1p, Sir3p and Sir4p ( 10 ). Depending on the specific region where silencing occurs, Sir2p cooperates with different partners. In association with Sir1p, Sir3p and Sir4p, Sir2p mediates the silencing at the mating type loci (HML and HMR) ( 10 ), while in association with Sir3p and Sir4p it is involved in telomeric silencing. The rDNA is a third genetic locus where transcriptional silencing occurs in S.cerevisiae . Here, Sir2p is the only Sir protein necessary for maintenance of the silenced state. At this locus, however, it requires the presence of Net1p (part of the RENT complex) ( 11 , 12 ), which is believed to be crucial for nucleolar localization.
|
17012273_p1
|
17012273
|
INTRODUCTION
| 4.478349 |
biomedical
|
Study
|
[
0.9993483424186707,
0.00027206895174458623,
0.0003795455559156835
] |
[
0.9957481026649475,
0.0012785455910488963,
0.002863041590899229,
0.00011041704419767484
] |
en
| 0.999997 |
In a sir2 Δ yeast strain, loss of transcriptional silencing is observed at the rDNA locus; this mutant is also characterized by hyper-accessible chromatin ( 13 , 14 ), hyper-recombination of ribosomal repeat units ( 15 ), and decreased life span associated with the accumulation of extrachromosomal circles (ERCs) ( 16 ).
|
17012273_p2
|
17012273
|
INTRODUCTION
| 4.231259 |
biomedical
|
Study
|
[
0.9995843768119812,
0.00018381733389105648,
0.00023173789668362588
] |
[
0.9985365867614746,
0.0008244442287832499,
0.0005543667357414961,
0.00008461370453005657
] |
en
| 0.999996 |
In addition, several lines of evidences link SIR2 to DNA replication. At the rDNA locus of S.cerevisiae , it has been reported that Sir2p is preferentially associated with non-replicating chromatin, exerting a form of negative control over origin firing ( 17 ); more recently, an additional report has shown that Sir2p negatively controls pre-replication complex (RC) formation by interfering with loading of the MCM complex on the replication origin ( 18 ).
|
17012273_p3
|
17012273
|
INTRODUCTION
| 4.275523 |
biomedical
|
Study
|
[
0.999503493309021,
0.00022594333859160542,
0.0002706022060010582
] |
[
0.9976601600646973,
0.0005496885278262198,
0.0017049852758646011,
0.00008520638948539272
] |
en
| 0.999998 |
Some of the phenotypes associated with the sir2 Δ mutation are potentially related to DNA supercoiling, an aspect of DNA organization. In particular, similar phenotypes have been reported for sir2 Δ and top1 Δ, such as hyper-recombination of rDNA repeat units ( 15 , 19 ), loss of transcriptional silencing at the rDNA locus ( 20 , 21 ) and alteration of histone acetylation ( 14 , 22 ). Since DNA topoisomerase I controls DNA superhelicity, we decided to investigate whether Sir2p may also interfere with the process of supercoiling.
|
17012273_p4
|
17012273
|
INTRODUCTION
| 4.115474 |
biomedical
|
Study
|
[
0.9994522929191589,
0.00023651335504837334,
0.00031124899396672845
] |
[
0.9993510842323303,
0.000293019664241001,
0.00029528012964874506,
0.00006050766023690812
] |
en
| 0.999998 |
In eukaryotic cells, DNA superhelicity arises mainly from the wrapping of DNA around nucleosome particles ( 23 ), resulting in approximately one negative superhelical turn per nucleosome ( 24 , 25 ). It has been reported that supercoiling of DNA elements (plasmids or excised DNA circles) containing silencer sequences is altered in mutants that affect transcriptional silencing ( 26 – 29 ). Moreover, changing the histone acetylation level has been reported to affect DNA supercoiling ( 30 , 31 ). By analogy, the reported alteration of chromatin structure ( 13 , 14 ) could lead to modification of DNA topology.
|
17012273_p5
|
17012273
|
INTRODUCTION
| 4.331343 |
biomedical
|
Study
|
[
0.9995239973068237,
0.00023242749739438295,
0.00024355173809453845
] |
[
0.9973167777061462,
0.00036928936606273055,
0.0022298935800790787,
0.00008400643127970397
] |
en
| 0.999996 |
Strains: W303-1a ; Y1422 (sir2Δ) kindly provided by J. Broach; AYH2.45 (Mata, ade2-101, his3-Δ200, leu2-3,-112, lys2-801, trp1-Δ901, ura3-52, adh4::URA3TelVII-L; STY30 (sir2Δ) isogenic to AYH2.45, sir2::TRP1; STY36 (sir4Δ) isogenic to AYH2.45, sir4::TRP1; LJY912 (Mata, ade2-101, his3-Δ200, leu2-3,-112, lys2-801, trp1-Δ901, ura3-52, thr tyr arg4-1, hhf1::HIS3; hhf2::HIS4/pLJ912(CEN3, ARS1, URA3, hhf2 K16Q) and its isogenic PKY501 (Mata, ade2-101, his3-Δ200, leu2-3,-112, lys2-801, trp1-Δ901, ura3-52, thr tyr arg4-1, hhf1::HIS3; hhf2::HIS4/pPK301 (CEN3, ARS1, URA3, HHF2) kindly provided by M. Grunstein. JRY4602(sir3Δ) kindly provided by Jasper Rine; YKL52 (Mcm7-td) (Mata, ade2-1, ura3-1, his3-11, 15 trp1-1, leu2-3112 can 1-100, MCM7::GAL-Ubiquitin-M-lac1 fragment-Myc-UBR1 (URA3) kindly provided by J. Diffley; WY69 (net1Δ): kindly provided by D. Moazed. AEY1958 (MATα HMRa-e ** hht1-hhf1Δ::LEU2 hht2-hhf2Δ::HIS3 pRS414-[HHT1-HHF1]) and AEY1956 (MATα HMRa-e ** hht1-hhf1Δ::LEU2 hht2-hhf2Δ::HIS3 pRS414-[HHT1 hhf1-21 (K16R)] kindly provided by A. E. Ehrenhofer-Murray.
|
17012273_p6
|
17012273
|
Yeast strains, plasmids and culture media
| 3.432145 |
biomedical
|
Other
|
[
0.9976640939712524,
0.0004730762157123536,
0.0018628519028425217
] |
[
0.4488028883934021,
0.5482401847839355,
0.001766691100783646,
0.0011901602847501636
] |
en
| 0.999998 |
YNZ1 (produced in this work) isogenic to PKY501, sir2:: kanMX; YNZ2 (produced in this work) as LJY912 , sir2: kanMX.
|
17012273_p7
|
17012273
|
Yeast strains, plasmids and culture media
| 2.050906 |
biomedical
|
Other
|
[
0.9843980669975281,
0.0009841956198215485,
0.01461778860539198
] |
[
0.06951109319925308,
0.9287925362586975,
0.0007355110719799995,
0.0009608881664462388
] |
en
| 0.999997 |
Plasmids: ypGM1 ( 32 ); pRS316 ( 33 ), p415Gal, p414Gal ( 34 ), pADH426 ( 35 ), yCp50 ( 36 ), pAR44 ( 37 ), pPK301 and pLJ912 ( 38 ).
|
17012273_p8
|
17012273
|
Yeast strains, plasmids and culture media
| 1.943494 |
biomedical
|
Other
|
[
0.987939715385437,
0.0010077118640765548,
0.01105261966586113
] |
[
0.040562115609645844,
0.9579925537109375,
0.0006752852350473404,
0.0007700134301558137
] |
en
| 0.85714 |
The culture media utilized for cell growth were complete YPD or minimal YNB ( 39 ), both supplemented with 2% glucose, or complete YPGal supplemented with 2% galactose when appropriate (see text).
|
17012273_p9
|
17012273
|
Culture media and conditions
| 3.619778 |
biomedical
|
Study
|
[
0.9992307424545288,
0.0002540198911447078,
0.000515225634444505
] |
[
0.9027749300003052,
0.09468945115804672,
0.0020859765354543924,
0.000449623737949878
] |
en
| 0.999996 |
Over-expression of Sir2p was achieved by transforming W303-1a with high-copy number plasmid pAR44 carrying the coding region for Sir2p under the GAL10 promoter as described ( 37 ).
|
17012273_p10
|
17012273
|
Culture media and conditions
| 4.00611 |
biomedical
|
Study
|
[
0.9992321729660034,
0.00028706356533803046,
0.000480783375678584
] |
[
0.993691086769104,
0.005613310262560844,
0.0005361133371479809,
0.00015949711087159812
] |
en
| 0.999996 |
Standard procedures ( 40 ) for SIR2 gene disruption have been employed utilizing the following oligonucleotides (5′–3′): TCGGTAGACACATTCAAACCATTTTTCCCTCATCGGCACATTAAAGCTGGCGGATCCCCGGGTTAATTAA (forward); GGCACTTTTAAATTATTAAATTGCCTTCTACTTAGAGGGTTTTGGGATGTGAATTCGAGCTCGTTTAAAC (reverse).
|
17012273_p11
|
17012273
|
Production of YNZ1 and YNZ2 mutants
| 4.032681 |
biomedical
|
Study
|
[
0.9994542002677917,
0.0002092901850119233,
0.00033646775409579277
] |
[
0.9806174635887146,
0.018459182232618332,
0.0006924712797626853,
0.00023078611411619931
] |
en
| 0.999996 |
Asynchronous cultures of mcm7-td UBR1 + and mcm7-td ubr1 Δ :: GAL-UBR1 strains were grown in YP + Glucose medium (GAL-UBR1 OFF) or YP + Galactose (GAL-UBR1 ON) at 24°C. Cells were then shifted to 37°C and samples were taken after 1 h to purify plasmid DNA.
|
17012273_p12
|
17012273
|
Induction of Mcm7p degradation
| 3.984888 |
biomedical
|
Study
|
[
0.9994300007820129,
0.00024512939853593707,
0.00032487191492691636
] |
[
0.9971799850463867,
0.0023715950082987547,
0.00034998447517864406,
0.00009844201122177765
] |
en
| 0.999998 |
Restriction enzymes and micrococcal nuclease were purchased from Roche; Taq polymerase from Perkin–Helmer; zymolyase from Seikagaku (Tokyo, Japan); nystatin from Sigma and radiochemicals from Amersham.
|
17012273_p13
|
17012273
|
Enzymes and chemicals
| 1.130366 |
biomedical
|
Other
|
[
0.9792869091033936,
0.001962604932487011,
0.018750499933958054
] |
[
0.04651373252272606,
0.9502488970756531,
0.0013562854146584868,
0.00188114820048213
] |
en
| 0.999997 |
Micrococcal nuclease treatment: cells (100–200 ml grown to 0.4 OD/ml) were pelleted and resupended in 10 ml of a buffer containing 1 M sorbitol, 50 mM Tris–HCl (pH 7.5), 10 mM β-mercaptoethanol, in the presence of 0.03 mg/3 × 10 7 cells of Zymolyase 100T, and incubated for 10 min at 30°C. The resulting spheroplasts were harvested, resuspended in Nystatin buffer [50 mM NaCl, 1.5 mM CaCl 2 , 20 mM Tris–HCl (pH 8.0), 0.9 M sorbitol and 100 µg/ml nystatin] ( 41 ) and divided into 0.4 ml aliquots. MNase (0.2, 0.4, 0.8 and 1.6 U) was added to each aliquot and the samples were incubated at 37°C for 15 min. The reaction was stopped with 1% SDS, 5 mM EDTA (final concentrations). Proteinase K (40 µg/sample) was added and the samples kept at 56°C for 2 h. The DNA was then purified by three phenol/chloroform extractions and ethanol precipitation. RNase treatment was also performed.
|
17012273_p14
|
17012273
|
Chromatin analysis
| 4.199863 |
biomedical
|
Study
|
[
0.999259889125824,
0.0005077066016383469,
0.00023244167095981538
] |
[
0.9926508069038391,
0.006395382806658745,
0.0007219546241685748,
0.0002319040213478729
] |
en
| 0.999998 |
Subsets and Splits
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