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1.
Two-dimensional (2-D) gel analysis of replication intermediates in the Chinese hamster dihydrofolate reductase domain has suggested that nascent chains can initiate at any of a large number of sites scattered throughout a ~50 kb “initiation locus” (although the level of initiation detected at any given site within this region was relatively low). This result contrasts markedly with data from anin vitro strand switching assay suggesting that >80% of initiations occur within a single 500 bp fragment lying within the initiation locus. In an effort to reconcile these two disparate views of the initiation reaction, we have questioned the validity of our 2-D gel data in several ways. We show here that: 1) the number of replication bubbles detected in the DHFR locus in the early S period is markedly increased when the cells are released from a synchronizing agent that inhibits initiationper se, rather than from aphidicolin, which is a chain elongation inhibitor; 2) initiation in the DHFR domain occurs only during the first 90 min of the S period, as would be expected of an early-firing origin; 3) a pulse of3H-thymidine moves through the structures observed on 2-D gels with the kinetics expected ofbonafide replication intermediates; and 4) preparations of replication intermediates that are subsequently analyzed on 2-D gels appear, by electron microscopy, to represent the typical theta structures and single-forked molecules expected of bidirectional origins of replication; no unusual structures (e.g., microbubbles) were seen. 相似文献
2.
The Chinese hamster dihydrofolate reductase origin consists of multiple potential nascent-strand start sites. 总被引:13,自引:6,他引:7
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Previous two-dimensional gel replicon-mapping studies on the amplified dihydrofolate reductase (DHFR) domain in CHOC 400 cells suggested that replication can initiate at any of a large number of sites scattered throughout a 55-kb region lying between two convergently transcribed genes. It could be argued that this unusual distributive initiation mode is unique to amplified chromosomal loci. In this paper, we report the first application of the two-dimensional gel techniques to the analysis of a single-copy locus in mammalian cells. Results obtained with both synchronized and exponentially growing CHO cells suggest that (i) initiation can also occur at any of a large number of sites distributed throughout the intergenic region in the nonamplified DHFR locus, (ii) initiation is confined to the first 2 to 2.5 h of the S period, and (iii) initiation occurs only in a fraction of the DHFR loci in each cell cycle. 相似文献
3.
Initiation sites are distributed at frequent intervals in the Chinese hamster dihydrofolate reductase origin of replication but are used with very different efficiencies
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Previous radiolabeling and two-dimensional (2-D) gel studies of the dihydrofolate reductase (DHFR) domain of Chinese hamster cells have suggested that replication can initiate at any one of a very large number of inefficient sites scattered throughout the 55-kb intergenic spacer region, with two broad subregions (ori-beta and ori-gamma) preferred. However, high-resolution analysis by a PCR-based nascent strand abundance assay of the 12-kb subregion encompassing ori-beta has suggested the presence of a relatively small number of fixed, highly efficient initiation sites distributed at infrequent intervals that correspond to genetic replicators. To attempt to reconcile these observations, two different approaches were taken in the present study. In the first, neutral-neutral 2-D gel analysis was used to examine replication intermediates in 31 adjacent and overlapping restriction fragments in the spacer, ranging in size from 1.0 to 18 kb. Thirty of 31 fragments displayed the complete bubble arcs characteristic of centered origins. Taking into account overlapping fragments, these data suggest a minimum of 14 individual start sites in the spacer. In the second approach, a quantitative early labeled fragment hybridization assay was performed in which radioactive origin-containing DNA 300 to 1,000 nucleotides in length was synthesized in the first few minutes of the S period and used to probe 15 clones distributed throughout the intergenic spacer but separated on average by more than 1,000 bp. This small nascent DNA fraction hybridized to 14 of the 15 clones, ranging from just above background to a maximum at the ori-beta locus. The only silent region detected was a small fragment lying just upstream from a centered matrix attachment region--the same region that was also negative for initiation by 2-D gel analysis. Results of both approaches suggest a minimum of approximately 20 initiation sites in the spacer (two of them being ori-beta and ori-gamma), with ori-beta accounting for a maximum of approximately 20% of initiations occurring in the spacer. We believe that the results of all experimental approaches applied to this locus so far can be fitted to a model in which the DHFR origin consists of a 55-kb intergenic zone of potential sites that are used with very different efficiencies and which are separated in many cases by a few kilobases or less. 相似文献
4.
Replication in the amplified dihydrofolate reductase domain in CHO cells may initiate at two distinct sites, one of which is a repetitive sequence element. 总被引:30,自引:22,他引:8
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To study initiation of DNA replication in mammalian chromosomes, we have established a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) that contains approximately 1,000 copies of the early replicating dihydrofolate reductase (DHFR) domain. We have previously shown that DNA replication in the prevalent 243-kilobase (kb) amplicon type in this cell line initiates somewhere within a 28-kb region located downstream from the DHFR gene. In an attempt to localize the origin of replication with more precision, we blocked the progress of replication forks emanating from origins at the beginning of the S phase by the introduction of trioxsalen cross-links at 1- to 5-kb intervals in the parental double-stranded DNA. The small DNA fragments synthesized under these conditions (which should be centered around replication origins) were then used as hybridization probes on digests of cosmids and plasmids from the DHFR domain. These studies suggested that in cells synchronized by this regimen, DNA replication initiates at two separate sites within the previously defined 28-kb replication initiation locus, in general agreement with results described in the accompanying paper (T.-H. Leu and J. L. Hamlin, Mol. Cell. Biol. 9:523-531, 1989). One of these sites contains a repeated DNA sequence element that is found at or near many other initiation sites in the genome, since it was also highly enriched in the early replicating DNA isolated from cross-linked CHO cells that contain only two copies of the DHFR domain. 相似文献
5.
Mapping of replication initiation sites in mammalian genomes by two-dimensional gel analysis: stabilization and enrichment of replication intermediates by isolation on the nuclear matrix. 总被引:31,自引:16,他引:15
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Two complementary two-dimensional gel electrophoretic techniques have recently been developed that allow initiation sites to be mapped with relative precision in eukaryotic genomes at least as complex as those of yeast and Drosophila melanogaster. We reported the first application of these mapping methods to a mammalian genome in a study on the amplified dihydrofolate reductase (DHFR) domain of the methotrexate-resistant CHO cell line CHOC 400 (J.P. Vaughn, P.A. Dijkwel, and J.L. Hamlin, Cell 61:1075-1087, 1990). Our results suggested that in this 240-kb domain, initiation of nascent DNA strands occurs at many sites within a 30- to 35-kb zone mapping immediately downstream from the DHFR gene. In the course of these studies, it was necessary to develop methods to stabilize replication intermediates against branch migration and shear. This report describes these stabilization methods in detail and presents a new enrichment protocol that extends the neutral/neutral two-dimensional gel mapping method to single-copy loci in mammalian cells. Preliminary analysis of replication intermediates purified from CHO cells by this method suggests that DNA synthesis may initiate at many sites within a broad zone in the single-copy DHFR locus as well. 相似文献
6.
Identification of Primary Initiation Sites for DNA Replication in the Hamster Dihydrofolate Reductase Gene Initiation Zone 总被引:15,自引:10,他引:5
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Takehiko Kobayashi Theo Rein Melvin L. DePamphilis 《Molecular and cellular biology》1998,18(6):3266-3277
Mammalian replication origins appear paradoxical. While some studies conclude that initiation occurs bidirectionally from specific loci, others conclude that initiation occurs at many sites distributed throughout large DNA regions. To clarify this issue, the relative number of early replication bubbles was determined at 26 sites in a 110-kb locus containing the dihydrofolate reductase (DHFR)-encoding gene in CHO cells; 19 sites were located within an 11-kb sequence containing ori-β. The ratio of ~0.8-kb nascent DNA strands to nonreplicated DNA at each site was quantified by competitive PCR. Nascent DNA was defined either as DNA that was labeled by incorporation of bromodeoxyuridine in vivo or as RNA-primed DNA that was resistant to λ-exonuclease. Two primary initiation sites were identified within the 12-kb region, where two-dimensional gel electrophoresis previously detected a high frequency of replication bubbles. A sharp peak of nascent DNA occurred at the ori-β origin of bidirectional replication where initiation events were 12 times more frequent than at distal sequences. A second peak occurred 5 kb downstream at a previously unrecognized origin (ori-β′). Thus, the DHFR gene initiation zone contains at least three primary initiation sites (ori-β, ori-β′, and ori-γ), suggesting that initiation zones in mammals, like those in fission yeast, consist of multiple replication origins. 相似文献
7.
The dihydrofolate reductase origin of replication does not contain any nonredundant genetic elements required for origin activity
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The Chinese hamster dihydrofolate reductase (DHFR) origin of replication consists of a broad zone of potential initiation sites scattered throughout a 55-kb intergenic spacer, with at least three sites being preferred (ori-beta, ori-beta', and ori-gamma). We previously showed that deletion of the most active site or region (ori-beta) has no demonstrable effect on initiation in the remainder of the intergenic spacer nor on the time of replication of the DHFR locus as a whole. In the present study, we have now deleted ori-beta', both ori-beta and ori-beta', an 11-kb region just downstream from the DHFR gene, or the central approximately 40-kb core of the spacer. The latter two deletions together encompass >95% of the initiation sites that are normally used in this locus. Two-dimensional gel analysis shows that initiation still occurs in the early S phase in the remainder of the intergenic spacer in each of these deletion variants. Even removal of the 40-kb core fails to elicit a significant effect on the time of replication of the DHFR locus in the S period; indeed, in the truncated spacer that remains, the efficiency of initiation actually appears to increase relative to the corresponding region in the wild-type locus. Thus, if replicators control the positions of nascent strand start sites in this complex origin, either (i) there must be a very large number of redundant elements in the spacer, each of which regulates initiation only in its immediate environment, or (ii) they must lie outside the central core in which the vast majority of nascent strand starts occur. 相似文献
8.
Replication initiation sites are distributed widely in the amplified CHO dihydrofolate reductase domain. 总被引:10,自引:3,他引:7
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In previous studies, we utilized a neutral/neutral two-dimensional (2-D) gel replicon mapping method to analyze the pattern of DNA synthesis in the amplified dihydrofolate reductase (DHFR) domain of CHOC 400 cells. Replication forks appeared to initiate at any of a large number of sites scattered throughout the 55 kb region lysing between the DHFR and 2BE2121 genes, and subsequently to move outward through the two genes. In the present study, we have analyzed this locus in detail by a complementary, neutral/alkaline 2-D gel technique that determines the direction in which replication forks move through a region of interest. In the early S period, forks are observed to travel in both directions through the intergenic region, but only outward through the DHFR gene. Surprisingly, however, replication forks also move in both directions through the 2BE2121 gene. Furthermore, in early S phase, small numbers of replication bubbles can be detected in the 2BE2121 gene on neutral/neutral 2-D gels. In contrast, replication bubbles have never been detected in the DHFR gene. Thus, replication initiates not only in the intergenic region, but also at a lower frequency in the 2BE2121 gene. We further show that only a small fraction of DHFR amplicons sustains an active initiation event, with the rest being replicated passively by forks from distant amplicons. These findings are discussed in light of other experimental approaches that suggest the presence of a much more narrowly circumscribed initiation zone within the intergenic region. 相似文献
9.
Mark G Alexandrow Marion Ritzi Alexander Pemov Joyce L Hamlin 《The Journal of biological chemistry》2002,277(4):2702-2708
Mini-chromosome maintenance (MCM) proteins were originally identified in yeast, and homologues have been identified in several other eukaryotic organisms, including mammals. These findings suggest that the mechanisms by which eukaryotic cells initiate and regulate DNA replication have been conserved throughout evolution. However, it is clear that many mammalian origins are much more complex than those of yeast. An example is the Chinese hamster dihydrofolate reductase (DHFR) origin, which resides in the spacer between the DHFR and 2BE2121 genes. This origin consists of a broad zone of potential sites scattered throughout the 55-kb spacer, with several subregions (e.g. ori-beta, ori-beta', and ori-gamma) being preferred. We show here that antibodies to human MCMs 2-7 recognize counterparts in extracts prepared from hamster cells; furthermore, co-immunoprecipitation data demonstrate the presence of an MCM2-3-5 subcomplex as observed in other species. To determine whether MCM proteins play a role in initiation and/or elongation in Chinese hamster cells, we have examined in vivo protein-DNA interactions between the MCMs and chromatin in the DHFR locus using a chromatin immunoprecipitation (ChIP) approach. In synchronized cultures, MCM complexes associate preferentially with DNA in the intergenic initiation zone early in S-phase during the time that replication initiates. However, significant amounts of MCMs were also detected over the two genes, in agreement with recent observations that the MCM complex co-purifies with RNA polymerase II. As cells progress through S-phase, the MCMs redistribute throughout the DHFR domain, suggesting a dynamic interaction with DNA. In asynchronous cultures, in which replication forks should be found at any position in the genome, MCM proteins were distributed relatively evenly throughout the DHFR locus. Altogether, these data are consistent with studies in yeast showing that MCM subunits localize to origins during initiation and then migrate outward with the replication forks. This constitutes the first evidence that mammalian MCM complexes perform a critical role during the initiation and elongation phases of replication at the DHFR origin in hamster cells. 相似文献
10.
Lagging-Strand, Early-Labelling, and Two-Dimensional Gel Assays Suggest Multiple Potential Initiation Sites in the Chinese Hamster Dihydrofolate Reductase Origin 总被引:5,自引:3,他引:2
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There is general agreement that DNA synthesis in the single-copy and amplified dihydrofolate reductase (DHFR) loci of CHO cells initiates somewhere within the 55-kb spacer region between the DHFR and 2BE2121 genes. However, results of lagging-strand, early-labelling fragment hybridization (ELFH), and PCR-based nascent-strand abundance assays have been interpreted to suggest a very narrow zone of initiation centered at a single locus known as ori-β, while two-dimensional (2-D) gel analyses suggest that initiation can occur at any of a large number of potential sites scattered throughout the intergenic region. The results of a leading-strand assay and two intrinsic labelling techniques are compatible with a broad initiation zone in which ori-β and a second locus (ori-γ) are somewhat preferred. To determine how these differing views are shaped by differences in experimental manipulations unrelated to the biology itself, we have applied the lagging-strand, ELFH, neutral-neutral, and/or neutral-alkaline 2-D gel assays to CHOC 400 cell populations synchronized and manipulated in the same way. In our experiments, the lagging-strand assay failed to identify a template strand switch at ori-β; rather, we observed a gradual, undulating change in hybridization bias throughout the intergenic spacer, with hybridization to the two templates being approximately equal near a centered matrix attachment region. In the ELFH assay, all of the fragments in the 55-kb intergenic region were labelled in the first few minutes of the S phase, with the regions encompassing ori-β and ori-γ being somewhat preferred. Under the same conditions, neutral-neutral and neutral-alkaline 2-D gel analyses detected initiation sites at multiple locations in the intergenic spacer. Thus, the results of all existing replicon-mapping methods that have been applied to the amplified DHFR locus in CHOC 400 cells are consistent with a model in which two somewhat preferred subzones reside in a larger zone of multiple potential initiation sites in the intergenic region. 相似文献
11.
1-beta-D-Arabinofuranosylcytosine (ara-C) inhibits nuclear DNA replication in Chinese hamster ovary cells by an efficient chain termination mechanism without affecting the rate at which cells traverse G1 and enter S [Heintz, N. H., & Hamlin, J. L. (1983) Biochemistry 22, 3557-3562]. Here we have employed ara-C to enrich for replication intermediates formed during initiation of DNA synthesis in synchronized CHOC 400 cells, a methotrexate-resistant derivative of Chinese hamster ovary cells that contains approximately 1000 copies of an early replicating 150-kb chromosomal domain. This highly amplified domain includes the gene for dihydrofolate reductase (DHFR). CHOC 400 cells were collected at the G1/S boundary of the cell cycle with aphidicolin prior to release into S in the presence of both [methyl-3H] thymidine and various concentrations of ara-C. Chromatographic fractionation of restriction endonuclease digests over benzoylated naphthoylated DEAE-cellulose (BND-cellulose) showed that high concentrations of ara-C inhibited the maturation of chromosomal replication intermediates containing ssDNA (replication forks) into dsDNA for up to 60 min. The effect of ara-C on the sequence complexity of replication intermediates formed during early S phase was determined by hybridizing purified intermediates labeled with 32P in vitro to Southern blots of genomic DNA derived from both methotrexate-sensitive and methotrexate-resistant Chinese hamster ovary cells. In the absence of ara-C, 32P-labeled ssDNA BND-cellulose fractions from cultures released into S for 30-60 min hybridized to a spectrum of restriction fragments encompassing 40-50 kb of the amplified DHFR domain.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
12.
Replication initiates in a broad zone in the amplified CHO dihydrofolate reductase domain 总被引:56,自引:0,他引:56
We have used two complementary two-dimensional gel electrophoretic methods to localize replication inititation sites and to determine replication fork direction in the amplified 240 kb dihydrofolate reductase domain of the methotrexate-resistant CHO cell line CHOC 400. Surprisingly, our analysis indicates that replication begins at many sites in several restriction fragments distributed throughout a previously defined 28 kb initiation locus, including a fragment containing a matrix attachment region. Initiation sites were not detected in regions lying upstream or downstream of this locus. Our results suggest that initiation reactions in mammalian chromosomal origins may be more complex than in the origins of simple microorganisms. 相似文献
13.
Multiple origins of replication in the dihydrofolate reductase amplicons of a methotrexate-resistant chinese hamster cell line. 总被引:9,自引:6,他引:3
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We recently showed that replication initiates in the early S period at two closely spaced zones in the 240-kilobase (kb) dihydrofolate reductase (DHFR) amplicon of the methotrexate-resistant Chinese hamster ovary cell line CHOC 400. Both of these initiation loci (ori-beta and ori-gamma) have previously been cloned in a recombinant cosmid. In this study, we identified a third early-firing initiation locus (ori-alpha) in the much larger DHFR amplicon of the independently isolated methotrexate-resistant Chinese hamster cell line DC3F-A3/4K (A3/4K). We describe the molecular cloning of this newly identified locus and demonstrate by chromosomal walking that ori-alpha lies approximately 240 kb upstream from ori-beta. Using overlapping cosmid clones for more than 450 kb of DNA sequence from this region of the DHFR domain, we have monitored the replication pattern of the amplicons in synchronized A3/4K cells. These studies suggest that ori-alpha, ori-beta, and ori-gamma are the only early-firing initiation sites in this 450-kb sequence. In addition, we have been able to roughly localize the termini between ori-alpha and ori-beta and between ori-alpha and the next origin in the 5' direction. Thus, we have now isolated the equivalent of three early-firing replicons (including their origins) from a well-characterized chromosomal domain. With these tools, it should be possible to determine those properties that are shared by the origins and termini of different replicons and which are therefore likely to be functionally significant. 相似文献
14.
High-resolution mapping of replication fork movement through the amplified dihydrofolate reductase domain in CHO cells by in-gel renaturation analysis. 总被引:30,自引:21,他引:9
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Utilizing an in vivo labeling method on synchronized cultures, we have previously defined a 28-kilobase (kb) replication initiation locus in the amplified dihydrofolate reductase domain of a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) (N. H. Heintz and J. L. Hamlin, Proc. Natl. Acad. Sci. USA 79:4083-4087, 1982; N. H. Heintz and J. L. Hamlin, Biochemistry 22:3552-3557, 1983; N. H. Heintz, J. D. Milbrandt, K. S. Greisen, and J. L. Hamlin, Nature [London] 302:439-441, 1983). To locate the origin of replication in this 243-kb amplicon with more precision, we used an in-gel renaturation procedure (I. Roninson, Nucleic Acids Res. 11:5413-5431, 1983) to examine the labeling pattern of restriction fragments from the amplicon in the early S phase. This method eliminates background labeling from single-copy sequences and allows quantitation of the relative radioactivity in individual fragments. We used this procedure to follow the movement of replication forks through the amplicons, to roughly localize the initiation locus, and to estimate the rate of fork travel. We also used a slight modification of this method (termed hybridization enhancement) to illuminate the labeling pattern of smaller restriction fragments derived solely from the initiation locus itself, thereby increasing resolution. Our preliminary results suggest that there are actually two distinct initiation sites in the amplicon that are separated by approximately 22 kb. 相似文献
15.
The neutral/neutral and neutral/alkaline two-dimensional gel electrophoretic techniques are sensitive physical mapping methods that have been used successfully to identify replication initiation sites in genomes of widely varying complexity. We present detailed methodology for the preparation of replication intermediates from mammalian cells and their analysis by both neutral/neutral and neutral/alkaline two-dimensional gel approaches. The methods described allow characterization of the replication pattern of single-copy loci, even in mammalian cells. When applied to metazoans, initiation is found to occur at multiple sites scattered throughout zones that can be as long as 50 kb, with some subregions being preferred. Although these observations do not rule out the possibility of genetically defined replicators, they offer the alternative or additional possibility that chromosomal context may play an important role in defining replication initiation sites in complex genomes. We discuss novel recombination strategies that can be used to test for the presence of sequence elements critical for origin function if the origin lies in the vicinity of a selectable gene. Application of this strategy to the DHFR locus shows that loss of sequences more than 25 kb from the local initiation zone can markedly affect origin activity in the zone. 相似文献
16.
Specific initiation at an origin of replication from Schizosaccharomyces pombe. 总被引:9,自引:2,他引:7
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Using a genetic assay for efficient autonomous replication, we have isolated from Schizosaccharomyces pombe a 6.2-kb fragment which shows the properties expected of an origin of DNA replication in S. pombe. A 2.8-kb subclone of the fragment has the same replication properties. Two-dimensional gel analysis of replication intermediates throughout plasmids carrying the 6.2- or 2.8-kb fragments shows that replication initiates only in a specific region, which can be localized to within several hundred base pairs, in the fragments. This region is also a site of replication initiation in the S. pombe chromosome where the fragments normally reside. These results provide strong evidence that initiation of replication in S. pombe is localized and mediated by specific DNA sequence signals. 相似文献
17.
18.
Matrix attachment regions are positioned near replication initiation sites, genes, and an interamplicon junction in the amplified dihydrofolate reductase domain of Chinese hamster ovary cells. 总被引:27,自引:11,他引:16
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Genomic DNA in higher eucaryotic cells is organized into a series of loops, each of which may be affixed at its base to the nuclear matrix via a specific matrix attachment region (MAR). In this report, we describe the distribution of MARs within the amplified dihydrofolate reductase (DHFR) domain (amplicon) in the methotrexate-resistant CHO cell line CHOC 400. In one experimental protocol, matrix-attached and loop DNA fractions were prepared from matrix-halo structures by restriction digestion and were analyzed for the distribution of amplicon sequences between the two fractions. A second, in vitro method involved the specific binding to the matrix of cloned DNA fragments from the amplicon. Both methods of analysis detected a MAR in the replication initiation locus that we have previously defined in the DHFR amplicon, as well as in the 5'-flanking region of the DHFR gene. The first of these methods also suggests the presence of a MAR in a region mapping approximately 120 kilobases upstream from the DHFR gene. Each of these MARs was detected regardless of whether the matrix-halo structures were prepared by the high-salt or the lithium 3,5-diiodosalicylate extraction protocols, arguing against their artifactual association with the proteinaceous scaffolding of the nucleus during isolation procedures. However, the in vitro binding assay did not detect the MAR located 120 kilobases upstream from the DHFR gene but did detect specific matrix attachment of a sequence near the junction between amplicons. The results of these experiments suggest that (i) MARs can occur next to different functional elements in the genome, with the result that a DNA loop formed between two MARs can be smaller than a replicon; and (ii) different methods of analysis detect a somewhat different spectrum of matrix-attached DNA fragments. 相似文献
19.
The Chinese hamster dihydrofolate reductase replication origin beta is active at multiple ectopic chromosomal locations and requires specific DNA sequence elements for activity
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To identify cis-acting genetic elements essential for mammalian chromosomal DNA replication, a 5.8-kb fragment from the Chinese hamster dihydrofolate reductase (DHFR) locus containing the origin beta (ori-beta) initiation region was stably transfected into random ectopic chromosomal locations in a hamster cell line lacking the endogenous DHFR locus. Initiation at ectopic ori-beta in uncloned pools of transfected cells was measured using a competitive PCR-based nascent strand abundance assay and shown to mimic that at the endogenous ori-beta region in Chinese hamster ovary K1 cells. Initiation activity of three ectopic ori-beta deletion mutants was reduced, while the activity of another deletion mutant was enhanced. The results suggest that a 5.8-kb fragment of the DHFR ori-beta region is sufficient to direct initiation and that specific DNA sequences in the ori-beta region are required for efficient initiation activity. 相似文献
20.
Lubelsky Y Sasaki T Kuipers MA Lucas I Le Beau MM Carignon S Debatisse M Prinz JA Dennis JH Gilbert DM 《Nucleic acids research》2011,39(8):3141-3155
Genome-scale mapping of pre-replication complex proteins has not been reported in mammalian cells. Poor enrichment of these proteins at specific sites may be due to dispersed binding, poor epitope availability or cell cycle stage-specific binding. Here, we have mapped sites of biotin-tagged ORC and MCM protein binding in G1-synchronized populations of Chinese hamster cells harboring amplified copies of the dihydrofolate reductase (DHFR) locus, using avidin-affinity purification of biotinylated chromatin followed by high-density microarray analysis across the DHFR locus. We have identified several sites of significant enrichment for both complexes distributed throughout the previously identified initiation zone. Analysis of the frequency of initiations across stretched DNA fibers from the DHFR locus confirmed a broad zone of de-localized initiation activity surrounding the sites of ORC and MCM enrichment. Mapping positions of mononucleosomal DNA empirically and computing nucleosome-positioning information in silico revealed that ORC and MCM map to regions of low measured and predicted nucleosome occupancy. Our results demonstrate that specific sites of ORC and MCM enrichment can be detected within a mammalian initiation zone, and suggest that initiation zones may be regions of generally low nucleosome occupancy where flexible nucleosome positioning permits flexible pre-RC assembly sites. 相似文献