PubMed Health⌕ Search

Biomedical subjects

P A Dijkwel

Publications and source records attributed to P A Dijkwel.

At least 19 recordsLinked to original sources
PubMed2005

Promiscuous initiation on mammalian chromosomal DNA templates and its possible suppression by transcription.

The Chinese hamster dihydrofolate reductase (DHFR) origin consists of many inefficient initiation sites scattered throughout a 55-kb intergenic spacer, with the ori-beta, ori-beta', and ori-gamma subregions being preferred. To test for the presence of genetic replicators within these subregions, fragments containing ori-beta, ori-beta', or, as a negative control, a fragment from the DHFR gene that never initiates in loco were placed at ectopic chromosomal positions. Two-dimensional gel analysis demonstrates that initiation occurs in all three fragments, including the fragment from the gene, and appears to occur at different positions within each fragment in different cells in the population. However, initiation is not detectable in the adjacent, transcribed, neomycin-resistance marker. When a cosmid containing an active DHFR gene was inserted into an ectopic chromosomal site, initiation could no longer be observed in the body of the gene, but was clearly evident in the transcriptionally-silent bacterial vector sequences. In the thirteen independent chromosomal positions into which the four different constructs were inserted, initiation occurred in early S-phase. Since these loci necessarily had to be permissive for transcription of the neo(r) marker or DHFR gene used to select transfectants, it is possible that early-firing origin activity and local transcription are somehow linked. In total, these data suggest that potential initiation sites are distributed at very frequent intervals in the mammalian genome, with usage being regulated both positively and negatively by local transcription.

Animals↗
PubMed2003

The matrix attachment region in the Chinese hamster dihydrofolate reductase origin of replication may be required for local chromatid separation.

Centered in the Chinese hamster dihydrofolate reductase origin of replication is a prominent nuclear matrix attachment region (MAR). Indirect lines of evidence suggested that this MAR might be required for origin activation in early S phase. To test this possibility, we have deleted the MAR from a Chinese hamster ovary variant harboring a single copy of the dihydrofolate reductase locus. However, 2D gel replicon mapping shows that removal of the MAR has no significant effect either on the frequency or timing of initiation in this locus. Rather, fluorescence in situ hybridization studies on cells swollen under either neutral or alkaline conditions show that deletion of the MAR interferes with local separation of daughter chromatids. This surprising result provides direct genetic evidence that at least a subset of MARs performs an important biological function, possibly related to chromatid cohesion and separation.

Animals↗
PubMed2003

The dihydrofolate reductase origin of replication does not contain any nonredundant genetic elements required for origin activity.

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.

Animals↗
PubMed2000

Dispersive initiation of replication in the Chinese hamster rhodopsin locus.

Several higher eukaryotic replication origins appear to be composed of broad zones of potential nascent strand start sites, while others are more circumscribed, resembling those of yeast, bacteria, and viruses. The most delocalized origin identified so far is approximately 55 kb in length and lies between the convergently transcribed dihydrofolate reductase (DHFR) and the 2BE2121 genes on chromosome 2 in the Chinese hamster genome. In some of our studies, we have utilized the rhodopsin origin as an early replicating internal standard for assessing the effects of deleting various parts of the DHFR locus on DHFR origin activity. However, it had not been previously established that the rhodopsin locus was located at a site far enough away to be immune to such deletions, nor had the mechanism of initiation at this origin been characterized. In the present study, we have localized the rhodopsin domain to a pair of small metacentric chromosomes and have used neutral/neutral 2-D gel replicon mapping to show that initiation in this origin is also highly delocalized, encompassing a region more than 50 kb in length that includes the nontranscribed rhodopsin gene itself. The initiation zone is flanked at least on one end by an actively transcribed gene that does not support initiation. Thus, the DHFR and rhodopsin origins belong to a class of complex, polydisperse origins that appears to be unique to higher eukaryotic cells.

Animals↗
PubMed1999

Radiation down-regulates replication origin activity throughout the S phase in mammalian cells.

An asynchronous culture of mammalian cells responds acutely to ionizing radiation by inhibiting the overall rate of DNA replication by approximately 50% for a period of several hours, presumably to allow time to repair DNA damage. At low and moderate doses, this S phase damage-sensing (SDS) pathway appears to function primarily at the level of individual origins of replication, with only a modest inhibition of chain elongation per se. We have shown previously that the majority of the inhibition observed in an asynchronous culture can be accounted for by late G1cells that were within 2-3 h of entering the S period at the time of irradiation and which then fail to do so. A much smaller effect was observed on the overall rate of replication in cells that had already entered the S phase. This raised the question whether origins of replication that are activated within S phase per se are inhibited in response to ionizing radiation. Here we have used a two-dimensional gel replicon mapping strategy to show that cells with an intact SDS pathway completely down-regulate initiation in both early- and late-firing rDNA origins in human cells. We also show that initiation in mid- or late-firing rDNA origins is not inhibited in cells from patients with ataxia telangiectasia, confirming the suggestion that these individuals lack the SDS pathway.

Animals↗
PubMed1999

Physical and genetic mapping of mammalian replication origins.

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.

Animals↗
PubMed1999

DNA replication during amplification of the C3 puff of Rhynchosciara americana initiates at multiple sites in a 6 kb region.

Two independent two-dimensional agarose gel electrophoresis methods have been used to map the origin of replication that directs amplification of the C3 DNA puff of Rhynchosciara americana. The results of neutral/neutral two-dimensional gel electrophoresis show that DNA replication initiates at multiple sites in a zone of at least 6 kb situated immediately upstream from the promoter of the main transcription unit of this puff. The complementary neutral/alkaline two-dimensional gel electrophoresis technique shows that, within the initiation zone, forks move in both directions. In contrast, unidirectional fork movement away from the initiation zone is observed at the ends of the region, implying that it is the only place in the amplified region of the C3 puff where initiations occur. Since the initiation zone coincides with the region that is most highly amplified, amplification of the C3 puff probably occurs by an onion skin-type mechanism.

Animals↗
PubMed1998

Distal sequences, but not ori-beta/OBR-1, are essential for initiation of DNA replication in the Chinese hamster DHFR origin.

In the Chinese hamster dihydrofolate reductase replication initiation zone, the ori-beta locus is preferred over other start sites. To test the hypothesis that ori-beta contains a genetic replicator, we restored a deletion in the 3' end of the DHFR gene with a cosmid that provides the missing sequence and simultaneously knocks out the downstream ori-beta locus. Replication initiates normally in ori-beta knockout cell lines, and the DHFR domain is still synthesized in early S phase. However, initiation is completely suppressed in the starting deletion variant lacking the 3' end of the gene. We conclude that ori-beta does not contain an essential replicator, but that distant sequence elements have profound effects on origin activity in this locus.

Animals↗
PubMed1998

Lagging-strand, early-labelling, and two-dimensional gel assays suggest multiple potential initiation sites in the Chinese hamster dihydrofolate reductase origin.

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-beta, 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-beta and a second locus (ori-gamma) 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-beta; 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-beta and ori-gamma 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.

Animals↗
PubMed1997

Mapping replication origins by neutral/neutral two-dimensional gel electrophoresis.

Neutral/neutral two-dimensional gel electrophoresis is a sensitive physical mapping technique that has been successfully used to unambiguously identify replication initiation sites in genomes of widely varying complexity in vivo. The technique exploits the fact that restriction fragments containing different classes of replicative intermediates (single forks, initiation bubbles, or termination structures) migrate to different and characteristic positions in agarose gels. The replication pattern of any region of interest can then be determined by sequential hybridization with appropriate radioactive probes from that region.

Animals↗
PubMed1996

Sequence and context effects on origin function in mammalian cells.

Jacob and Brenner proposed a model for control of DNA replication in which a trans-acting initiator protein binds to a cis-acting replicator to effect initiation of nascent DNA chains at a fixed locus. Although replicators have been identified in prokaryotic and simple eukaryotic genomes, it has been much more difficult to demonstrate their presence in mammalian chromosomes. Owing to the lack of genetic approaches for identifying mammalian replicators, investigators have directed attention to localizing nascent strand start sites, which should lie close to replicators. Toward this end, a variety of clever techniques have been invented for analyzing replication intermediates, but only rarely have more than one of these techniques been applied to a single locus. However, virtually all have been used to analyze the dihydrofolate reductase locus in CHO cells. The picture that has developed in this locus is that initiation can occur at any of a large number of sites scattered throughout a broad zone, but somewhat more frequently near two sites that may correspond to true genetic replicators. Furthermore, it appears that local transcriptional activity, as well as appropriate torsional stress (as imparted by local attachment to the nuclear matrix), may have profound effects on origin activity.

Animals↗
PubMed1996

Characterizing replication intermediates in the amplified CHO dihydrofolate reductase domain by two novel gel electrophoretic techniques.

Using neutral/neutral and neutral/alkaline two-dimensional (2-D) gel techniques, we previously obtained evidence that initiation can occur at any of a large number of sites distributed throughout a broad initiation zone in the dihydrofolate reductase (DHFR) domain of Chinese hamster ovary (CHO) cells. However, other techniques have suggested a much more circumscribed mode of initiation in this locus. This dichotomy has raised the issue whether the patterns of replicating DNA on 2-D gels have been misinterpreted and, in some cases, may represent such noncanonical replication intermediates as broken bubbles or microbubbles. In an accompanying study (R. F. Kalejta and J. L. Hamlin, Mol. Cell. Biol. 16:4915-4922, 1996), we have shown that broken bubbles migrate to unique positions in three different gel systems and therefore are not likely to be confused with classic replication intermediates. Here, we have applied a broken bubble assay developed from that study to an analysis of the amplified DHFR locus in CHO cells. This assay gives information about the number and positions of initiation sites within a fragment. In addition, we have analyzed the DHFR locus by a novel stop-and-go-alkaline gel technique that measures the size of nascent strands at all positions along each arc in a neutral/neutral 2-D gel. Results of these analyses support the view that the 2-D gel patterns previously assigned to classic, intact replication bubbles and single-forked structures indeed correspond to these entities. Furthermore, potential nascent-strand start sites appear to be distributed at very frequent intervals along the template in the intergenic region in the DHFR domain.

Animals↗
PubMed1995

Yeast and mammalian replication intermediates migrate similarly in two-dimensional gels.

In the budding yeast, Saccharomyces cerevisiae, DNA replication initiates at specific, discrete chromosomal locations. At each initiation site, a single small replication bubble is generated, which subsequently expands at Y-like replication forks. We wanted to know whether other eukaryotic organisms utilize similar initiation mechanisms. For this purpose, replication intermediates (RIs) from three different organisms (Schizosaccharomyces pombe, Chinese hamster and human) were mixed individually with RIs from S. cerevisiae and then subjected to two-dimensional (2D) gel electrophoresis under conditions known to resolve molecules having different structures. All of the RIs detected by the hybridization probes we used for each organism migrated nearly identically to specific RIs of similar size from S. cerevisiae, implying that the detected RIs from all the studied organisms have very similar structures and may therefore employ the same basic initiation mechanism.

Animals↗
PubMed1995

On the nature of replication origins in higher eukaryotes.

Establishing whether DNA replication in higher eukaryotic cells is regulated by genetic replicators has been one of the more challenging problems in cell biology. Several important replicon-mapping techniques have been developed in the past decade that have opened up new windows on replication origins. In the past few years, the application of these strategies has identified a large number of origins in a variety of different loci and organisms. Comparison of sequence motifs and chromosomal milieu, as well as genetic manipulation, should begin to uncover the secrets of these illusive regulatory elements.

Amino Acid Sequence↗
PubMed1995

Origins of replication and the nuclear matrix: the DHFR domain as a paradigm.

The eukaryotic genome appears to be organized in a loopwise fashion by periodic attachment to the nuclear matrix. The proposal that a chromatin loop corresponds to a functional domain has stirred interest in the properties of the DNA sequences at the bases of these loops, the matrix-attached regions (MARs). Evidence has been presented suggesting that certain MARs act as boundary elements isolating domains from their chromosomal context. MARs have also been found in the vicinity of promoters and enhancers and they could act by displacing these cis-regulatory elements into the proper nuclear subcompartment. Attachment to the matrix might also play a role in DNA replication. A large body of evidence indicates that replication occurs on the nuclear matrix. This implies that any DNA sequence will be attached to the matrix at a certain time during the cell cycle. This transient mode of attachment contrasts with the proposed permanent attachment of origins of DNA replication with the nuclear matrix. While some data exist that support this suggestion, the current lack of understanding of the mammalian replication origin precludes definitive conclusions regarding the role of MARs in the initiation process.

Animals↗
PubMed1995

The Chinese hamster dihydrofolate reductase origin consists of multiple potential nascent-strand start sites.

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.

Animals↗
PubMed1994

Replication initiation sites are distributed widely in the amplified CHO dihydrofolate reductase domain.

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.

Animals↗