共查询到20条相似文献,搜索用时 31 毫秒
1.
SCC30 cells (derived from a single cell from the Chinese hamster ovary CHO-K1 cell line, selected on the basis of a stable chromosome complement) were used to select cell variants with hypomethylated DNA. Cells were treated with 5-aza-2'-deoxycytidine (5azadCyd) at 0.1, 1, or 5 microM for two weeks with the medium and drug renewed twice weekly. From the few surviving cells, 25 random single cell-derived clones were grown for freezing cell stocks, and for DNA isolation for 5-methyldeoxycytidine (5medCyd) estimations. After a minimum of one month's recovery from the drug, these cells showed a continuum of 5medCyd levels ranging from ones with the same as the parental clone (2.93%) to ones having lost almost 50% of their DNA methylation. The modal value corresponded to a loss of one third to one quarter of methylated sites. Five subclones with hypomethylated DNA were grown from the frozen stocks. These cells were shown not to be 5azaCyd-resistant cell variants. By the time sufficient cells had been grown to determine DNA methylation levels, the average percentage of 5medCyd had increased to 76% of the SCC30 value compared to 67% at the time of freezing cell stocks. However, this level of DNA hypomethylation remained constant over two months of continuous culture. Cells of one of these hypomethylated subclones were subjected to a second cycle of 5azaCyd treatment. Six random clones from the survivors showed a further decrease averaging 11% in the level of DNA methylation but, by two months in continuous culture, 5medCyd levels had returned to that present before the second cycle of selection. Hence, cell variants can be readily obtained which have lost some 8-10 million methylated sites (pairs of methylated deoxycytidines), and this loss does not compromise cell viability in in vitro culture. This is consistent with mammalian genomes containing a high level of background methylation in non-essential sites. The usefulness of such single cell-derived clones with stably hypomethylated genomes is discussed in relation to understanding the functions of deoxycytidine methylation in mammalian DNA. 相似文献
2.
David M. Woodcock Jillian K. Adams Ian A. Cooper 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1982,696(1):15-22
In mammalian cells, inhibitors of DNA replication have been shown to induce chromosomal aberrations, cell death and changes in gene control. Inhibition of DNA synthesis has been reported to induce hypermethylation of mammalian DNA (enzymatic postsynthetic formation of 5-methylcytosine). These 5-methylcytosines in mammalian DNA have variously been suggested to be important in gene control, DNA repair, and control of DNA replication. In establishing the normal characteristics of enzymatic DNA methylation, we have demonstrated that, in asynchronously growing cells of both human and hamster origin, some cytosine methylation is delayed for several hours after strand synthesis and that this delayed methylation is completed before the DNA strand acts as a template for DNA replication in the next S-phase. Further, in testing whether the deleterious effects on mammalian cells of DNA synthesis inhibitors might be mediated via changes in enzymatic DNA methylation, we have found, contrary to some previous findings, no evidence for any change in the level of DNA methylation in DNA strands synthesized during 6 h of treatment of cells of human origin with high concentrations of four different inhibitors of DNA replication or during the 4 h following the 6 h treatment. Almost totally blocking DNA replication had no effect on the small amount of delayed methylation of DNA strands not involved in semi-conservative replication during the time of the experiment. This lack of effect on DNA methylation was obtained when the labelling medium contained normal, undialysed serum. In contrast, if dialysed serum was used in the labelling medium in order to maximize l-[Me-3H]methionine utilization, highly variable, totally irreproducible patterns of apparent DNA hypermethylation were obtained. 相似文献
3.
Sequence specificity of cytosine methylation in the DNA of the Chinese hamster ovary (CHO-K1) cell line 总被引:1,自引:0,他引:1
We have determined the DNA renaturation kinetics for those DNA sequences of the Chinese hamster ovary (CHO-K1) cells in which enzymatic cytosine methylation occurred immediately after strand synthesis and for those in which methylation was delayed after strand synthesis. DNA sequences showing immediate or delayed methylation were found to be distributed throughout all repetition classes of the DNA of these cells, with a slight concentration of immediate methylation in moderately repetitive sequences and with delayed methylation being slightly over-represented in the highly repetitive fraction. However, DNA sequences showing both classes of methylation were represented equally in unique DNA sequences. We interpret these data to mean that the methylase acting near the replication forks (the 'immediate' methylase) is a relatively inefficient enzyme, missing some 20% of hemimethylated sites produced by DNA replication in these cells. We suggest that the methylase performing maintenance methylation at sites remote from the replication forks (the 'delayed' methylase) is simply a back-up enzyme for the first and that it has no true sequence specificity. The implications of this for the function(s) of DNA methylation in mammalian cells are discussed. 相似文献
4.
D.M. Woodcock P.J. Crowther D.L. Simmons I.A. Cooper 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1984,783(3):227-233
We have determined the DNA renaturation kinetics for those DNA sequences of the Chinese hamster ovary (CHO-K1) cells in which enzymatic cytosine methylation occurred immediately after strand synthesis and for those in which methylation was delayed after strand synthesis. DNA sequences showing immediate or delayed methylation were found to be distributed throughout all repetition classes of the DNA of these cells, with a slight concentration of immediate methylation in moderately repetitive sequences and with delayed methylation being slightly over-represented in the highly repetitive fraction. However, DNA sequences showing both classes of methylation were represented equally in unique DNA sequences. We interpret these data to mean that the methylase acting near the replication forks (the ‘immediate’ methylase) is a relatively inefficient enzyme, missing some 20% of hemimethylated sites produced by DNA replication in these cells. We suggest that the methylase performing maintenance methylation at sites remote from the replication forks (the ‘delayed’ methylase) is simply a back-up enzyme for the first and that it has no true sequence specificity. The implications of this for the function(s) of DNA methylation in mammalian cells are discussed. 相似文献
5.
Salamon D Takacs M Myöhänen S Marcsek Z Berencsi G Minarovits J 《Biological chemistry》2000,381(2):95-105
Latent episomal genomes of Epstein-Barr virus, a human gammaherpesvirus, represent a suitable model system for studying replication and methylation of chromosomal DNA in mammals. We analyzed the methylation patterns of CpG dinucleotides in the latent origin of DNA replication of Epstein-Barr virus using automated fluorescent genomic sequencing of bisulfite-modified DNA samples. We observed that the minimal origin of DNA replication was unmethylated in 8 well-characterized human cell lines or clones carrying latent Epstein-Barr virus genomes as well as in a prototype virus producer marmoset cell line. This observation suggests that unmethylated DNA domains can function as initiation sites or zones of DNA replication in human cells. Furthermore, 5' from this unmethylated region we observed focal points of de novo DNA methylation in nonrandom positions in the majority of Burkitt's lymphoma cell lines and clones studied while the corresponding CpG dinucleotides in viral genomes carried by lymphoblastoid cell lines and marmoset cells were completely unmethylated. Clustering of highly methylated CpG dinucleotides suggests that de novo methylation of unmethylated double-stranded episomal viral genomes starts at discrete founder sites in vivo. This is the first comparative high-resolution methylation analysis of a latent viral origin of DNA replication in human cells. 相似文献
6.
7.
Spreading of DNA methylation across integrated foreign (adenovirus type 12) genomes in mammalian cells. 总被引:6,自引:0,他引:6
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The establishment of de novo-generated patterns of DNA methylation is characterized by the gradual spreading of DNA methylation (I. Kuhlmann and W. Doerfler, J. Virol. 47:631-636, 1983; M. Toth, U. Lichtenberg, and W. Doerfler, Proc. Natl. Acad. Sci. USA 86:3728-3732, 1989; M. Toth, U. Müller, and W. Doerfler J. Mol. Biol. 214:673-683, 1990). We have used integrated adenovirus type 12 (Ad12) genomes in hamster tumor cells as a model system to study the mechanism of de novo DNA methylation. Ad12 induces tumors in neonate hamsters, and the viral DNA is integrated into the hamster genome, usually nearly intact and in an orientation that is colinear with that of the virion genome. The integrated Ad12 DNA in the tumor cells is weakly methylated at the 5'-CCGG-3' sequences. These sequences appear to be a reliable indicator for the state of methylation in mammalian DNA. Upon explantation of the tumor cells into culture medium, DNA methylation at 5'-CCGG-3' sequences gradually spreads across the integrated viral genomes with increasing passage numbers of cells in culture. Methylation is reproducibly initiated in the region between 30 and 75 map units on the integrated viral genome and progresses from there in either direction on the genome. Eventually, the genome is strongly methylated, except for the terminal 2 to 5% on either end, which remains hypomethylated. Similar observations have been made with tumor cell lines with different sites of Ad12 DNA integration. In contrast, the levels of DNA methylation do not seem to change after tumor cell explanation in several segments of hamster cell DNA of the unique or repetitive type. Restriction (HpaII) and Southern blot experiments were performed with selected cloned hamster cellular DNA probes. The data suggest that in the integrated foreign DNA, there exist nucleotide sequences or structures or chromatin arrangements that can be preferentially recognized by the system responsible for de novo DNA methylation in mammalian cells. 相似文献
8.
9.
10.
11.
12.
Alterations of DNA methylation patterns in germ cells and Sertoli cells from developing mouse testis
Coffigny H Bourgeois C Ricoul M Bernardino J Vilain A Niveleau A Malfoy B Dutrillaux B 《Cytogenetics and cell genetics》1999,87(3-4):175-181
In situ alterations of DNA methylation were studied between 14 d postcoitum and 4 d postpartum in Sertoli cells and germ cells from mouse testis, using anti-5-methylcytosine antibodies. Compared to cultured fibroblasts, Sertoli cells display strongly methylated juxtacentromeric heterochromatin, but hypomethylated chromatids. Germ cells always possess hypomethylated heterochromatin, whereas their euchromatin passes from a demethylated to a strongly methylated status between days 16 and 17 postcoitum. This hypermethylation occurs in the absence of DNA replication, germ cells being blocked in the G(0)-G(1) phase from day 15 postcoitum to birth. The DNA hypermethylation of germ cells is maintained until birth and could be visualized on both chromatids of metaphase chromosomes at the first postpartum cell division. Subsequently, the DNA hypermethylation is lost semiconservatively, being replaced by a methylation pattern recalling the typical fibroblast pattern. These alterations of DNA methylation follow a strict chronology, are chromosome structure and cell-type dependent, and may underlie profound changes of genome function. 相似文献
13.
T Rein T Kobayashi M Malott M Leffak M L DePamphilis 《The Journal of biological chemistry》1999,274(36):25792-25800
In Escherichia coli, DNA methylation regulates both origin usage and the time required to reassemble prereplication complexes at replication origins. In mammals, at least three replication origins are associated with a high density cluster of methylated CpG dinucleotides, and others whose methylation status has not yet been characterized have the potential to exhibit a similar DNA methylation pattern. One of these origins is found within the approximately 2-kilobase pair region upstream of the human c-myc gene that contains 86 CpGs. Application of the bisulfite method for detecting 5-methylcytosines at specific DNA sequences revealed that this region was not methylated in either total genomic DNA or newly synthesized DNA. Therefore, DNA methylation is not a universal component of mammalian replication origins. To determine whether or not DNA methylation plays a role in regulating the activity of origins that are methylated, the rate of remethylation and the effect of hypomethylation were determined at origin beta (ori-beta), downstream of the hamster DHFR gene. Remethylation at ori-beta did not begin until approximately 500 base pairs of DNA was synthesized, but it was then completed by the time that 4 kilobase pairs of DNA was synthesized (<3 min after release into S phase). Thus, DNA methylation cannot play a significant role in regulating reassembly of prereplication complexes in mammalian cells, as it does in E. coli. To determine whether or not DNA methylation plays any role in origin activity, hypomethylated hamster cells were examined for ori-beta activity. Cells that were >50% reduced in methylation at ori-beta no longer selectively activated ori-beta. Therefore, at some loci, DNA methylation either directly or indirectly determines where replication begins. 相似文献
14.
Eukaryotic DNA methylation: facts and problems 总被引:5,自引:0,他引:5
W Doerfler M Toth S Kochanek S Achten U Freisem-Rabien A Behn-Krappa G Orend 《FEBS letters》1990,268(2):329-333
Patterns of DNA methylation in complex genomes like those of mammalian cells have been viewed as indicators of different levels of genetic activities. It is as yet unknown how these complicated patterns are generated and maintained during cell replication. There is evidence from many different biological systems that the sequence-specific methylation of promoters in higher eukaryotes is one of the important factors in controlling gene activity at a long-term level. In general, the fifth nucleotide 5-methyldeoxycytidine can be considered as a modulator of protein-DNA interactions. The degree and direction of this modulation has to be assessed experimentally in each individual instance. The establishment of de novo patterns of DNA methylation is characterized by the gradual non-random spreading of DNA methylation by an essentially unknown mechanism. In this review, some of the general concepts of DNA methylation in mammalian systems are presented, and research currently performed in the authors' laboratory has been summarized. 相似文献
15.
Diane P. Genereux 《PLoS genetics》2009,5(6)
Asymmetric strand segregation has been proposed as a mechanism to minimize effective mutation rates in epithelial tissues. Under asymmetric strand segregation, the double-stranded molecule that contains the oldest DNA strand is preferentially targeted to the somatic stem cell after each round of DNA replication. This oldest DNA strand is expected to have fewer errors than younger strands because some of the errors that arise on daughter strands during their synthesis fail to be repaired. Empirical findings suggest the possibility of asymmetric strand segregation in a subset of mammalian cell lineages, indicating that it may indeed function to increase genetic fidelity. However, the implications of asymmetric strand segregation for the fidelity of epigenetic information remain unexplored. Here, I explore the impact of strand-segregation dynamics on epigenetic fidelity using a mathematical-modelling approach that draws on the known molecular mechanisms of DNA methylation and existing rate estimates from empirical methylation data. I find that, for a wide range of starting methylation densities, asymmetric—but not symmetric—strand segregation leads to systematic increases in methylation levels if parent strands are subject to de novo methylation events. I found that epigenetic fidelity can be compromised when enhanced genetic fidelity is achieved through asymmetric strand segregation. Strand segregation dynamics could thus explain the increased DNA methylation densities that are observed in structured cellular populations during aging and in disease. 相似文献
16.
Concurrent Replication and Methylation at Mammalian Origins of Replication 总被引:7,自引:1,他引:6
下载免费PDF全文
![点击此处可从《Molecular and cellular biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Felipe D. Araujo J. David Knox Moshe Szyf Gerald B. Price Maria Zannis-Hadjopoulos 《Molecular and cellular biology》1998,18(6):3475-3482
Observations made with Escherichia coli have suggested that a lag between replication and methylation regulates initiation of replication. To address the question of whether a similar mechanism operates in mammalian cells, we have determined the temporal relationship between initiation of replication and methylation in mammalian cells both at a comprehensive level and at specific sites. First, newly synthesized DNA containing origins of replication was isolated from primate-transformed and primary cell lines (HeLa cells, primary human fibroblasts, African green monkey kidney fibroblasts [CV-1], and primary African green monkey kidney cells) by the nascent-strand extrusion method followed by sucrose gradient sedimentation. By a modified nearest-neighbor analysis, the levels of cytosine methylation residing in all four possible dinucleotide sequences of both nascent and genomic DNAs were determined. The levels of cytosine methylation observed in the nascent and genomic DNAs were equivalent, suggesting that DNA replication and methylation are concomitant events. Okazaki fragments were also demonstrated to be methylated, suggesting that the rapid kinetics of methylation is a feature of both the leading and the lagging strands of nascent DNA. However, in contrast to previous observations, neither nascent nor genomic DNA contained detectable levels of methylated cytosines at dinucleotide contexts other than CpG (i.e., CpA, CpC, and CpT are not methylated). The nearest-neighbor analysis also shows that cancer cell lines are hypermethylated in both nascent and genomic DNAs relative to the primary cell lines. The extent of methylation in nascent and genomic DNAs at specific sites was determined as well by bisulfite mapping of CpG sites at the lamin B2, c-myc, and β-globin origins of replication. The methylation patterns of genomic and nascent clones are the same, confirming the hypothesis that methylation occurs concurrently with replication. Interestingly, the c-myc origin was found to be unmethylated in all clones tested. These results show that, like genes, different origins of replication exhibit different patterns of methylation. In summary, our results demonstrate tight coordination of DNA methylation and replication, which is consistent with recent observations showing that DNA methyltransferase is associated with proliferating cell nuclear antigen in the replication fork. 相似文献
17.
18.
19.
20.
In order to elucidate whether data about the fast regulation of DNA replication in dependence on oxygen supply and on a functioning protein synthesis, previously elaborated with Ehrlich ascites cells, are valid for human cells too, we repeated key experiments with CCRF-CEM and HeLa cells. The most important techniques employed were DNA fibre autoradiography and alkaline sedimentation analyses of growing (pulse-labeled) daughter strand DNA. It was found that CCRF-CEM and HeLa cells responded to transient hypoxia and to transient inhibition of protein synthesis in an almost identical fashion. Scheduled replicon initiations were reversibly suppressed and the progress rates of replication forks, which were already active before the respective inhibitory conditions were established, were reversibly slowed down. The inclusion of the fork progress rate in the response differs from Ehrlich ascites cells, which respond only by suppressing initiation. Further circumstances of the fast oxygen dependent response, concerning the behaviour of ribonucleotide reductase and of the dNTP pools, revealed no significant differences among the three cell lines. The striking identity of the response of each of the cell lines to hypoxia and to inhibited protein synthesis prompts the suspicion that converging fast regulatory pathways act on the cellular replication machinery. The phenomena as such seem to be rather widespread among mammalian cells. 相似文献