共查询到20条相似文献,搜索用时 0 毫秒
1.
2.
A high-resolution electron microscopy study of nucleosomes from simian virus 40 chromatin 总被引:4,自引:0,他引:4
The DNA sequence changes of 31 mutations altering the attenuation control mechanism of the histidine operon are presented. These mutations are discussed in terms of a model for operon regulation that involves a his leader peptide gene whose translation regulates formation of alternative stem-loop structures in the his leader messenger RNA. Three suppressible mutations generate nonsense codons (ochre and UGA) in the his leader peptide gene, demonstrating that translation of this gene is essential for operon expression. Eight mutations presumably reduce the efficiency of translation initiation of the his leader peptide gene, causing reduced levels of operon expression. Five of these mutations directly alter the leader peptide gene initiator codon (AUG). Three mutations alter sequences just in front of the initiator codon and presumably alter the ribosome recognition site. Fourteen mutations reduce the stability of the his leader mRNA stem-loop structures that are alternatives to the attenuator stem. The properties of these mutations provide support for the role of these stem-loop structures in preventing formation of the attenuator stem. Finally, we show that mutations that alter the attenuator stem suppress hisO mutations. This lends support to the proposal that these hisO mutations cause reduced levels of operon expression due to excessive attenuator stem formation. The properties of these 31 mutations provide substantial support for the model of his operon regulation described in this paper. 相似文献
3.
4.
Specific association of simian virus 40 tumor antigen with simian virus 40 chromatin 总被引:22,自引:15,他引:7
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)
Simian virus 40 tumor antigen (SV40 T antigen) was bound to both replicating and fully replicated SV40 chromatin extracted with a low-salt buffer from the nuclei of infected cells, and at least a part of the association was tight specific. T antigen cosedimented on sucrose gradients with SV40 chromatin, and T antigen-chromatin complexes could be precipitated from the nuclear extract specifically with anti-T serum. From 10 to 20% of viral DNA labeled to steady state with [3H]thymidine for 12 h late in infection or 40 to 50% of replicating viral DNA pulse-labeled for 5 min was associated with T antigen in such immunoprecipitates. After reaction with antibody, most of the T antigen-chromatin complex was stable to washing with 0.5 M NaCl, but only about 20% of the DNA label remained in the precipitate after washing with 0.5 M NaCl-0.4% Sarkosyl. This tightly bound class of T antigen was associated preferentially with a subfraction of pulse-labeled replicating DNA which comigrated with an SV40 form I marker. A tight binding site for T antigen was identified tentatively by removing the histones with dextran sulfate and heparin from immunoprecipitated chromatin labeled with [32P]phosphate to steady state and then digesting the DNA with restriction endonucleases HinfI and HpaII. The site was within the fragment spanning the origin of replication, 0.641 to 0.725 on the SV40 map. 相似文献
5.
Mapping the in vivo arrangement of nucleosomes on simian virus 40 chromatin by the photoaddition of radioactive hydroxymethyltrimethylpsoralen. 总被引:6,自引:1,他引:5
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)
Intracellular simian virus 40 (SV40) chromatin was photoreacted with a 3H-labeled psoralen derivative, hydroxymethyltrimethylpsoralen (HMT), at 48 h postinfection. Psoralen compounds have been shown to readily penetrate intact cells and, in the presence of long-wavelength UV light, form covalent adducts to DNA, preferentially at regions unprotected by nucleosomes. The average distribution pattern of [3H]HMT on the SV40 genome was determined by specific activity measurements of the DNA fragments generated by HindIII plus HpaII or by AtuI restriction enzyme digestion. At levels of 1 to 10 [3H]HMT photoadducts per SV40 molecule, the radiolabel was found to be distributed nonrandomly. Comparison of the labeling pattern in vivo with that of purified SV40 DNA labeled in vitro revealed one major difference. A region of approximately 400 base pairs, located between 0.65 and 0.73 on the physical map, was preferentially labeled under in vivo conditions. This finding strongly suggests that the highly accessible region near the origin of replication, previously observed on isolated SV40 "minichromosomes," exists on SV40 chromatin in vivo during a lytic infection. 相似文献
6.
Dispersive segregation of nucleosomes during replication of simian virus 40 chromosomes 总被引:9,自引:0,他引:9
The distribution of preformed ("old") histone octamers between the two arms of DNA replication forks was analyzed in simian virus 40(SV40)-infected cells following treatment with cycloheximide to prevent nucleosome assembly from nascent histones. Viral chromatin synthesized in the presence of cycloheximide was shown to be deficient in nucleosomes. Replicating SV40 DNA (wild-type 800 and capsid assembly mutant, tsB11) was radiolabeled in either intact cells or nuclear extracts supplemented with cytosol. Nascent nucleosomal monomers were then released by extensive digestion of isolated nuclei, nuclear extracts or isolated viral chromosomes with micrococcal nuclease. The labeled nucleosomal DNA was purified and found to hybridize to both strands of SV40 DNA restriction fragments taken from each side of the origin of DNA replication, whereas Okazaki fragments hybridized only to the strand representing the retrograde DNA template. In addition, isolated, replicating SV40 chromosomes were digested with two strand-specific exonucleases that excised nascent DNA from either the forward or the retrograde side of replication forks. Pretreatment of cells with cycloheximide did not result in an excess of prenucleosomal DNA on either side of replication forks, but did increase the amount of internucleosomal DNA. These data are consistent with a dispersive model for nucleosome segregation in which "old" histone octamers are distributed to both arms of DNA replication forks. 相似文献
7.
Ultraviolet irradiation inhibits encapsidation of simian virus 40 chromatin. 总被引:2,自引:1,他引:1
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)
During normal maturation and majority of pulse-labeled simian virus 40 DNA progresses from chromatin to previrions and virions within 5 h. UV light inhibits this progression. In heavily irradiated cultures (108 J m-2) most of the simian virus 40 DNA synthesized immediately before irradiation remains as chromatin for at least 5 h. This inhibition of maturation seems to be a result of the inhibition of protein synthesis. The data suggest that the pool of proteins required for maturation is sufficient to convert one-third of the simian virus 40 DNA molecules labeled in a 10-min pulse (at 33 h postinfection) from chromatin to previrions and virions and is exhausted within 1 h. 相似文献
8.
Unique positioning of reconstituted nucleosomes occurs in one region of simian virus 40 DNA 总被引:4,自引:0,他引:4
A Stein 《The Journal of biological chemistry》1987,262(8):3872-3879
A direct end label method was used to study the positioning of nucleosome arrays on several long (greater than 2200 base pairs) SV40 DNA fragments reconstituted in vitro with core histones. Comparison of micrococcal nuclease cutting sites in reconstituted and naked DNA fragments revealed substantial differences in one DNA region. When sufficient core histones were annealed with the DNA to form closely spaced nucleosomes over most of the molecule, a uniquely positioned array of four nucleosomes could be assigned, by strict criteria, to a 610-base pair portion of the SV40 "late region," with a precision of about +/- 20 base pairs. In some other DNA regions, a number of alternative nucleosome positions were indicated. The uniquely positioned four-nucleosome array spanned the same 610 nucleotides on two different DNA fragments that possessed different ends. Removal of a DNA region that had contained a terminal nucleosome of the array, by truncation of the fragment before reconstitution, did not affect the positioning of the other three nucleosomes. As the core histone to DNA ratio was lowered, evidence for specific positioning of nucleosomes diminished, except within the region where the four uniquely positioned nucleosomes formed. This region, however, does not appear to have a higher affinity for core histones than other regions of the DNA. 相似文献
9.
10.
11.
Exonucleases specific for either 3' ends (Escherichia coli exonuclease III) or 5' ends (bacteriophage T7 gene 6 exonuclease) of nascent DNA chains have been used to determine the number of nucleotides from the actual sites of DNA synthesis to the first nucleosome on each arm of replication forks in simian virus 40 (SV40) chromosomes labeled with [3H]thymidine in whole cells. Whereas each enzyme excised all of the nascent [3H]DNA from purified replicating SV40 DNA, only a fraction of the [3H]DNA was excised from purified replicating SV40 chromosomes. The latter result was attributable to the inability of either exonuclease to digest nucleosomal DNA in native replicating SV40 chromosomes, as demonstrated by the following observations: (i) digestion with either exonuclease did not reduce the amount of newly synthesized nucleosomal DNA released by micrococcal nuclease during a subsequent digestion period; (ii) in briefly labeled molecules, as much as 40% of the [3H]DNA was excised from long nascent DNA chains; (iii) the fraction of [3H]DNA excised by exonuclease III was reduced in proportion to the actual length of the radiolabeled DNA; (iv) the effects of the two exonucleases were additive, consistent with each enzyme trimming only the 3' or 5' ends of nascent DNA chains without continued excision through to the opposite end. When the fraction of nascent [3H]DNA excised from replicating SV40 DNA by exonuclease III was compared with the fraction of [32P]DNA simultaneously excised from an SV40 DNA restriction fragment, the actual length of nascent [3H]DNA was calculated. From this number, the fraction of [3H]DNA excised from replicating SV40 chromosomes was converted into the number of nucleotides. Accordingly, the average distance from either 3' or 5' ends of long nascent DNA chains to the first nucleosome on either arm of replication forks was found to be 125 nucleotides. Furthermore, each exonuclease excised about 80% of the radiolabel in Okazaki fragments, suggesting that less than one-fifth of the Okazaki fragments were contained in nucleosomes. On the basis of these and other results, a model for eukaryotic replication forks is presented in which nucleosomes appear rapidly on both the forward and retrograde arms, about 125 and 300 nucleotides, respectively, from the actual site of DNA synthesis. In addition, it is proposed that Okazaki fragments are initiated on nonnucleosomal DNA and then assembled into nucleosomes, generally after ligation to the 5' ends of long nascent DNA chains is completed. 相似文献
12.
A single-stranded DNA-dependent ATPase from monkey kidney tissue culture cells (CV-1) has been found associated with SV40 chromatin. This ATPase activity is distinguishable from the ATPase activity of T-antigen by the following properties: the Km for ATP, elution from phosphocellulose, and stimulation of the ATPase activity by single-stranded DNA but not by double-stranded DNA. The ATPase has been isolated and characterized from the nuclei of uninfected cells. ATP hydrolysis is dependent on single-stranded DNA and a divalent cation. The km values for ATP and single-stranded DNA are 0.024 mM and 0.09 microgram/ml, respectively. The affinity of the ATPase for single-stranded DNA is sufficiently high that the enzyme co-sediments with single-stranded DNA in glycerol gradients. The binding of single-stranded DNA is independent of ATP and MgCl2; however, ATP hydrolysis increases the exchange of enzyme between different DNA molecules. Form I (superhelical) SV40 DNA is also a substrate for ATPase binding, but relaxed Form I, Form II (nicked circular), and double-stranded linear SV40 DNAs are not substrates. Because the DNA helix within chromatin is not under the same kind of tortional strain as Form I DNA, we hypothesize that the ATPase is bound to the single-stranded regions of replication forks in the SV40 chromatin. 相似文献
13.
Phasing of nucleosomes in SV40 chromatin reconstituted in vitro 总被引:4,自引:0,他引:4
14.
DNA replication and chromatin structure of simian virus 40 insertion mutants. 总被引:25,自引:10,他引:15
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)
Insertion of DNA segments into the nuclease-sensitive region of simian virus 40 alters both replication efficiency and chromatin structure. Mutants containing large insertions between the simian virus 40 origin of replication (ori site) and the 21-base-pair repeated sequences replicated poorly when assayed by transfection into COS-1 cells. Replication of mutants with shorter insertions was moderately reduced. This effect was cis-acting and independent of the nucleotide sequence of the insert. The nuclease-sensitive chromatin structure was retained in these mutants, but the pattern of cleavage sites was displaced in the late direction from the ori site. New cleavage sites appeared within the inserted sequences, suggesting that information specifying the nuclease-sensitive chromatin structure is located on the late side of the inserts. Accessibility to BglI (which cleaves within the ori site) was reduced in the larger insertion mutants. These results support the conclusion that efficient function of the viral origin of replication is correlated with its proximity to an altered chromatin structure. 相似文献
15.
The structure of simian virus 40 (SV40) chromatin was probed by treatment with single- and multiple-site bacterial restriction endonucleases. Approximately the same fraction of the chromatin DNA was cleaved by each of three different single-site endonucleases, indicating that the nucleosomes do not have unique positions with regard to specific nucleotide sequences within the population of chromatin molecules. However, the extent of digestion was found to be strongly influenced by salt concentration. At 100 mM NaCl-5 mM MgCl2, only about 20% of the simian virus 40 (SV40) DNA I in chromatin was converted to linear SV40 DNA III. In contrast, at lower concentrations of NaCl (0.05 or 0.01 M), an additional 20 to 30% of the DNA was cleaved. These results suggest that at 100 mM NaCl only the DNA between nucleosomes was accessible to the restriction enzymes, whereas at the lower salt concentrations, DNA within the nucleosome regions became available for cleavage. Surprisingly, when SV40 chromatin was digested with multiple-site restriction enzymes, less than 2% of the DNA was digested to limit digest fragment, whereas only a small fraction (9 to 15%) received two or more cuts. Instead, the principal digest fragment was full-length linear SV40 DNA III. The failure to generate limit digest fragments was not a consequence of reduced enzyme activity in the reaction mixtures or of histone exchange. When the position of the principal cleavage site was mapped after HpaI digestion, it was found that this site was not unique. Nevertheless, all sites wree not cleaved with equal probability. An additional finding was that SV40 chromatin containing nicked-circular DNA II produced by random nicking of DNA I was also resistant to digestion by restriction enzymes. These results suggest that the initial cut which causes relaxation of topological constraint in SV40 chromatin DNA imparts resistance to further digestion by restriction enzymes. We propose that this may be accomplished by either "winding" of the internucleosomal DNA into the body of the nucleosome, or as suggested by others, by successive right-hand rotation of nucleosomes. 相似文献
16.
Sites in simian virus 40 chromatin which are preferentially cleaved by endonucleases. 总被引:66,自引:0,他引:66
Simian virus 40 (SV40) chromatin isolated from infected BSC-1 cell nuclei was incubated with deoxyribonuclease I, staphylococcal nuclease or an endonuclease endogenous to BSC-1 cells under conditions selected to introduce one doublestrand break into the viral DNA. Full-length linear DNA was isolated, and the distribution of sites of initial cleavage by each endonuclease was determined by restriction enzyme mapping. Initial cleavage of SV40 chromatin by deoxyribonuclease I or by endogenous nuclease reduced the recovery of Hind III fragment C by comparison with the other Hind III fragments. Similarly, Hpa I fragment B recovery was reduced by comparison with the other Hpa I fragments. When isolated SV40 DNA rather than SV40 chromatin was the substrate for an initial cut by deoxyribonuclease I or endogenous nuclease, the recovery of all Hind III or Hpa I fragments was approximately that expected for random cleavage. Initial cleavage by staphylococcal nuclease of either SV40 DNA or SV40 chromatin occurred randomly as judged by recovery of Hind III or Hpa I fragments. These results suggest that, in at least a portion of the SV40 chromatin population, a region located in Hind III fragment C and Hpa I fragment B is preferentially cleaved by deoxyribonuclease I or by endogenous nuclease but not by staphylococcal nuclease.Complementary information about this nuclease-sensitive region was provided by the appearance of clusters of new DNA fragments after restriction enzyme digestion of DNA from viral chromatin initially cleaved by endogenous nuclease. From the sizes of new fragments produced by different restriction enzymes, preferential endonucleolytic cleavage of SV40 chromatin has been located between map positions 0.67 and 0.73 on the viral genome. 相似文献
17.
Deletion mutants which affect the nuclease-sensitive site in simian virus 40 chromatin. 总被引:10,自引:8,他引:2
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)
A short segment of simian virus 40 (SV40) chromatin on the late side of the origin of replication is hypersensitive to nuclease cleavage. The role of DNA sequence information in this nuclease-sensitive feature was examined by constructing deletion mutations in this region. Deletions were introduced into the inserted segment of in(Or)-1411 (a viable, partially duplicated variant of SV40), and nuclease sensitivity of the inserted segment was compared with that of the unaltered sequences in their normal location in the viral genome. Extended deletions (118 to 161 base pairs) essentially abolished nuclease sensitivity within the inserted segment. Shorter deletions (21 to 52 base pairs) at separate locations retained the nuclease-sensitive feature. In some short-deletion mutants nuclease susceptibility was substantially reduced. We conclude that more than one genetic element in this region contributes to the organization of the nuclease-sensitive feature and that the SV40 72-base repeat is not, in itself, sufficient signal for this feature. 相似文献
18.
In 8 M CsCl, a fraction of the wild-type previrions and tsB228 nucleoprotein complexes lose their core histones but retain their capsid. These histone-depleted complexes appear in the electron microscope as a protein shell attached to supercoiled DNA. Consistent with this result, we find that in 1 M NaCl, the wild-type previrions dissociate into two populations of nucleoprotein complexes. One population sediments between 50 and 140 S and morphologically resembles the shell-DNA complexes isolated in CsCl gradients. The other population is comprised primarily of nucleoproteins which sediment at 40 S. 相似文献
19.
The DNA in replicating simian virus 40 chromatin and cellular chromatin was labeled with short pulses of [3H]thymidine. The structure of pulse-labeled nucleoprotein complexes was studied by micrococcal nuclease digestion. It was found that in both newly replicated viral and cellular chromatin, a structural state appears which is characterized by an increased sensitivity to nuclease and a faster than usual rate of cleavage to DNA fragments of monomeric nucleosome size and smaller. Pulse-chase experiments show that each of these effects requires a characteristic time to disappear in both systems, suggesting the existence of different sub-processes of chromatin maturation. One of these processes, detectable by the reversion of the unusually fast production of subnucleosomal fragments, is delayed in SV40 chromatin replication. 相似文献
20.
In our previous work we have shown by comparison of experimental and computational data that the positions of the histone
octamers bound to the DNA molecule appear to be completely sequence-dependent. This provides a convenient and quick method
for locating the nucleosomes along the DNA molecule, as soon as the nucleotide sequence is known. Using this computational
approach, the complete nucleosomal map of the SV40 minichromosome has been constructed. The map consists of 25 nucleosomes,
with their coordinates (centers) being specified with high accuracy. The map is found to be in remarkable agreement with available
experimental data. 相似文献