共查询到20条相似文献,搜索用时 62 毫秒
1.
Integrated Simian Virus 40 DNA: Nucleotide Sequences at Cell-Virus Recombinant Junctions 总被引:25,自引:10,他引:15
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)
James R. Stringer 《Journal of virology》1981,38(2):671-679
DNA fragments containing the integrated viral DNA present in the simian virus 40 (SV40)-transformed rat cell lines SVRE9 and SVRE17 were cloned in procaryotic vectors, and the DNA sequences linking SV40 and cell DNA were determined. Comparison of the DNA sequences at the SV40-cell junctions in SVRE9 and SVRE17 cells with those of a previously characterized viral insertion from SV14B cells shows that no specific viral or cellular sequences occur at SV40-cell junctions and that the cellular DNA sequences adjacent to integrated SV40 DNA do not display the direct repeat structure characteristic of transposons and retrovirus proviruses. 相似文献
2.
The structure and expression of the integrated viral DNA in mouse cells transformed by simian virus 40 总被引:5,自引:0,他引:5
P W Rigby W Chia C E Clayton M Lovett 《Proceedings of the Royal Society of London. Series B, Containing papers of a Biological character. Royal Society (Great Britain)》1980,210(1180):437-450
3.
Cloning and structural analysis of integrated woodchuck hepatitis virus sequences from a chronically infected liver 总被引:12,自引:8,他引:4
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)
We have isolated and determined the structure of a recombinant clone in lambda phage Charon 30 which contains woodchuck hepatitis virus sequences integrated in woodchuck genomic DNA sequences. This clone, in contrast to previously reported clones (Ogston et al., Cell 29:385-394, 1982), was isolated from a chronically infected liver which never developed hepatocellular carcinoma. Southern blot analysis of viral sequences in the clone in conjunction with electron microscope heteroduplex analysis showed that the integrated viral sequences did not contain internal rearrangements, as have those from hepatomas, but were colinear with the cloned viral genome except for the deletion of approximately 500 base pairs of viral sequences (between positions 1,000 and 1,550 on the viral map). Therefore, the integration was probably a defective genome incapable of supporting viral replication. However, the complete open reading frames coding for the viral X, core, presurface , and surface antigen genes were present, indicating that the viral sequences could code for viral antigens. Southern blot analysis of the normal cellular flanking sequences, using flanking sequence probes from the clone, showed that no detectable rearrangements of cellular DNA (less than 50 base pairs) had occurred at the site of viral integration. 相似文献
4.
5.
Long terminal repeat of murine retroviral DNAs: sequence analysis, host-proviral junctions, and preintegration site. 总被引:69,自引:32,他引:37
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 nucleotide sequence of the long terminal repeat (LTR) of three murine retroviral DNAs has been determined. The data indicate that the U5 region (sequences originating from the 5' end of the genome) of various LTRs is more conserved than the U3 region (sequences from the 3' end of the genome). The location and sequence of the control elements such as the 5' cap, "TATA-like" sequences, "CCAAT-box," and presumptive polyadenylic acid addition signal AATAAA in the various LTRs are nearly identical. Some murine retroviral DNAs contain a duplication of sequences within the LTR ranging in size from 58 to 100 base pairs. A variant of molecularly cloned Moloney murine sarcoma virus DNA in which one of the two LTRs integrated into the viral DNA was also analyzed. A 4-base-pair duplication was generated at the site of integration of LTR in the viral DNA. The host-viral junction of two molecularly cloned AKR-murine leukemia virus DNAs (clones 623 and 614) was determined. In the case of AKR-623 DNA, a 3- or 4-base-pair direct repeat of cellular sequences flanking the viral DNA was observed. However, AKR-614 DNA contained a 5-base-pair repeat of cellular sequences. The nucleotide sequence of the preintegration site of AKR-623 DNA revealed that the cellular sequences duplicated during integration are present only once. Finally, a striking homology between the sequences flanking the preintegration site and viral LTRs was observed. 相似文献
6.
Titration of integrated simian virus 40 DNA sequences, using highly radioactive, single-stranded DNA probes. 总被引:2,自引:0,他引:2
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)
Nick-translated simian virus 40 (SV40) [32P]DNA fragments (greater than 2 X 10(8) cpm/micrograms) were resolved into early- and late-strand nucleic acid sequences by hybridization with asymmetric SV40 complementary RNA. Both single-stranded DNA fractions contained less than 0.5% self-complementary sequences; both included [32P]-DNA sequences that derived from all regions of the SV40 genome. In contrast to asymmetric SV40 complementary RNA, both single-stranded [32P]DNAs annealed to viral [3H]DNA at a rate characteristic of SV40 DNA reassociation. Kinetics of reassociation between the single-stranded [32P]DNAs indicated that the two fractions contain greater than 90% of the total nucleotide sequences comprising the SV40 genome. These preparations were used as hybridization probes to detect small amounts of viral DNA integrated into the chromosomes of Chinese hamster cells transformed by SV40. Under the conditions used for hybridization titrations in solution (i.e., 10- to 50-fold excess of radioactive probe), as little as 1 pg of integrated SV40 DNA sequence was assayed quantitatively. Among the transformed cells analyzed, three clones contained approximately one viral genome equivalent of SV40 DNA per diploid cell DNA complement; three other clones contained between 1.2 and 1.6 viral genome equivalents of SV40 DNA; and one clone contained somewhat more than two viral genome equivalents of SV40 DNA. Preliminary restriction endonuclease maps of the integrated SV40 DNAs indicated that four clones contained viral DNA sequences located at a single, clone-specific chromosomal site. In three clones, the SV40 DNA sequences were located at two distinct chromosomal sites. 相似文献
7.
Cloned mouse mammary tumor virus DNA exhibits glucocorticoid-dependent expression in simian virus 40-transformed mink cells. 总被引:3,自引:0,他引:3
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)
Mink lung epithelial cells were transfected with two cloned mouse mammary tumor virus (MMTV) DNAs, a 9-kilobase clone derived from an unintegrated exogenous viral genome and a 14-kilobase clone containing an integrated endogenous provirus along with cellular flanking sequences. Mink lung cells were chosen because they do not contain endogenous MMTV sequences. On the basis of our observation that simian virus 40 DNA efficiently transforms these cells, we isolated cell clones containing MMTV DNA by using transformation with simian virus 40 DNA as a selective marker in cotransfection experiments. Levels of the 9-kilobase MMTV mRNA representing the entire viral genome and of the spliced 4.4-kilobase mRNA which codes for the viral envelope proteins were glucocorticoid dependent in transformed cells. Expression of low levels of Pr77gag, the precursor of the group-specific viral core proteins, and of gPr73env, the precursor of the viral envelope proteins, was also hormone dependent. We conclude that these cloned MMTV DNAs contain all the information necessary for the synthesis of normal viral RNAs and proteins. These findings also provide further evidence that the DNA sequences involved in the hormone responsiveness of MMTV expression are contained within the viral genome. 相似文献
8.
Adeno-associated virus (AAV) is a prevalent human virus whose replication requires factors provided by a coinfecting helper virus. AAV can establish latent infections in vitro by integration of the AAV genome into cellular DNA. To study the process of integration as well as the rescue of AAV replication in latently infected cells after superinfection with a helper virus, we established a panel of independently derived latently infected cell clones. KB cells were infected with a high multiplicity of AAV in the absence of helper virus, cloned, and passaged to dilute out input AAV genomes. AAV DNA replication and protein synthesis were rescued from more than 10% of the KB cell clones after superinfection with adenovirus type 5 (Ad5) or herpes simplex virus types 1 or 2. In the absence of helper virus, there was no detectable expression of AAV-specific RNA or proteins in the latently infected cell clones. Ad5 superinfection also resulted in the production of infectious AAV in most cases. All mutant adenoviruses tested that were able to help AAV DNA replication in a coinfection were also able to rescue AAV from the latently infected cells, although one mutant, Ad5hr6, was less efficient at AAV rescue. Analysis of high-molecular-weight cellular DNA indicated that AAV sequences were integrated into the cell genome. The restriction enzyme digestion patterns of the cellular DNA were consistent with colinear integration of the AAV genome, with the viral termini present at the cell-virus junction. In addition, many of the cell lines appeared to contain head-to-tail concatemers of the AAV genome. The understanding of the integration of AAV DNA is increasingly important since AAV-based vectors have many advantages for gene transduction in vitro and in vivo. 相似文献
9.
10.
Chromosomal translocation and inverted duplication associated with integrated hepatitis B virus in hepatocellular carcinomas. 总被引:11,自引:5,他引:6
T Tokino S Fukushige T Nakamura T Nagaya T Murotsu K Shiga N Aoki K Matsubara 《Journal of virology》1987,61(12):3848-3854
Integrated hepatitis B virus (HBV) DNA is found in hepatocellular carcinomas which develop in HBV carriers. Presented here are the results of analyses of four integrants that show chromosomal rearrangements associated with the integrated HBV DNA. Two clones (p4 and C15) were found to have large inverted repeating structures, each consisting of HBV genome along with flanking cellular sequences. The structure must have arisen by duplication of the primary integrant, including the flanking cellular DNA, followed by recombination within the viral DNA. One of the two viral arms in each clone joins to the other viral arm at the "cohesive end region." Two clones (DA2-2 and DA2-6) were found to have integrated HBV sequences, each flanked by cellular DNAs from different chromosomes (chromosome X joined to 17 and chromosome 5 joined to 9). They must be the products of cellular DNA translocations using the integrated HBV DNA as the switch point. The viral DNA in each clone is a continuous stretch of a single virus genome with one end in the cohesive end region. These complex structures seem to have been produced by activation of the cohesive end of an integrant viral genome, followed by its recombination with another chromosomal DNA. 相似文献
11.
Characterization of flat revertant cells isolated from simian virus 40-transformed mouse and rat cells which contain multiple copies of viral genomes. 总被引:11,自引:9,他引:2
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)
Eight clones of flat revertants were isolated by negative selection from simian virus 40 (SV40)-transformed mouse and rat cell lines in which two and six viral genome equivalents per cell were integrated, respectively. These revertants showed either a normal cell phenotype or a phenotype intermediate between normal and transformed cells as to cellular morphology and saturation density and were unable to grow in soft agar medium. One revertant derived from SV40-transformed mouse cells was T antigen positive, whereas the other seven revertants were T antigen negative. SV40 could be rescued only from the T-antigen-positive revertant by fusion with permissive monkey cells. The susceptibility of the revertants to retransformation by wild-type SV40 was variable among these revertants. T-antigen-negative revertants from SV40-transformed mouse cells were retransformed at a frequency of 3 to 10 times higher than their grandparental untransformed cells. In contrast, T-antigen-negative revertants from SV40-transformed rat cells could not be retransformed. The arrangement of viral genomes was analyzed by digestion of cellular DNA with restriction enzymes of different specificity, followed by detection of DNA fragments containing a viral sequence and rat cells were serially arranged within the length of about 30 kilobases, with at least two intervening cellular sequences. A head-to-tail tandem array of unit length viral genomes was present in at least one insertion site in the transformed rat cells. All of the revertants had undergone a deletion(s), and only a part of the viral genome was retained in T-antigen-negative revertants. A relatively high frequency of reversion in the transformed rat cells suggests that reversion occurs by homologous recombination between the integrated viral genomes. 相似文献
12.
Structural and biological analysis of integrated polyoma virus DNA and its adjacent host sequences cloned from transformed rat cells. 总被引:17,自引:7,他引:10
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)
EcoRI fragments containing integrated viral and adjacent host sequences were cloned from two polyoma virus-transformed cell lines (7axT and 7axB) which each contain a single insert of polyoma virus DNA. Cloned DNA fragments which contained a complete coding capacity for the polyoma virus middle and small T-antigens were capable of transforming rat cells in vitro. Analysis of the flanking sequences indicated that rat DNA had been reorganized or deleted at the sites of polyoma virus integration, but none of the hallmarks of retroviral integration, such as the duplication of host DNA, were apparent. There was no obvious similarity of DNA sequences in the four virus-host joins. In one case the virus-host junction sequence predicted the virus-host fusion protein which was detected in the transformed cell line. DNA homologous to the flanking sequences of three out of four of the joins was present in single copy in untransformed cells. One copy of the flanking host sequences existed in an unaltered form in the two transformed cell lines, indicating that a haploid copy of the viral transforming sequences is sufficient to maintain transformation. The flanking sequences from one cell line were further used as a probe to isolate a target site (unoccupied site) for polyoma virus integration from uninfected cellular DNA. The restriction map of this DNA was in agreement with that of the flanking sequences, but the sequence of the unoccupied site indicated that viral integration did not involve a simple recombination event between viral and cellular sequences. Instead, sequence rearrangements or alterations occurred immediately adjacent to the viral insert, possibly as a consequence of the integration of viral DNA. 相似文献
13.
The arrangement of simian virus 40 sequences in the DNA of transformed cells. 总被引:156,自引:0,他引:156
High molecular weight DNA, isolated from eleven cloned lines of rat cells independently transformed by SV40, was cleaved with various restriction endonucleases. The DNA was fractionated by electrophoresis through agarose gels, denatured in situ, transferred directly to sheets of nitrocellulose as described by Southern (1975), and hybridized to SV40 DNA labeled in vitro to high specific activity. The location of viral sequences among the fragments of transformed cell DNA was determined by autoradiography. The DNAs of seven of the cell lines contained viral sequences in fragments of many different sizes. The remaining four cell lines each contain a single insertion of viral DNA at a different chromosomal location. The junctions between viral and cellular sequences map at different places on the viral genome. 相似文献
14.
Integration site of polyoma virus DNA in the inducible LPT line of polyoma-transformed rat cells 总被引:8,自引:3,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)
The structure of the polyoma virus (Py) integration site in the inducible LPT line of Py-transformed rat cells was determined by biochemical methods of gene mapping. LPT cell DNA was digested with various restriction enzymes. The digestion products were electrophoresed in agarose gels and transferred onto nitrocellulose sheets by Southern blotting. Fragments containing viral or cell DNA sequences, or both, were identified by hybridization with Py DNA or with a cloned flanking cell DNA probe. Cleavage of LPT DNA with enzymes that restrict the Py genome once generated linear Py DNA molecules and two fragments containing both cell and viral DNA sequences. Cleavage of LPT DNA with enzymes which do not restrict Py DNA generated series of fragments whose lengths were found to differ by increments of a whole Py genome; the smallest fragment in each series was found to be longer than the viral genome. These data indicate that LPT cultures contain Py insertions of various lengths integrated into the same chromosomal site in all the cells. The length heterogeneity of the viral insertions is due to the presence of 0, 1, 2, 3. . . Py genomes arranged in a direct tandem repeat within invariable sequences of viral DNA. Double-digestion experiments were also carried out with the above enzymes and with enzymes that cleave the Py genome at multiple sites. The data obtained in these experiments were used to construct a physical map of the integration site. This map showed that the early region of the virus remained intact even in the smallest insertion (which contains no whole duplicated genomes), whereas the late region was partially duplicated and split during integration. The smallest insertion is colinear with the Py physical map over a region including the entire Py genome and at least a part of the duplicated segment. This structure could give rise to nondefective circular viral DNA molecules by single homologous recombination events. Similar recombination events may occur at a higher frequency in the longer insertions, which include longer regions of homology, and may yield many more free viral genomes. The presence of these insertions in LPT cells could thus be one of the factors which account for the high inducibility of the LPT line. 相似文献
15.
Analysis of integrated human papillomavirus type 16 DNA in cervical cancers: amplification of viral sequences together with cellular flanking sequences. 总被引:11,自引:2,他引:9
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)
We have isolated four clones of integrated human papillomavirus type 16 (HPV-16) DNA from four different primary cervical cancer specimens. All clones were found to be monomeric or dimeric forms of HPV-16 DNA with cellular flanking sequences at both ends. Analysis of the viral sequences in these clones showed that E6/E7 open reading frames and the long control region were conserved and that no region specific for the integration was detected. Analysis of the cellular flanking sequences revealed no significant homology with any known human DNA sequences, except Alu sequences, and no homology among the clones, indicating no cellular sequence specific for the integration. By probing with single-copy cellular flanking sequences from the clones, it was demonstrated that the integrated HPV-16 DNAs, with different sizes in the same specimens, shared the same cellular flanking sequences at the ends. Furthermore, it was shown that the viral sequences together with cellular flanking sequences were amplified. The possible process of viral integration into cell chromosomes in cervical cancer is discussed. 相似文献
16.
In the LPT line of polyoma (Py)-transformed rat cells, amplification of the integrated viral DNA and of cell nucleotide sequences flanking the viral integration site, can be induced either spontaneously or by treatment with carcinogens. We show here that the amplified DNA includes interspersed viral and cellular sequences generated by 'illegitimate' recombination events. Genomic libraries have been prepared in phage lambda vectors from LPT cells treated with the inducing agent mitomycin C and from untreated LPT cells. Four phages, including viral-cell DNA recombinants, have been isolated from these libraries. Sequencing through the recombination sites revealed the following characteristics: (i) The crossover points map at four different positions in the viral DNA and at four different positions in the flanking cell DNA. (ii) There are very short homologous sequences of 1, 2, or 4 bp, at the recombination sites. (iii) Aside from the exchanges between the viral and the cellular DNA, no further rearrangements occurred around the new viral-cellular DNA junctions. (iv) Next to the recombination sites, there are blocks of homopurine-homopyrimidine sequences, which may assume a structure that differs from the Watson-Crick double helix. (v) Clustered homologous sequence blocks of up to 10 bp are present less than 200 bp away from the recombination sites. These homologies are not in register. Based on these results, we propose a model that may account for these recombination events and, more generally, for recombination events that occur during gene amplification in mammalian cells. 相似文献
17.
Adeno-associated virus general transduction vectors: analysis of proviral structures. 总被引:35,自引:25,他引:10
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)
We used two kinds of adeno-associated virus (AAV) vectors to transduce the neomycin resistance gene into human cells. The first of these (dl52-91) retains the AAV rep genes; the second (dl3-94) retains only the AAV terminal repeats and the AAV polyadenylation signal (428 base pairs). Both vectors could be packaged into AAV virions and produced proviral structures that were essentially the same. Thus, the AAV sequences that are required in cis for packaging (pac), integration (int), rescue (res), and replication (ori) of viral DNA are located within a 284-base-pair sequence that includes the terminal repeat. Most of the G418r cell lines (73%) contained proviruses which could be rescued (Res+) when the cells were superinfected with the appropriate helper viruses. Some produced high yields of viral DNA; other rescued at a 50-fold lower level. Most of the lines that were Res+ (79%) contained a tandem repeat of the AAV genome (2 to 20 copies) which was integrated randomly with respect to cellular DNA. Junctions between two consecutive AAV copies in a tandem array contained either one or two copies of the AAV terminal palindrome. Junctions between AAV and cellular sequences occurred predominantly at or within the AAV terminal repeat, but in some cases at internal AAV sequences. Two lines were seen that contained free episomal copies of AAV DNA. Res+ clones contained deleted proviruses or tandem repeats of a deleted genome. Occasionally, flanking cellular DNA was also amplified. There was no superinfection inhibition of AAV DNA integration. Our results suggest that AAV sequences are amplified by DNA replication either before or after integration and that the mechanism of replication is different from the one used during AAV lytic infections. In addition, we have described a new AAV general transduction vector, dl3-94, which provides the maximum amount of room for insertion of foreign DNA and integrates at a high frequency (80%). 相似文献
18.
Cloning and sequencing of viral integration site in human fibroblasts immortalized by simian virus 40. 总被引:1,自引:0,他引:1
O Yano H Hirano Y Karasaki K Higashi H Nakamura S Akiya S Gotoh 《Cell structure and function》1991,16(1):63-71
We have analyzed cellular DNA sequences at the viral genome integration site in a human fibroblast cell line VA13 immortalized by simian virus 40 (SV40). The computer analysis of the junctional cellular DNA sequences did not show any homology to the DNA sequences previously reported. This suggests that immortalization by SV40 was not induced by the destruction of any known oncogene or anti-oncogene at the integration site. We did not find the precise substantial sequence homology at the junctional site between the cellular DNA and SV40 DNA, indicating that the recombination mechanism involved does not require precise sequence homology and therefore, SV40 genome was probably not integrated by homologous recombination. Short direct and inverted repeats of 5 to 29 nucleotides were found in the junctional cellular and SV40 DNA. Cellular DNA abutting SV40 DNA was found by the Northern blot analysis to be expressed in diploid human fibroblasts and SV40-transformed cells. The nature of this RNA is now under study. 相似文献
19.
Using the approach described by Botchan, Topp, and Sambrook (Cell 9:269-287, 1976), we analyzed the organization of the integrated viral sequences in five clonal isolates from the same permissive, inducible cell line (Cyp line) transformed by the tsP155 mutant of polyoma virus. In all five clones, viral sequences were found that could be assigned to a common integration site, as they were joined to the cellular DNA in the same fashion in every instance. However, the sequences comprised between these points differed markedly from clone to clone, as if cell propagation had been accompanied by amplification or recombination or both within the viral insertion. When the clones were compared, no correlation could be found between the abundance, or the organization, of the integrated viral sequences and the amount, or the nature, of the free viral DNA molecules produced during induction. Altogether, our findings suggest that specific events, occurring during either the excision or the subsequent replication of the integrated viral sequences, are responsible for the predominant production of nondefective viral DNA molecules by permissive transformed cells, such as Cyp cells. 相似文献
20.
In mouse cells transformed by a temperature-sensitive polyoma virus (Py) genome, the integrated viral genome recombines with adjacent chromosomal DNA to yield a small cyclic molecule (RmI) with defined viral and cellular components. We have cloned the cellular component (Ins), determined its sequence, and examined its distribution in normal mouse DNA. The sequence of Ins displays several homologies with that surrounding the replication origin (ori) of Py or SV40 DNA. 相似文献