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1.
K Hirai 《Microbiology and immunology》1977,21(5):267-278
Several clones of SV40 transformed CV-1 cells have been characterized for the production of T- and V-antigens and for the state of viral genome. The transformed CV-1 cells failed to produce infectious virions as assayed after sonication or cocultivation and fusion with normal CV-1 cells, and were resistant to super-infection by SV40. Some clones of the transformed cells contained V-antigens. The population of V-antigen positive cells varied from 0 to 100% depending on the passage number while the T-antigen positive cells were always 100%. The virions isolated from the transformed cells were similar in morphology to complete SV40, but lighter in density than complete SV40. In one clone, a small amount of SV40 DNA was detectable in a free state while a large proportion of the DNA hybridizable with SV40 3H cRNA was linearly integrated into the cell DNA. The free SV40 DNA was noninfectious, closed circular DNA with a size smaller than infectious SV40 DNA component I. Since the cell extracts of the transformed cells contained an agent(s) which induced T- and V-antigens in normal CV-1 cells, it was suggested that the SV40 transformed CV-1 cells contained free as well as integrated defective SV40 genomes responsible for the synthesis of T- and V-antigens. 相似文献
2.
Induction of cellular DNA synthesis by a simian virus 40 mutant defective in nuclear transport of T antigen. 总被引:1,自引:1,他引:0
The simian virus 40 (SV40) (cT)-3 mutant [SV40(cT)-3], which is defective in nuclear transport of T antigen, was utilized to determine whether cellular DNA synthesis can be stimulated by SV40 in the absence of detectable nuclear T antigen. Cellular DNA synthesis was examined in the temperature-sensitive cell cycle mutants, BHK ts13 and BHK tsAF8, after microinjection of quiescent cells with plasmid DNA containing cloned copies of wild-type SV40 or SV40(cT)-3. The efficiency of induction of cellular DNA synthesis was identical for both wild-type SV40 and SV40(cT)-3 in both cell lines. The results suggest that cell surface-associated T antigen, either alone or possibly in combination with minimal amounts of nuclear T antigen below our limit of detection, is able to stimulate cellular DNA synthesis. 相似文献
3.
Evidence for the lack of deoxyribonucleic acid dark-repair in Halobacterium cutirubrum. 总被引:2,自引:0,他引:2
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1. Halobacterium cutirubrum does not perform dark-repair of DNA either after u.v. irradiation or during normal growth. 2. Cultures irradiated with u.v. are readily photoreactivated, but do not recover viability in the dark. 3. No increase in the rate of DNA synthesis is observed in the surviving cells after u.v. irradiation. 4. At early times during normal semiconservative replication, newly incorporated thymidine is found only in the hybrid DNA. 5. It is suggested that these bacteria may be useful in the study of DNA replication and photoreactivation. 相似文献
4.
An immortalized xeroderma pigmentosum, group C, cell line which replicates SV40 shuttle vectors 总被引:5,自引:1,他引:4
We have established and characterized an immortalized xeroderma pigmentosum (XP), group C, cell line. Transformation of the human fibroblasts was carried out with a recombinant plasmid, pLAS-wt, containing SV40 DNA encompassing the entire early region with a defective origin of DNA replication. The transformed XP cell line, XP4PA-SVwt, and the normal transformed fibroblasts AS3-SVwt, both express SV40 T antigen together with enhanced levels of the transformation-associated cellular protein, p53. XP4PA-SVwt retains the XP UV-repair defective phenotype as demonstrated by low levels of unscheduled DNA synthesis and by the reduced survival of irradiated SV40 virus. Analysis of cellular DNA shows a single major, stable, integration site of pLAS-wt in the XP4PA-SVwt cells. The T antigen in these cells supports efficiently the replication of SV40 based shuttle vectors and should prove suitable for the introduction, expression and selection of genes related to DNA repair and to the study of mutagenesis using defined molecular probes. 相似文献
5.
Simian virus 40 gene A regulation of cellular DNA synthesis. I. In permissive cells. 总被引:8,自引:5,他引:3
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The kinetics of host cellular DNA stimulation by simian virus 40 (SV40) tsA58 infection was studied by flow microfluorometry and autoradiography in two types of productively infected monkey kidney cells (AGMK, secondary passage, and the TC-7 cell line). Prior to infection, the cell populations were maintained predominantly in G0-G1 hase of the cell cycle by low (0.25%) serum concentration. Infection of TC-7 or AGMK cells by wild-type SV40, viable deletion mutant dl890, or by SV40 tsA58 at 33 degrees C induced cells through S phase after which they were blocked with a 4N DNA content in the G2 phase. The infection of TC-7 cells by tsA58 at 41 degrees C, which was a nonpermissive temperature for viral DNA replication, induced a round of cell DNA synthesis in approximately 30% of the cell population. These cells proceeded through S phase but then re-entered the G1 resting state. In contrast, infection of AGMK cells by tsA58 at 41 degrees C induced DNA synthesis in approximately 50% of the cells, but this population remained blocked in the G2 phase. These results indicate that the mitogenic effect of the A gene product upon cellular DNA is more heat resistant than its regulating activity on viral DNA synthesis and that the extent of induction of cell DNA synthesis by the A gene product may be influenced by the host cell. 相似文献
6.
Isolation and characterization of defective simian virus 40 genomes which complement for infectivity. 总被引:1,自引:1,他引:0
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A new variant of simian virus 40 (EL SV40), containing the complete viral DNA separated into two molecules, was isolated. One DNA species contains nearly all of the early (E) SV40 sequences, and the other DNA contains nearly all of the late (L) viral sequences. Each genome was encircled by reiterated viral origins and termini and migrated in agarose gels as covalently closed supercoiled circles. EL SV40 or its progenitor appears to have been generated in human A172 glioblastoma cells, as defective interfering genomes during acute lytic infections, but was selected during the establishment of persistently infected (PI) green monkey cells (TC-7). PI TC-7/SV40 cells contained EL SV40 as the predominant SV40 species. EL SV40 propagated efficiently and rapidly in BSC-1, another line of green monkey cells, where it also formed plaques. EL SV40 stocks generated in BSC-1 cells were shown to be free of wild-type SV40 by a number of criteria. E and L SV40 genomes were also cloned in the bacterial plasmid pBR322. When transfected into BSC-1 cell monolayers, only the combination of E and L genomes produced a lytic infection, followed by the synthesis of EL SV40. However, transfection with E SV40 DNA alone did produce T-antigen, although at reduced frequency. 相似文献
7.
A DNA replication-positive mutant of simian virus 40 that is defective for transformation and the production of infectious virions. 总被引:8,自引:7,他引:1
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K W Peden S L Spence L C Tack C A Cartwright A Srinivasan J M Pipas 《Journal of virology》1990,64(6):2912-2921
Simian virus 40 (SV40) mutant 5002 carries base pair substitutions of C-5109----T and C-5082----T. These mutations lie in a region of the genome that encodes amino acids common to the large and small viral tumor antigens (T and t antigens, respectively) and result in amino acid substitutions of Leu-19----Phe and Pro-28----Ser. In contrast to wild-type SV40, which produces large plaques that are clearly visible 8 days postinfection, mutant 5002 is defective for productive infection, producing tiny plaques that arise at around 21 days postinfection. However, 5002 is capable of replicating viral DNA and producing normal amounts of capsid proteins, indicating that the mutations alter an activity of T antigen that is required subsequent to DNA synthesis, such as maturation, viral assembly, or release of virions. The mutant T antigen has normal ATPase activity, is phosphorylated in a manner that is indistinguishable from that of the wild-type T antigen, and retains the ability to oligomerize. 5002 complements mutants defective in T antigen host range-adenovirus helper function for productive infection. Thus, T antigen encodes two activities that affect at least two different steps in viral infection other than DNA replication, one inactivated by mutations in the host range-adenovirus helper domain and one inactivated by the mutations present in 5002. The 5002-encoded T antigen is also defective for transformation of REF52 cells when expressed from the normal SV40 early promoter, although this defect can be partially overcome by expressing the protein from stronger promoters. 相似文献
8.
Phenotypic complementation of the SV40 tsA mutant defect in viral DNA synthesis following microinjection of SV40 T antigen. 总被引:9,自引:0,他引:9
African green monkey cells (CV-1P) were microinjected with highly purified SV40 T antigen using protein-loaded red cell ghosts and polyethylene glycol as fusagen. The microinjected cells were infected with a temperature-sensitive mutant of SV40 (tsA209) which is defective in the initiation of viral DNA synthesis. Using in situ hybridization as an assay method, we found that PEG-microinjection of both partially and highly purified T antigen resulted in an increase in the amount of viral DNA sequences in the monolayer. Moreover, 3H-thymidine-labeled and unlabeled Hirt supernatant from microinjected, tsA209-injected cells contained significantly more SV40 DNA than comparable extracts from sham-injected, tsA209-infected or uninfected cells, which were tested in parallel. Thus the introduction of highly purified, "large" SV40 T antigen led to phenotypic complementation of the tsA defect in viral DNA synthesis. 相似文献
9.
Topological requirements for homologous recombination among DNA molecules transfected into mammalian cells. 总被引:64,自引:31,他引:33
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Cultured animal cells rearrange foreign DNA very efficiently by homologous recombination. The individual steps that constitute the mechanism(s) of homologous recombination in transfected DNA are as yet undefined. In this study, we examined the topological requirements by using the genome of simian virus 40 (SV40) as a probe. By assaying homologous recombination between defective SV40 genomes after transfection into CV1 monkey cells, we showed that linear molecules are preferred substrates for homologous exchanges, exchanges are distributed around the SV40 genome, and the frequency of exchange is not diminished significantly by the presence of short stretches of non-SV40 DNA at the ends. These observations are considered in relation to current models of homologous recombination in mammalian cells, and a new model is proposed. The function of somatic cell recombination is discussed. 相似文献
10.
Genetic and biochemical analysis of transformation-competent, replication-defective simian virus 40 large T antigen mutants. 总被引:7,自引:27,他引:7
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To study the role of the biochemical and physiological activities of simian virus 40 (SV40) large T antigen in the lytic and transformation processes, we have analyzed DNA replication-defective, transformation-competent T-antigen mutants. Here we describe two such mutants, C8/SV40 and T22/SV40, and also summarize the properties of all of the mutants in this collection. C8/SV40 and T22/SV40 were isolated from C8 and T22 cells (simian cell lines transformed with UV-irradiated SV40). Early regions encoding the defective T antigens were cloned into a plasmid vector to generate pC8 and pT22. The mutations responsible for the defects in viral DNA replication were localized by marker rescue, and subsequent DNA sequencing revealed missense and one nonsense mutation. The T22 mutation predicts a change of histidine to glutamine at residue 203. C8 has two mutations, one predicts lysine224 to glutamamic acid and the other changes the codon for glutamic acid660 to a stop codon; therefore, C8 T antigen lacks the 49 carboxy-terminal amino acids. pC8A and pC8B were constructed to contain the C8 mutations separately. Plasmids pT22, pC8, pC8A, and pC8B were able to transform primary rodent cell cultures. T22 T antigen is defective in binding to the SV40 origin. C8B (49-amino-acid truncation) is a host-range mutant defective in a late function in CV-1 but not BSC cells. Analysis of T antigens in mutant SV40-transformed mouse cells suggests that the replicative function of T antigen is important in generating SV40 DNA rearrangements that allow the expression of "100K" variant T antigens in the transformants. 相似文献
11.
Virogenic Properties of Bromodeoxyuridine-sensitive and Bromodeoxyuridine-resistant Simian Virus 40-transformed Mouse Kidney Cells 总被引:20,自引:12,他引:8
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When simian virus 40 (SV40)-transformed mouse kidney cells (mKS) were grown in the presence of susceptible indicator cells, SV40 was readily recovered from: (i) 15 transformed cell lines, (ii) transformed cells subcultured 45 times over a 7-month period in medium containing antiviral serum and bromodeoxyuridine (dBU), (iii) 45 of 46 clonal lines isolated in the presence of antiviral serum, (iv) 19 of 19 secondary clones isolated from two clonal lines, and (v) dBU-resistant transformed cell lines. dBU-resistant SV40-transformed mouse kidney cell lines were selected and shown to contain the T antigen and to have normal levels of thymidylate kinase and deoxyribonucleic acid (DNA) polymerase, but to be deficient in thymidine (dT) kinase. Radioautographic and biochemical experiments demonstrated that very little (3)H-dT was incorporated into DNA of dBU-resistant cells during a 6-hr labeling period. After infection of dT kinase-deficient mKS cells with vaccinia virus, high levels of dT kinase were induced. The properties of SV40 recovered from dBU-sensitive and dBU-resistant cells were studied. SV40 recovered from transformed cells was shown to express in CV-1 cells at least six functions characteristic of parental virus: synthesis of capsid antigen, synthesis of T antigen, synthesis of viral DNA, induction of dT kinase, induction of DNA polymerase, and induction of host cell DNA synthesis. In addition, SV40 recovered from the transformed cells induced T antigen, dT kinase, deoxycytidylate deaminase, thymidylate kinase, and DNA polymerase in abortively infected mouse kidney cultures, and the virus was also capable of transforming primary cultures of mouse kidney cells. 相似文献
12.
Ultraviolet-irradiated simian virus 40 activates a mutator function in rat cells under conditions preventing viral DNA replication 总被引:1,自引:0,他引:1
The UV-irradiated temperature-sensitive early SV40 mutant tsA209 is able to activate at the nonpermissive temperature the expression of mutator and recovery functions in rat cells. Unirradiated SV40 activates these functions only to a low extent. The expression of these mutator and recovery functions in SV40-infected cells was detected using the single-stranded DNA parvovirus H-1 as a probe. Because early SV40 mutants are defective in the initiation of viral DNA synthesis at the nonpermissive temperature, these results suggest that replication of UV-damaged DNA is not a prerequisite for the activation of mutator and recovery functions in mammalian cells. The expression of the mutator function is dose-dependent, i.e., the absolute number of UV-irradiated SV40 virions introduced per cell determines its level. Implications for the interpretation of mutation induction curves in the progeny of UV-irradiated SV40 in permissive host cells are discussed. 相似文献
13.
Molecular analysis of mutagenesis in mammalian cells 总被引:1,自引:0,他引:1
A Sarasin F Bourre A Benoit L Daya-Grosjean A Gentil 《International journal of radiation biology and related studies in physics, chemistry, and medicine》1985,47(5):479-488
Mammalian cells are constantly facing various types of mutagens. However, due to the high complexity of the cell genome, the molecular analysis of mutagenesis has not yet been possible. Therefore, we have used simian virus 40 (SV40) as a biological and molecular probe to characterize mutagenesis at the nucleotide level. By using a reversion assay from a temperature-sensitive phenotype towards a wild-type phenotype, we have analysed mutagenesis induced by u.v.-light and by apurinic sites (Ap sites). We report here experiments allowing us to quantify and to compare the mutagenic efficiency of various DNA lesions measured on the SV40 genome. The Ap sites are very mutagenic in this type of assay. The molecular analysis of u.v.-induced mutagenesis reveals that mutations correspond to single base-pair substitutions always located opposite Py-Py lesions. The mutations are almost equally distributed between transition and transversion types, and between the 5' and the 3' side of the Py-Py targets. These results demonstrate for the first time in animal cells the existence of targeted mutations induced by u.v.-light. We propose therefore, the use of SV40 as an efficient biological and molecular probe for assaying mutagenic pathways in mammalian cells. 相似文献
14.
Deoxyribonucleic Acid Replication in Simian Virus 40-Infected Cells IV. Two Different Requirements for Protein Synthesis During Simian Virus 40 Deoxyribonucleic Acid Replication 总被引:4,自引:4,他引:0
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The replication of simian virus 40 (SV40) deoxyribonucleic acid (DNA) was inhibited by 99% 2 hr after the addition of cycloheximide to SV40-infected primary African green monkey kidney cells. The levels of 25S (replicating) and 21S (mature) SV40 DNA synthesized after cycloheximide treatment were always lower than those observed in an infected untreated control culture. This is consistent with a requirement for a protein(s) or for protein synthesis at the initiation step in SV40 DNA replication. The relative proportion of 25S DNA as compared with 21S viral DNA increased with increasing time after cycloheximide treatment. Removal of cycloheximide from inhibited cultures allowed the recovery of viral DNA synthesis to normal levels within 3 hr. During the recovery period, the ratio of 25S DNA to 21S DNA was 10 times higher than that observed after a 30-min pulse with (3)H-thymidine with an infected untreated control culture. The accumulation of 25S replicating SV40 DNA during cycloheximide inhibition or shortly after its removal is interpreted to mean that a protein(s) or protein synthesis is required to convert the 25S replicating DNA to 21S mature viral DNA. Further evidence of a requirement for protein synthesis in the 25S to 21S conversion was obtained by comparing the rate of this conversion in growing and resting cells. The conversion of 25S DNA to 21S DNA took place at a faster rate in infected growing cells than in infected confluent monolayer cultures. A temperature-sensitive SV40 coat protein mutation (large-plaque SV40) had no effect on the replication of SV40 DNA at the nonpermissive temperature. 相似文献
15.
Deoxyribonucleic Acid Replication in Simian Virus 40-Infected Cells: III. Comparison of Simian Virus 40 Lytic Infection in Three Different Monkey Kidney Cell Lines
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A comparative study of simian virus 40 (SV40) lytic infection in three different monkey cell lines is described. The results demonstrate that viral deoxyribonucleic acid (DNA) synthesis and infectious virus production begin some 10 to 20 hr earlier in CV-1 cells and primary African green monkey kidney (AGMK) cells than in BSC-1 cells. Induction of cellular DNA synthesis by SV40 was observed in CV-1 and AGMK cells but not with BSC-1 cells. Excision of large molecular weight cellular DNA to smaller fragments was easily detectable late in infection of AGMK cells. Little or no excision was observed at comparable times after infection of CV-1 and BSC-1 cells. The different kinds of responses of these three monkey cell lines during SV40 lytic infection suggest the involvement of cellular functions in the virus-directed induction of cellular DNA synthesis and the excision of this DNA from the genome. 相似文献
16.
Response of Simian Virus 40-Transformed Cell Lines and Cell Hybrids to Superinfection with Simian Virus 40 and Its Deoxyribonucleic Acid 总被引:13,自引:9,他引:4
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Peter Swetly Giuseppe Barbanti Brodano Barbara Knowles Hilary Koprowski 《Journal of virology》1969,4(4):348-355
Whereas normal human and monkey cells were susceptible both to intact simian virus 40 (SV40) and to SV40 deoxyribonucleic acid (DNA), human and monkey cells transformed by SV40 were incapable of producing infectious virus after exposure to SV40, but displayed susceptibility to SV40 DNA. On the other hand, mouse and hamster cells, either normal or SV40-transformed, were resistant both to the virus and to SV40 DNA. Hybrids between permissive and nonpermissive parental cells revealed a complex response: whereas most hybrids tested were resistant, three of them produced a small amount of infectious virus upon challenge with SV40 DNA. All were resistant to whole virus challenge. The persistence of infectious SV40 DNA in permissive and nonpermissive cells up to 96 hr after infection was ascertained by cell fusion. The decay kinetics proved to be quite different in permissive and nonpermissive cells. Adsorption of SV40 varied widely among the different cell lines. Very low adsorption of SV40 was detected in nonsusceptible cells with the exception of the mKS-BU100 cell line. A strong increase in SV40 adsorption was produced by pretreating cells with polyoma virus. In spite of this increased adsorption, the resistance displayed by SV40-transformed cells to superinfection with the virus was maintained. 相似文献
17.
Edward L. Gershey 《Journal of virology》1979,30(1):76-83
Both exponentially growing and serum-arrested subcloned CV-1 cell cultures were infected with simian virus 40 (SV40). By 24 h after infection 96% of the nuclei of these permissive cells contained SV40 T-antigen. Analysis of the average DNA content per cell at various times after infection indicated that by 24 h most of the cells contained amounts of DNA similar to those normally found in G(2) cells. Analysis of cell cycle distributions indicated that a G(2) DNA complement was maintained by over 90% of the cells in the infected populations 24 to 48 h postinfection. Cells continued to synthesize SV40 DNA during the first 50 h after infection, and cytopathic effect was first observed 60 h after inoculation. After infection the number of mitotic cells that could be recovered by selective detachment decreased precipitously and was drastically reduced by 24 h. A study of the kinetics of decline in the number of mitotic cells suggests that this decline is related to an event during the cell cycle at or near the G(1)-S-phase border upon which commencement of SV40 DNA replication apparently depends. It was concluded that after SV40 infection, stationary cells are induced to cycle, and cycling cells complete one round of cellular DNA synthesis but do not divide. Although the infected cells continue to synthesize viral DNA, they do not appear able to reinitiate cellular DNA replication units. These results imply that the abundance of T-antigen (produced independently of cell cycle phase) in the presence of the enzymes required for continued DNA synthesis is not sufficient for reinitiation of cellular DNA synthesis. 相似文献
18.
Fate of Infecting Simian Virus 40-Deoxyribonucleic Acid in Nonpermissive Cells: Integration into Host Deoxyribonucleic Acid 总被引:4,自引:4,他引:0
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Nonpermissive 3T3 cells were infected with purified superhelical simian virus 40 (SV40) deoxyribonucleic acid I (DNA I). One hour after infection, approximately 60% of the intracellular SV40 DNA was converted to relaxed forms. One day after infection, all intracellular SV40 DNA was present as slow-sedimenting material, and no SV40 DNA I was detectable. At 2 days after infection there appeared viral DNA sequences cosedimenting with cellular DNA during alkaline velocity centrifugation. Furthermore, by both alkaline equilibrium gradient centrifugation and by DNA-ribonucleic acid hybridization analysis, covalent linkage of viral DNA sequences to cellular DNA was demonstrated. Integration of SV40 DNA into cellular DNA did not appear to require DNA synthesis, although DNA synthesis followed by mitotic division of the cells enhanced the amount of viral DNA integrated. Based on data obtained by two different methods, it was calculated that 1,100 to 1,200 SV40 DNA equivalents must be integrated per cell by 48 hr after infection. 相似文献
19.
Histone messenger RNA has been identified in CV-1 monkey kidney cells and its synthesis during the simian virus 40 (SV40) productive cycle has been correlated with the synthesis of cellular DNA and viral DNA. In cultures of CV-1 cells that have reached confluence, infection with SV40/5 (a high-yield clone of SV40) promotes an increase in the rate of cellular DNA synthesis followed by a decline. During this decline the rate of viral DNA synthesis continues to rise and eventually surpasses that of cellular DNA.The synthesis of histone mRNA rises concomitantly with the increase in the synthesis of cellular DNA. This occurs in a fashion similar to that observed when confluent CV-1 cultures are stimulated by the addition of fresh serum to the growth medium. However, whereas in cells stimulated with serum the synthesis of histone mRNA closely parallels that of cellular DNA, in cells infected with SV40, histone mRNA synthesis continues at a high rate even after the decline of cellular DNA synthesis. The rate of histone mRNA synthesis thus appears to he coupled to the total (cellular plus viral) DNA synthesis and not to the synthesis of the host DNA alone. The high rate of synthesis of the F1 histone at late times after infection suggests that histone genes are transcribed co-ordinately. 相似文献
20.
Establishment and characterization of a permanent pSV ori--transformed ataxia-telangiectasia cell line 总被引:11,自引:0,他引:11
A permanent ataxia-telangiectasia (A-T) cell line has been established from the fibroblast strain AT2SF after transfection with the bacterial plasmid pSV ori-, which contains replication origin-defective SV40 sequences. The original transfection frequency, as measured by transformed foci, was markedly reduced in two A-T strains when compared with either normal or xeroderma pigmentosum fibroblasts. As with SV40 virion-transformed fibroblasts, pSV ori--transformed cells entered a crisis phase, from which about one-fourth of the original clones from A-T and normal fibroblasts recovered. Both the pSV ori--transformed TAT2SF cell line and an SV40 virion-transformed AT5BI (GM5489) cell line retained their characteristic sensitivity to the lethal effects of ionizing radiation, as well as their X ray-resistant DNA synthesis. Southern blot analysis of cellular SV40 sequences demonstrated a single major integration site of pSV ori- in the AT2SF cells. In contrast, AT5BI cells transformed with SV40 virions demonstrated a high degree of heterogeneity of integrated viral sequences. Neither the TAT2SF nor the GM5489 transformed cell line contains any detectable freely replicating SV40 viral sequences, which are seen in many other semipermissive SV40-transformed cells. 相似文献