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1.
We have developed an in vitro system for packaging of mature bacteriophage DNA. DNA purified from phage T3, when incubated in a reaction mixture containing binary combinations of extracts prepared from Escherichia coli cells infected with T3 amber mutants of genes involved in DNA replication (genes 3, 4, 5, and 6), is packaged intact and fully conserved into infectious particles. The products of genes 3, 4, 5, 6, and 19 (necessary for DNA maturation in vivo) are required for packaging in vitro. Other requirements are Mg2+, spermidine, and either ATP or GTP. The packaging efficiency decreases with decreasing concentration of exogenous DNA. Exogenous DNA was converted into concatemeric forms in the reaction mixture, depending upon the products of genes 3, 4, 5, and 6. These results suggest that the exogenous DNA proceeds by way of concatemeric intermediates before being packaged. Genetic recombination also occurs in the in vitro packaging system. Recombination is accompanied by physical exchange of sequences between exogenous DNA molecules. ATP is required for in vitro recombination; GTP cannot be substituted for ATP in this reaction. 相似文献
2.
Mature, headful-sized DNA extracted from the Salmonella phages P22 and L, and P22/L-hybrid phages can be encapsulated in vitro by means of a packaging system for exogenous DNA. The probability of packaging reaches about 10?3 per headful-sized molecule. The absence of in vitro recombination was demonstrated, to eliminate the possibility that such a process had created concatemers. The endonucleolytic cut at the pac site, which initiates sequential packaging in vivo, does not occur with the mature DNA substrate in vitro. The position of pac on the molecule is not important but the pac-recognizing phage protein gp3 is indispensable for in vitro encapsulation. 相似文献
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4.
Specific mRNA complementary to the adenovirus type 12 (AM) transforming gene was selected from Ad12-infected cells by hybridization with EcoRI-C (0–16.5 map units) and AccI-H (0–4.7 map units) DNA restriction fragments, and translated in vitro in [35S]methio-nine-containing reticulocyte lysate. The products were analyzed by two-dimensional gel electrophoresis. The result showed that the EcoRI-C region encoded at least three polypeptides with molecular weights of 40,000 (40K), 38K, and 10K, while the AccI-H region encoded only 40K and 38K polypeptides. According to molecular weights and pI values, it is found that 40K or 38K and 10K polypeptides correspond to T antigen g and f, respectively. The result indicates that T antigen g is coded for by the left end of the transforming gene (0–4.7 map units, region la) and suggests that T antigen f is coded by the rest region (4.7–6.8 map units, region Ib). The size of mRNA for T antigen g was examined by translation with mRNA fractionated by sedimentation through a sucrose gradient and electrophoresis of 32P-labeled mRNA selected by the Accl-H fragment. The results showed that the size of mRNA for T antigen g was about 13 S. The structure of the 13 S mRNA was examined by nuclease S1 mapping described by Berk and Sharp, and two :kinds of mRNA were detected. These two mRNAs are considered to be translated into two species of T antigen g polypeptides (40K and 38K). 相似文献
5.
Specific interaction of the SV40 T antigen-cellular p53 protein complex with SV40 DNA 总被引:8,自引:0,他引:8
Three forms of the simian virus 40 (SV40) large tumor antigen (T antigen), free T antigen from infected monkey cells, T antigen-p53 complex from virus-infected mouse cells, and T antigen-p53 complex from SV40-transformed mouse cells, were examined for their abilities to bind specifically to SV40 DNA. Employing an immunoassay that detected T antigen and/or p53 protein bound to restriction fragments of viral DNA, it was shown that all three of these forms of SV40 T antigen bind preferentially to the restriction fragment of SV40 DNA containing the nucleotide sequence at the origin of viral DNA replication. Free p53 protein alone failed to bind to SV40 DNA in this assay. These results indicate that the SV40 T antigen-p53 protein complex displays the same SV40 DNA binding specificity as free T antigen obtained from a productive infection. 相似文献
6.
Alternative interactions of the SV40 A protein with DNA 总被引:30,自引:0,他引:30
We have characterized the DNA binding properties of SV40 A protein (T antigen) from permissive cells. The viral protein protects 110- to 115-, 60- to 65-, 40- to 45-, and 30- to 35-bp fragments of SV40 DNA from an excess of DNase 1. The 60- to 65-bp fragment extends some 30–35 by to either side of the BglI site at the origin of replication. DNase footprinting confirms the location of this binding region and shows that it is the most completely protected DNA binding site under our conditions. The A protein selectively recognizes the 60- to 65-bp origin fragment in the complete absence of adjacent DNA and binds to it in three alternative ways that protect 60–65, 40–45, or 30–35 by from nuclease. Thus these three interactions with isolated origins recapitulate the binding of viral protein to intact SV40 DNA. We propose that the alternative interactions of SV40 A protein with the 60- to 65-bp origin fragment may represent three different conformational alignments of protein with DNA rather than a simple repetition of a single kind of binding interaction. Viral protein also protects pBR322 and λ phage DNA from nuclease to a limited extent. Similar interactions could possibly alter the function of DNA control regions of host cells. 相似文献
7.
Plaques produced by our P? mutants of vesicular stomatitis virus (VSV), which are defective in the inhibition of total protein synthesis in infected cells, stop increasing in size after several days of incubation under conditions where those produced by P+ mutants increase linearly in size. The basis for the arrest in size of P? plaques has been shown to be due to the induction of interferon, and the phenotype is termed PIF+ for “plaque interferon positive.” Thus P? plaques can inhibit the increase in size of adjacent P+ plaques and the factor responsible has the biological and physical properties of interferon. Also P? mutants, when plaqued on VERO cells which cannot be induced to produce interferon, produce plaques which increase linearly in size like P+ plaques. Finally, the inclusion of anti-interferon antibody in the overlay medium also causes P? mutants to produce plaques like P+ mutants. VERO cells were found to be useful to separate the effects of is mutations on plaque size from the interferon effect. Using other cell types the latter effect (PIF assay) can be used as an assay for the ability of viruses to induce interferon, for the isolation of PIF+ mutants from PIF? virus, and as a test for the ability of cells to respond to interferon induction by PIF+ viruses. The assay can be increased in sensitivity through the use of specific cell types and of cell cultures preincubated for several days in the stationary phase of growth. In its most sensitive form, the assay could detect PIF+ behavior in certain ts mutants of VSV at permissive temperatures and in VSV mutants emerging from persistent infection. The assay has also been used to isolate novel mutants of VSV which show alterations in the viral P function. 相似文献
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We have analyzed minichromosomes of SV40 mutants with the electron microscope. In two insertion mutants, containing the SV40 DNA fragment from 0.66 to 0.715 map unit duplicated diametrically opposite to its original site in two alternative orientations, one nucleosome-free region (“gap”) was visualized in 25% of the molecules of each mutant with equal frequency either at the original position or at the insertion point. In a host-substituted mutant (F161F), where the viral DNA sequences spanning 0.67–0.73 map unit are interspersed as four copies between cellular sequences, one gap was visualized in almost 100% of the molecules in the vicinity of the viral DNA sequences. These observations indicate that gap formation is dependent upon SV40 DNA sequences located between 0.67 and 0.715 map unit. 相似文献
10.
Inhibition of SV40-induced cellular DNA synthesis by microinjection of monoclonal antibodies 总被引:5,自引:0,他引:5
The region of the SV40 large T-antigen molecule recognized by a panel of monoclonal antibodies has been determined using hybrid Adeno-SV40 viruses, and manual microinjection of cloned deletion mutants. In addition, an investigation was made of how monoclonal antibodies microinjected into the nucleus can affect the ability of the T-antigen coding gene to stimulate cell DNA synthesis. The monoclonal antibody Pab 14, that recognized the -COOH terminal half of large T, was comicroinjected into quiescent cells together with plasmid pCl-1. This plasmid contains only that part of the T-antigen coding gene that extends from nucleotide residue 120, counterclockwise to nucleotide residue 4002, and makes a truncated T antigen 33,000 in molecular weight and missing the last 435 amino acids on the -COOH terminal side. Monoclonal antibody Pab 14 did not inhibit the stimulation of cellular DNA synthesis caused by microinjection of pCl-1, although it did inhibit cell DNA synthesis induced by microinjection of pSV2G, a recombinant plasmid that contains the entire T-antigen coding gene of SV40. 相似文献
11.
The effect of butyrate on DNA synthesis, histone synthesis, and virus production in SV40-infected monkey cells has been studied. When cells in which DNA and histone synthesis have been blocked by treatment with butyrate are infected with SV40, DNA synthesis is induced to a level at least equal to that in untreated infected cells. Both viral and cellular DNA sequences are replicated. Viral DNA synthesized is predominantly SV40 Form I. The amount of viral DNA which accumulates is reduced by 10-fold in the presence of butyrate, and the yield of infectious virus is lowered 100-fold. Histone synthesis in these cells is stimulated concomitantly with DNA synthesis. The hyperacetylation of histones characteristic of butyrate-treated cells is unchanged by SV40 infection; the newly synthesized histones are hyperacetylated. Thus the mechanism by which butyrate blocks DNA and histone synthesis is not directly related to the hyperacetylation of histones. 相似文献
12.
The isolation of SV40 tsA/deletion, double mutants and the induction of host DNA synthesis 总被引:6,自引:0,他引:6
Simian virus 40 mutants were constructed that contain both a tsA mutation leading to temperature sensitivity of the 92K T-antigen, and deletions of 20–200 base pairs leading to a loss in the expression of the 20K t-antigen. As expected, these mutants were temperature sensitive for viral growth and viral DNA replication in lytically infected cells. At nonpermissive temperatures, the ts/deletion mutants stimulated the incorporation of nucleosides into host DNA as did the tsA mutant alone. This induction of incorporation by the tsA mutants resulted from semiconservative DNA replication, not repair synthesis. At 200 μg/ml caffeine the induction of host DNA by A209 was inhibited by 30 to 50%, whereas induction by the ts/deletion mutants was abolished. 相似文献
13.
Inhibition of nuclear migration of wild-type SV40 tumor antigen by a transport-defective mutant of SV40-adenovirus 7 hybrid virus 总被引:8,自引:0,他引:8
A mutant of the defective SV40-adenovirus 7 hybrid, PARA, induces the synthesis of SV40 large T-antigen (T-ag) that is not transported to the nucleus and accumulates in the cytoplasm of infected or transformed cells. The effect of this cT mutation on the transport of wild-type (WT) T-ag was examined by coinfection experiments. Immunofluorescence staining for T-ag revealed that when SV40-transformed green monkey kidney cells were infected with the cT mutant of PARA [PARA(2cT)] there was a total loss of nuclear T-ag (nT-ag) reactivity concomitant with the appearance of cytoplasmic T-ag (CT-ag) reactivity. The dominance of the cT-ag phenotype was also manifest in cells coinfected with PARA(2cT) and either SV40 or WT PARA, both of which induce nT-ag during single infection. Two lines of evidence indicate that the absence of nuclear T-ag reactivity in coinfected cells is due to a failure of migration of WT T-ag into the nucleus, rather than an inhibition of its synthesis. First, coinfection of cells with SV40 and either helper adenovirus or PARA-adenovirus populations, at the multiplicities of infection employed in these studies, did not reduce the yields of infectious SV40. Second, cells coinfected with PARA(2cT) and deletion mutants of SV40 which encode T-ag polypeptides of reduced molecular weight expressed the cT-ag phenotype, while the presence of the deleted forms of T-ag was confirmed by immunoprecipitation and SDS-polyacrylamide gel electrophoresis. The observation that the replication of infectious SV40 is not inhibited in cells coinfected with PARA(2cT) and expressing the cT phenotype indicates that levels of nuclear T-ag below the limits of detection by immunofluorescence are sufficient to promote SV40 DNA synthesis. The dominant effect of the cT mutation was specific for transport of SV40 T-ag, since normal migration of adenovirus tumor and virion antigens as well as SV40 virion antigen occurred. Several possible mechanisms for the dominance of the CT-ag phenotype are presented. 相似文献
14.
Monoclonal antibody to SV40 T-antigen blocks lysis of cloned cytotoxic T-cell line specific for SV40 TASA 总被引:5,自引:0,他引:5
The lysis of SV40-transformed target cells by a cloned cytotoxic T cell line (CTB6) that is SV40 TASA-specific and H-2Kb-restricted is blocked by a monoclonal antibody reactive with the SV40 T-antigen. The blocking is maximized at high antibody concentrations and low effector-to-target cell ratios. These data indicate that at least one antigenic determinant of the SV40 T-antigen molecule is expressed at the cell surface and is either recognized by this cytotoxic T lymphocyte or is proximate to that determinant recognized by this cytotoxic T lymphocyte. 相似文献
15.
Human interferon inhibits the synthesis of SV40 transplantation rejection antigen (TrAg) in SV40-infected but not in SV40-transformed monkey cells. The synthesis of SV40 tumor antigen as detected by the indirect immunofluorescence test and the large and small T antigens as detected by the immunoprecipitation with sera from tumor-bearing hamsters and electrophoresis in SDS-gels was similarly affected in SV40-infected monkey cells. These results suggest that the induction of SV40-specific TrAg in the cytolytic cycle depends upon a viral, rather than a host, message. 相似文献
16.
According to the base sequence homology of the gene coding for the nonstructural (NS) protein the influenza A viruses can be divided into at least three groups. Within each group the homology is about 90% or higher. The avian influenza A viruses fall at least into two groups between which the homology is about 40%. All human strains tested belong to another group. Influenza viruses of other species might comprise their own group(s). The related regions of the NS gene among the two groups of avian influenza viruses overlap completely and they are highly conserved. The results are discussed in terms of a selection pressure concerning the function exerted by the host during the evolution of the different NS genes, which also might explain a certain species specificity concerning the NS gene of influenza A viruses. 相似文献
17.
Human fibroblasts transformed by the early region of SV40 DNA: analysis of "free" viral DNA sequences 总被引:9,自引:0,他引:9
Human fibroblastic cells (HF) were transformed with the early region of the simian virus 40 genome (0.15 – 0.73 map units) by using the DNA-calcium phosphate coprecipitation technique of F. L. Graham and A. J. Van der Eb (1973, Virology 52, 456–467). Transformation resulted in altered morphology and ability to grow in agarose. The SV40-transformed human fibroblasts (SVHF-A) have a limited life span and reach “senescence” after 10–11 passages. Analysis of the low molecular weight DNA extracted from SVHF-A cells shows a relatively high amount of free viral DNA sequences in circular supercoiled form. These circular molecules are very heterogeneous in size and contain sequences corresponding to the early region of the SV40 genome. Part of them may contain cellular DNA sequences as well. In situ hybridization experiments indicate that a minority of the SVHF-A cells (2–3%) are spontaneously induced to synthesize free viral DNA molecules and their frequency is increased by mitomycin C treatment. Immunofluorescence staining for SV40 T antigens also indicates that the cells producing free viral DNA contain higher T-antigen levels than the rest of the population. Our data suggest that the “free” viral DNA molecules derive from integrated viral sequences following replication in a minority of the cells rather than originating from a persistent extrachromosomal replication in every cell. 相似文献
18.
Association of SV40 large tumor antigen and cellular proteins on the surface of SV40-transformed mouse cells 总被引:17,自引:0,他引:17
A cellular protein with a molecular weight of about 53,000 (53K) and histocompatibility antigens (mouse H-2 antigens) have been reported to be associated with viral-specified proteins in transformed cells. We investigated whether such associations could be detected on the surface of SV40-transformed mouse cells. A differential immunoprecipitation technique was adapted so that surface-associated antigens could be detected independently from intracellular antigens. Cells grown as monolayers were enzymatically labeled with 125I-Na using a lactoperoxidase-catalyzed reaction, or metabolically labeled with either [35S]methionine or 32Pi, and were then incubated with antisera against mouse H-2 antigens or SV40 large T-antigen (T-ag) or with monoclonal antibodies against mouse 53K nonviral T-antigen (nvT-ag). The cells were then disrupted with an NP40 solution, the extracts were clarified by centrifugation, and the immune complexes in the supernatant fluids adsorbed with protein A-containing Staphylococcus aureus. Internal antigens, present in the cell lysates, were precipitated by a second incubation with antiserum and the antigen-antibody complexes collected again with immunoadsorbent. The precipitated proteins were eluted and analyzed by SDS-polyacrylamide gel electrophoresis. Reconstruction experiments established that T-ag released from the nucleus during the extraction procedure was not combining with free antigen-binding sites on antibodies bound to the cell surface in the external reaction, that nuclear unbound T-ag was not exchanging with bound surface antigen during extraction, and that the surface reaction was not due to nuclear T-ag released from dead cells and nonspecifically adsorbed onto the surface of living cells. Iodinated 94K T-ag was specifically immunoprecipitated by T antibody during the external reaction; an iodinated 53K polypeptide was coprecipitated. Conversely, labeled T-ag and 53K were coprecipitated from surface-iodinated transformed cells by monoclonal antibodies against mouse 53K nvT-ag. Thus, it appears that SV40 large T-ag and cellular 53K protein are associated on the surface as well as within SV40-transformed mouse cells. In contrast, no detergent-stable complex between T-ag and mouse H-2 antigens was detected on the transformed cells. The possibility that molecular interactions between viral- and cell-coded proteins could be involved in determining some of the observed transformation-related cellular phenotypic changes is discussed. 相似文献
19.
Viral ribonucleoprotein (RNP) particles isolated from vesicular stomatitis virus (VSV)-infected cells synthesized genome-length, complementary viral RNA, in addition to viral messenger RNA, in the presence of uninfected HeLa S10 extracts. The newly synthesized viral RNA was assembled into an RNP-like structure. RNA replication in vitro ceased when protein synthesis was blocked with pactamycin. Antibody raised against VSV NS protein inhibited in vitro RNA replication as well as mRNA synthesis. Anti-N protein also inhibited RNA replication, although it has no effect on the synthesis of mRNAs. Anti-G and anti-M IgG had no effect on either reaction. Anti-L IgG stimulated RNA replication 1.5- to 2-fold, lthough the synthesis of mRNA was inhibited. 相似文献
20.
SV40 DNA synthesis was inhibited by approximately 50% following a 3-hr treatment with 100 μg/ml 5-azacytidine (5-AzaCR). Cesium chloride-ethidium bromide analysis of SV40 DNA from drug-treated infected cultures demonstrated that SV40 DNA I banded at a higher buoyant density and therefore had a decreased ability to bind ethidium bromide. Sedimentation analysis in sucrose gradients containing various concentrations of ethidium bromide indicated that these molecules were deficient in superhelical terms. Treatment of infected cultures with 5-AzaCR was shown to inhibit protein synthesis which preceeded the inhibition of DNA synthesis. It is concluded that inhibition of SV40 DNA synthesis and the conformational alterations in DNA I result from the inhibition of protein synthesis. 相似文献