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1.
BK virus-transformed inbred hamster brain cells. I. Status of the viral DNA and the association of BK virus early antigens with purified plasma membranes 总被引:2,自引:0,他引:2
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E Beth G Giraldo R Schmidt-Ullrich M M Pater A Pater G di Mayorca 《Journal of virology》1981,40(1):276-284
Inbred LSH hamster brain cells were transformed in vitro by the GS strain of BK virus (BKV), and transplantable tumors classified as undifferentiated glioblastomas were induced in the syngeneic host. The viral status in the transformed cells, designated LSH-BR-BK, was established. About 46 genome equivalents per cell of viral DNA was detected, with the majority of sequences in a free form. The transformed cells expressed large quantities of tumor (T) antigen as well as surface (S) antigen as demonstrated by indirect immunofluorescence. Sixty-three percent of tumor-bearing hamsters produced high-titer antibodies against T, whereas 3 of 14 (21%) hamsters also produced antibodies against the BKV-specific S antigen. Furthermore, the relatedness of BKV early gene products, including T, S, and tumor-specific transplantation antigen, was established by the production of a rabbit antiserum against highly purified plasma membranes of LSH-BR-BK cells and by the induction of a BKV-specific tumor-specific transplantation antigen response by these plasma membranes in the syngeneic host. 相似文献
2.
Expression of viral early functions in rat 3Y1 cells infected with human papovavirus BK. 总被引:6,自引:6,他引:0
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A plaque morphology mutant (pm-522) of BK virus (BKV) with a small deletion at map unit 0.72 can readily transform rat 3Y1 cells, but wild-type BKV (wt-501) cannot. We examined the expression of the viral early functions in BKV (wt-501 or pm-522)-infected 3Y1 cells within a 2-week period after infection, before foci of transformed cells became detectable, to know how the difference between the two BKVs occurs. After a high-multiplicity infection, comparable amounts of free viral DNA (forms I and II) were found by Southern blotting analyses to persist in the nuclei of the cells infected with wt and pm BKVs. Whereas the proportion of T antigen-positive cells, as revealed by the indirect immunofluorescence method with complement, remained at a level of 60% in pm BKV infection, the level of T antigen-positive cells in wt BKV infection decreased from the initial 45% to 1% on day 9. The results obtained by the immunoprecipitation analyses of radiolabeled proteins from the infected cells were consistent with the immunofluorescence data. Viral early mRNA was detectable on day 2 and increased on day 9 in pm BKV infection, but in wt BKV infection, the low level of early mRNA detected on day 2 disappeared on day 9. Cell DNA synthesis and cell growth were enhanced more in pm BKV infection than in wt BKV infection. The low level of viral DNA synthesis that occurred in the infected rat cells was more prominent in pm BKV infection than in wt BKV infection. These data indicate that the expression of viral early functions continued much longer in pm BKV-infected rat cells than in wt BKV-infected rat cells, where the expression was probably repressed soon after infection. Continued T antigen production directed by the unintegrated viral genomes appears to be required for efficient transformation of rat cells by BKV. 相似文献
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Michaël Katinka 《Biochimie》1984,66(11-12):681-691
Two defective polyoma virus genomes, deleted in the nucleotide sequences coding the N-termini of the tumor antigens, were introduced into Fisher 3T3 rat cells by DNA-mediated gene transfer (transfection). The resulting integrated genomes were incapable of conferring a transformed phenotype to the cells. However, after transfection of these lines with small polyoma fragments overlapping the deleted sequences, transformed clones were isolated. These clones were analyzed by Southern genomic blot hybridization and by isolation in E. coli of plasmids containing viral sequences excised following fusion with mouse polyoma growth-permissive cells. In all cases at least one intact copy of the early region of the polyoma genome was found. Furthermore, restriction sites adjacent to the initial inactive insertion remained unchanged in many of the transformed lines. These results show that functional restoration of the defective polyoma early region involves homologous recombination between the deleted viral genomes integrated in the cellular DNA and the transfecting viral fragments. 相似文献
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Transformation of rodent cells by a cloned DNA fragment of herpes simplex virus type 2. 总被引:22,自引:9,他引:13
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Transformation of rodent cells with isolated restriction endonuclease fragments of herpes simplex virus type 2 DNA identified a region of the genome located between map positions 0.58 and 0.62. These sequences were cloned into pBR322, and the recombinant plasmid was used to transform primary rat embryo cells and NIH 3T3 cells. The transformants were selected for their ability to form dense foci on a monolayer or to form colonies in semisolid medium. In contrast to the parental rat or mouse cells, cell lines transformed with the cloned herpes simplex virus type 2 fragment grow to high saturation densities, replicate in medium containing 1% serum, form colonies in dilute methylcellulose, show reduced levels of fibronectin, and are tumorigenic in nude mice and in their syngeneic hosts. Southern blot hybridizations have detected sequences homologous to the viral fragment in high-molecular-weight DNA from the transformed cell lines that are not present in DNA from normal rodents. In all cases, the plasmid DNA was present in less than one copy per cell, and the patterns of viral sequences changed with passage of the cell line in vivo. 相似文献
8.
The complementary strands of fragments of 32P-labelled adenovirus 2 DNA generated by cleavage with restriction endonucleases EcoRI or Hpa1 were separated by electrophoresis. Saturation hybridization reactions were performed between these fragment strands and unlabelled RNA extracted from the cytoplasm of adenovirus 2-transformed rat embryo cells or from human cells early after adenovirus 2 infection. The fraction of each fragment strand complementary to RNA from these sources was measured by chromatography on hydroxylapatite. Maps of the viral DNA sequences complementary to messenger RNA in different lines of transformed cells and early during lytic infection of human cells were constructed.Five lines of adenovirus 2-transformed cells were examined. All contained the same RNA sequences, complementary to about 10% of the light strand of EcoRI fragment A. DNA sequences coding for this RNA were more precisely located using Hpa1 fragments E and C and mapped at the left-hand end of the genome. Thus any viral function expressed in all adenovirus 2-transformed cells, tumour antigen, for example, must be coded by this region of the viral genome. Two lines, F17 and F18, express only these sequences; two others, 8617 and REM, also contain mRNA complementary to about 7% of the heavy strand of the right-hand end of adenovirus 2 DNA; a fifth line, T2C4, contains these and many additional viral RNA sequences in its cytoplasm.The viral RNA sequences found in all lines of transformed cells are also present in the cytoplasm of human cells during the early phase of a lytic adenovirus infection. The additional cytoplasmic sequences in the 8617 and REM cell lines also correspond to “early” RNA sequences. 相似文献
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The patterns of integration of the viral genome have been analyzed in four hamster cell lines transformed by adenovirus type 12 (Ad12). It has previously been shown that in each of the cell lines HA12/7, T637, A2497-2 and A2497-3, the viral genome persists in multiple copies, and that different parts of the viral DNA are represented non-stoichiometrically (Fanning and Doerfler, 1976). All four cell lines are oncogenic when injected into hamsters.The DNA from each of the cell lines was extracted and cleaved in different experiments with restriction endonucleases Bam HI, Bgl II, Eco RI, Hind III, Hpa II or Sma I. The DNA fragments were separated on 1% agarose slab gels and transferred to nitrocellulose filters by the Southern technique. Ad12 DNA sequences were detected by hybridization to Ad12 DNA, which was 32P-labeled by nick translation, and by subsequent autoradiography. In some experiments, the 32P-labeled Eco RI restriction endonuclease fragments of Ad12 DNA were used to investigate the distribution of specific segments of the viral genome in the cellular DNA.For each cell line, a distinct and specific pattern of integrated viral DNA sequences is observed for each of the restriction endonucleases used. Moreover, viral sequences complementary to the isolated Eco RI restriction endonuclease fragments are also distributed in patterns specific for each cell line. There are striking differences in integration patterns among the four different lines; there are also similarities. Because the organization of cellular genes in virus-transformed as compared to normal cells has not yet been determined, conclusions about the existence or absence of specific integration sites for adenovirus DNA appear premature. Analysis of the integration patterns of Ad12 DNA in the four hamster lines investigated reveals that some of the viral DNA molecules are fragmented prior to or during integration. Analysis with specific restriction endonuclease fragments demonstrates that the Eco RI B, D and E fragments, comprising a contiguous segment from 0.17–0.62 fractional length units of the viral DNA, remain intact during integration in a portion of the viral DNA molecules. Although each cell line carries multiple copies of Ad12 DNA, the viral DNA sequences are concentrated in a small number of distinct size classes of fragments. This finding is compatible with, but does not prove, the notion that at least a portion of the viral DNA sequences is integrated into repetitive sequences, or else that the integrated viral sequences have been amplified after integration.In the three cell lines which were tested, the integration pattern is stable over many generations, with continuous passage-twice weekly-of cells for 6–7 months. In the three cell lines which were examined, the integration pattern is identical in a number of randomly isolated clones. Hence it can be concluded that the patterns of integration are identical among all cells in a population of a given line of transformed cells. 相似文献
10.
Topography of polyoma virus messenger RNA molecules. 总被引:32,自引:0,他引:32
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Loss of integrated viral DNA sequences in polyomatransformed cells is associated with an active viral A function. 总被引:48,自引:0,他引:48
Rat cells transformed by polyoma virus contain, in addition to integrated viral DNA, a small number of nonintegrated viral DNA molecules. The free viral DNA originates from the integrated form through a spontaneous induction of viral DNA replication in a minority of the cell population. Its presence is under the control of the viral A locus. To determine whether the induction of free viral DNA replication was accompanied by a loss of integrated viral DNA molecules in a phenomenon similar to the "curing" of lysogenic bacteria, we selected for revertants arising in the transformed rat populations and determined whether these cells had lost integrated viral genomes. We further investigated whether the viral A function was necessary for "curing" by determining the frequency of cured cells in populations of rat cells transformed by the ts-a mutant of polyoma virus following propagation at the permissive or nonpermissive temperature. A large proportion of the revertants isolated were negative or weakly positive when assayed by immunofluorescence for polyoma T antigen and were unable to produce infectious virus upon fusion with permissive mouse cells. The T antigen-negative, virus rescue-negative clones can be retransformed by superinfection and appear to have lost a considerable proportion of integrated viral DNA sequences. Restriction enzyme analysis of the integrated viral DNA sequences shows that the parental transformed lines contain tandem repeats of integrated viral molecules, and that this tandem arrangement is generally lost in the cured derivatives. While cells transformed by wild-type virus undergo "curing" with about the same frequency at 33 degrees or 39 degrees C, cells transformed by the ts-a mutant contain a much higher frequency of cured cells after propagation at 33 degrees than at 39 degrees C. Our results indicate that in polyoma-transformed rat cells, loss of integrated viral DNA can occur at a rather high rate, producing (at least in some cases) cells which have reverted partially or completely to a normal phenotype. Loss of integrated viral DNA is never total and appears to involve an excision event. The polyoma A function (large T antigen) is necessary for such excision to occur. In the absence of a functional A gene product, the association of the viral DNA with the host DNA appears to be very stable. 相似文献
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McDonough feline sarcoma virus: characterization of the molecularly cloned provirus and its feline oncogene (v-fms). 总被引:50,自引:17,他引:33
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The genetic structure of the McDonough strain of feline sarcoma virus (SM-FeSV) was deduced by analysis of molecularly cloned, transforming proviral DNA. The 8.2-kilobase pair SM-FeSV provirus is longer than those of other feline sarcoma viruses and contains a transforming gene (v-fms) flanked by sequences derived from feline leukemia virus. The order of genes with respect to viral RNA is 5'-gag-fms-env-3', in which the entire feline leukemia virus env gene and an almost complete gag sequence are represented. Transfection of NIH/3T3 cells with cloned SM-FeSV proviral DNA induced foci of morphologically transformed cells which expressed SM-FeSV gene products and contained rescuable sarcoma viral genomes. Cells transformed by viral infection or after transfection with cloned proviral DNA expressed the polyprotein (P170gag-fms) characteristic of the SM-FeSV strain. Two proteolytic cleavage products (P120fms and pp55gag) were also found in immunoprecipitates from metabolically labeled, transformed cells. An additional polypeptide, detected at comparatively low levels in SM-FeSV transformants, was indistinguishable in size and antigenicity from the envelope precursor (gPr85env) of feline leukemia virus. The complexity of the v-fms gene (3.1 +/- 0.3 kilobase pairs) is approximately twofold greater than the viral oncogene sequences (v-fes) of Snyder-Theilen and Gardner-Arnstein FeSV. By heteroduplex, restriction enzyme, and nucleic acid hybridization analyses, v-fms and v-fes sequences showed no detectable homology to one another. Radiolabeled DNA fragments representing portions of the two viral oncogenes hybridized to different EcoRI and HindIII fragments of normal cat cellular DNA. Cellular sequences related to v-fms (designated c-fms) were much more complex than c-fes and were distributed segmentally over more than 40 kilobase pairs in cat DNA. Comparative structural studies of the molecularly cloned proviruses of Synder-Theilen, Gardner-Arnstein, and SM-FeSV showed that a region of the feline-leukemia virus genome derived from the pol-env junction is represented adjacent to v-onc sequences in each FeSV strain and may have provided sequences preferred for recombination with cellular genes. 相似文献
13.
Regulatory mechanism of simian virus 40 gene expression in permissive and in nonpermissive cells. 总被引:10,自引:4,他引:6
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Primary or continuous lines of mouse cells (3T3) are nonpermissive for simian virus 40 (SV40). Abortively infected cells synthesize tumor antigen (T antigen but not viral DNA and virus capsid protein (V antigen). V antigen, however, was obtained when SV40 DNA was injected into 3T3 cells. This late gene expression also appears to be correlated with the quantity of injected DNA molecules per 3T3 cell. T antigen formation can be detected after microinjection of only 1 to 2 DNA molecules, but the intensity of intranuclear T antigen fluorescence is significantly brighter with injection of higher concentrations of viral DNA. In permissive cells (TC7), early and late SV40 gene expression is directly related to the number of injected molecules. Microinjection of 1DNA molecule induced T and V antigen formation with the same efficiency as microinjection of 2,000 to 4,000 molecules. The question of weather late SV40 gene expression is directly related to the quantity of an early virus-specific product was approached by microinjection of early SV40 complementary RNA together with small amounts of viral DNA. V antigen was obtained in a high proportion of recipient 3T3 cells at conditions where microinjection of viral DNA alone induced T but not V antigen synthesis. 相似文献
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Transformation of primary rat kidney cells by DNA fragments of weakly oncogenic adenoviruses. 总被引:8,自引:6,他引:2
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Primary cultures of baby rat kidney (BRK) cells were transformed by intact DNA and DNA fragments of weakly oncogenic human adenovirus types 3 and 7. The smallest fragment found to contain transforming activity was the left-terminal 4% endo R.HindIII fragment (for both adenovirus type 3 and 7 DNAs). The efficiency of transformation of this fragment was low, and no permanent cell line could be established. Left-terminal fragments ranging from 84 to 4,5% of the viral genome could all transform BRK cells with the same efficiency as intact viral DNA. A number of adenovirus type 7 DNA fragment-transformed lines were established and were found to contain persistent viral DNA sequences and adenovirus subgroup B-specific T antigen. Consequently, the transforming functions of adenovirus types 3 and 7 are located at the extreme left-hand end of the genome, and the minimum size for a DNA fragment with transforming activity is 1.0 X 10(6) daltons. These results do not rule out the possibility that viral genes located outside the transforming region may also influence transformation. 相似文献
15.
Expression of the gene encoding the adenovirus DNA terminal protein precursor in productively infected and transformed cells. 总被引:4,自引:2,他引:2
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The major product of in vitro translation of early RNA prepared from H5ts125-infected cells and selected by hybridization to adenoviral DNA fragments spanning the region from 14.7 to 31.5 map units had been shown to be identical to the 87-kilodalton terminal protein precursor. A 72- to 75-kilodalton polypeptide whose rRNA can be selected by DNA from this same region and made in the presence of anisomycin was indistinguishable from the 72-kilodalton single-stranded DNA-binding protein encoded by the region from 60.1 to 66.6 map units. The accumulation of cytoplasmic RNA sequences complementary to these l-strand genes under various conditions of infection and in certain lines of transformed cells has been investigated by solution hybridization of cytoplasmic RNA to the separated strands of restriction endonuclease fragments of adenoviral DNA. During the early phase, RNA sequences complementary to the region from 11.6 to 36.7 map units were present at a concentration of 10 to 60 copies per cell, regardless of the nature of the block used to inhibit viral DNA synthesis. By 24 h after infection in the absence of any such block, sequences complementary to the regions from 11.6 to 18.2 map units (IVa2) and from 18.6 to 36.7 map units (E2B) accumulated to concentrations of 4,800 and 280 copies per cell, respectively. The ratio of cytoplasmic E2A RNA sequences to E2B RNA sequences remained close to 10:1 throughout the time period investigated. Of the transformed cell lines which retained E2B DNA sequences that were examined, only the T2C4 line expressed these sequences in cytoplasmic RNA. The implications of these observations for regulation of expression of the adenoviral early l-strand genes are discussed. 相似文献
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Cells transformed by Polyoma virus (Py) can undergo a high rate of excision or amplification of integrated viral DNA sequences, and these phenomena require the presence of homology (i.e., repeats) within the viral insertion as well as a functional viral large T antigen (T-Ag). To determine whether the main role of large T-Ag in excision and amplification was replicative or recombination-promoting, we studied transformed rat cell lines containing tandem insertions of a ts-a Py molecule (encoding a thermolabile large T-Ag) with a deletion of the origin of viral DNA replication. Culturing of these cells at the temperature permissive for large T-Ag function did not result in any detectable excision or amplification of integrated Py sequences. We then introduced into origin-defective lines a recombinant plasmid containing the viral origin of replication and the gene coding for resistance to the antibiotic G418. All G418-resistant clones analyzed readily amplified the integrated plasmid molecules when grown under conditions permissive for large T-Ag function, showing that these cells produced viral large T-Ag capable of promoting amplification in trans of DNA sequences containing the Py origin. These observations strongly suggest that Polyoma large T antigen promotes excision or amplification of viral DNA by initiating replication at the integrated origin, providing a favorable substrate for subsequent recombination. 相似文献
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We examined the affinities of SV40 large T antigen for unique viral DNA sequences by binding SV40 Bst NI DNA fragments in extracts of infected or transformed cells, and then immunoprecipitating the T antigen-DNA complex. The G fragment, which spans the viral origin of replication (ori) was quantitatively bound to T antigen. A T-antigen-specific monoclonal antibody (McI 7), which recognized only 5%-10% of the T antigen from infected or transformed cells, immunoprecipitated the majority of the ori-binding activity. This suggests that only a minor subclass of wild-type T antigen is active in binding to the origin. C6 cells contain a replication-defective mutant T antigen that when tested in the DNA-binding immunoassay, showed no affinity for the ori fragment. McI 7 not only failed to immunoprecipitate ori binding in C6 cells, but also did not detect any labeled C6 T antigen whatever. Thus McI 7 recognizes an immunologically distinct subset of wild-type 7 antigen that comprises the origin-binding form of the viral protein, which is absent in the C6 T antigen population. McI 122, which recognizes a 53 kilodalton host protein that complexes with T antigen, immunoprecipitated ori-binding activity from extracts of infected or transformed cells, but not from C6 cells. Thus wild-type T antigen can bind ori sequences even when complexed to the host protein. These data suggest that T antigen consists of different subpopulations with different functions. 相似文献
19.
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
20.
We analyzed the state of the genomic DNA of the papovavirus SV40 in human keratinocytes as viral-infected cells gradually acquired a transformed phenotype over time. Initially, the vast majority of the viral DNA is maintained either in a full-length supercoiled form or as truncated subgenomic fragments with little evidence of integration. However, analyses of clonal populations revealed great heterogeneity and instability of the viral DNA, and we were able to isolate one clonal subpopulation in which integrated forms of the virus appeared to predominate. Similarly, uncloned populations eventually ceased production of the "free" viral DNA after several years in culture and instead came to display tandemly repeated SV40 copies at a single host integration site. Interestingly, Bg1 II digestion of host DNA generated restriction fragments containing the integrated SV40 DNA, which were of differing sizes in cultures at the 144th vs the 163rd serial passage suggesting modification or rearrangement of sequences at or near the integration site. Host sequences flanking the integrated viral DNA at the 163rd serial passage have been isolated on restriction fragments generated by Eco RI, Bam HI, and Hpa II digestion. These analyses suggest that the integrated virus is linearized near the Bg1 I site and contains a large deletion in the SV40 early region at one of the viral-host junctions. 相似文献