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1.
Polyoma large T antigen regulates the integration of viral DNA sequences into the genome of transformed cells 总被引:34,自引:0,他引:34
The major coat protein of coliphage M13 is an integral protein of the E. coli plasma membrane prior to its assembly into new virus particles. It is generated from its precursor, procoat, by a membrane-bound leader peptidase. We now describe the reconstitution of a highly purified preparation of this enzyme into vesicles of E. coli phospholipids. These vesicles bind procoat made in vitro and procoat isolated from in vitro synthesis. Both the crude and the purified substrates were converted posttranslationally to coat protein. A significant proportion of the coat protein becomes inserted into the vesicle bilayer, with the N terminus facing the vesicle interior and the C terminus exposed to the external medium. These results strongly suggest that highly purified leader peptidase from E. coli and phospholipids are the only components necessary to mediate the binding, processing and insertion of this integral membrane protein. 相似文献
2.
The uptake of L-alanine into BHK21-C13 cells in culture has been studied. This amino acid appears to be transported essentially via a relatively low affinity, high capacity, sodium ion dependent transport system. Inhibition studies using other amino acids or their analogues provided information about the specificity of this system. This alanine transport system was shown to exhibit a broad substrate specificity and appeared to be capable of transporting most naturally occurring neutral alpha-amino acids. Kinetic studies of the inhibition of L-alanine uptake also indicated the presence of a second neutral amino acid transport system capable of transporting this amino acid. However, it is unlikely that this second uptake system contributes greatly to L-alanine uptake. Inhibition of the uptake of L-leucine indicated that this transport system has a similar specificity to the "L"-system initially described for Ehrlich ascites carcinoma cells. 相似文献
3.
Treatment of hamster embryo cells with diverse classes of chemical carcinogens enhances transformation by a carcinogenic simian adenovirus, SA7. Virus transformed foci selected from plates pretreated with 3-methyl-cholanthrene (MCA), methyl methanesulfonate (MMS) or 7,12-dimethylbenz[a]anthracene (DMBA) and established as cell lines in culture, contained equivalent amounts of SA7 viral genome. However, hamster embryo cultures treated with MMS or nickel sulfate had increased amounts of SA7 DNA integrated into cellular DNA when examined 2--9 days after chemical treatment and viral inoculation. An increased uptake of SA7 DNA was demonstrated in hamster cells treated with MMS during DNA repair synthesis in cells retricted in scheduled DNA synthesis by amino acid deprivation; addition of virus after the repair period did not result in an increased integration of viral DNA. These data suggest that enhancement of viral oncogenesis by chemical carcinogens or mutagens may be related to the formation of additional attachment sites in cellular DNA for insertion of viral DNA, thereby increasing the probability of viral transformation. 相似文献
4.
Selective sites of adenovirus (foreign) DNA integration into the hamster genome: changes in integration patterns. 总被引:4,自引:2,他引:2
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We investigated whether, upon the integration of multiple copies of adenovirus type 12 (Ad12) DNA into an established mammalian (hamster) genome, the pattern of foreign DNA insertion would remain stable or change with consecutive passages of cells in culture. By the injection of purified Ad12 into newborn hamsters, tumors were induced, cells from these tumors were cultivated, and five independent cell lines, HT5, H201/2, H201/3, H271, and H281, were established. These cell lines carried different copy numbers of Ad12 DNA per cell in an integrated form and differed in morphology. Cell line HT5 had been passed twice through hamsters as tumor cells and was subsequently passaged in culture. Patterns of Ad12 DNA integration were determined by restriction cleavage of the nuclear DNA with BamHI, EcoRI, HindIII, MspI, or PstI followed by Southern blot hybridization using 32P-labeled Ad12 DNA or its cloned terminal DNA fragments as hybridization probes. In this way, the off-size fragments, which represented the sites of linkage between Ad12 and cellular DNAs, were determined. At early passage levels in culture, the integration sites of Ad12 DNA in the hamster genome, as characterized by the positions of off-size fragments in agarose or polyacrylamide gel electrophoresis, were different in the five different tumor cell lines. Upon repeated passage, however, the off-size fragment patterns generated by the five restriction endonucleases became very similar in the five tumor cell lines. This surprising result indicates that under cell culture conditions, Ad12-transformed tumor cell lines that carry the foreign (Ad12) genome in selective, probably very similar sites of the cellular genome evolve. 相似文献
5.
6.
Polyoma and hamster papovavirus large T antigen-mediated replication of expression shuttle vectors in Chinese hamster ovary cells. 总被引:12,自引:1,他引:11
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Eukaryotic expression vectors have been used successfully in viral LT-expressing cell lines (ie. COS) to clone cDNAs encoding proteins that can be detected through their bio-activity or reactivity with specific antibodies. Since Chinese hamster ovary cells (CHO) have been used extensively for the isolation and characterization of somatic cell mutants, we felt it would be an advantage to develop an expression cloning system in CHO cells. We have modified the eukaryotic expression vector CDM8 by replacing the polyoma and SV40 origins of replication with the 427bp non-coding region of the Syrian hamster papovavirus. Wild-type CHO cells and the CHO glycosylation-mutant Lec4A were transfected with plasmids bearing the early genes of either polyoma virus or hamster papovavirus in order to establish stable, LT antigen-expressing cell lines designated CHOP or CHOH, respectively. CHOP cell lines expressing polyoma LT antigen supported efficient replication of CDM8, but replicated pMH poorly. Conversely, CHOH cells expressing the hamster papovavirus LT antigen supported replication of pMH, and at a lower efficiency, CDM8. Replication of CDM8 and pMH vectors were equally efficient in selected CHOP and CHOH cell lines, respectively and comparable to that of CDM8 replication in COS-1 cells. A bacterial beta-galactosidase fusion gene inserted into the multiple cloning site of a CDM8 derivative was efficiently expressed when transiently transfected into CHOP and CHOH cells but not CHO cells since only the former supports autonomous plasmid replication. These results show that expression-cloning in CHO cells expressing either polyoma virus or hamster papovavirus LT antigens is possible using either the CDM8 or the pMH vectors, respectively. 相似文献
7.
L S Popov 《Biokhimii?a (Moscow, Russia)》1989,54(11):1815-1820
The integration of temperature-sensitive SV40 mutant DNA (tsA239) into the Chinese hamster cellular genome at an early stage of infection was studied. The content of single-stranded DNA structures in the infected and control cells at a non-permissive temperature (40 degrees C) differed drastically from that in control cells at permissive temperatures (33 degrees C, 37 degrees C). The role of single-stranded structures in the integration of the SV40 genome into cellular DNA was shown by blot hybridization. The integration mechanism is discussed. 相似文献
8.
Cytopathogenicity of border disease virus is correlated with integration of cellular sequences into the viral genome. 总被引:7,自引:4,他引:3
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Two border disease virus (BDV) pairs each consisting of cytopathogenic (cp) and non-cp viruses have been analyzed at the molecular level. Within the NS2-3 (p125) encoding region of both cp viruses, insertions of cellular sequences were identified which were absent in the corresponding non-cp isolates. A comparative sequence analysis revealed that within each pair the cp and non-cp viruses are almost identical. This strongly suggests that the cp BDV isolates developed from the non-cp viruses by RNA recombination between the viral genome and cellular sequences. Nonstructural protein NS3 (p80) was demonstrated after infection with both cp BDV strains. In addition, fusion proteins composed of cellular and viral sequences were identified. In contrast, expression of NS3 and the fusion proteins was not found after infection with the respective non-cp counterparts. 相似文献
9.
William T. Melvin Julian F. Burke Alison A. Slater Hamish M. Keir 《Journal of cellular physiology》1979,98(1):73-79
Removal of serum from BHK-21/C13 cells in culture results in a decline in thymidine incorporation extending over five days. Additional removal of any of several amino acids results in a rapid decrease in incorporation of thymidine to negligible levels by 24 hours. Replacement by complete medium then provokes a synchronous wave of DNA synthesis after only ten hours with DNA synthesis first increased at six hours. Starvation for glutamine results in a rapid decline in protein synthesis over the 24 hour period when DNA synthesis is falling. However, there is considerable degradation of total protein during this period, and RNA degradation is also greatly increased. Concurrently, synthesis of RNA falls to less than 10% of that in control cells. 相似文献
10.
Patterns of integration of viral DNA in adenovirus type 2-transformed hamster cells 总被引:14,自引:0,他引:14
The patterns of integration of viral DNA in five lines of adenovirus type 2-transformed hamster cells have been investigated. Cell lines HE1 to HE5 were obtained by in vitro transformation of hamster embryo cells by ultraviolet light-inactivated Ad22. In all lines, segments in the central parts of the viral genome are missing. The lines HE1, HE2, HE3, HE4 and HE5 contain 2 to 4, 2 to 4, 6 to 10, about 10, and 2 to 3 genome fragment equivalents per cell, respectively.The patterns of integration in lines HE2 and HE3 are identical; however, the viral genome has been amplified in these cell lines to different extents. This result provides evidence for the post-integrational amplification of inserted viral genomes. It is also conceivable that line HE2 may have undergone losses of integrated Ad2 genomes. The persisting Ad2 genomes in lines HE2 and HE3 have deletions in parts of the EcoRI F and D fragments. The remainders of these fragments are linked to cellular DNA. The termini of the segments of the viral genome have been inverted and linked to each other. This linkage could have occurred via a circular intermediate in integration or via tandemly integrated viral genomes with subsequent deletion events. The linkage of the termini of viral DNA might be mediated by short sequences of cellular DNA.In line HE5, approximately 40% of the Ad2 genome is deleted, and the truncated segments, again comprising the terminal Ad2 DNA fragments, have been fused. The termini of the viral DNA are linked to cellular DNA. In lines HE1 and HE4 complex deletion and fusion events have altered the inserted Ad2 genomes. 相似文献
11.
Episomal amplification or chromosomal integration of the viral genome: alternative pathways in hamster polyomavirus-induced lymphomas.
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The state and expression of the hamster polyomavirus genome in a large panel of virus-induced lymphomas have been investigated. The viral genome is present within tumor cells either as abundant nonrandomly deleted extrachromosomal copies or as a single copy integrated into cellular DNA. We show that these two physical states are likely to be functionally equivalent: first, deletion and integration of the viral genome both inactivate the late coding region; second, the amount of viral early RNAs yielded by a single integrated copy appears to be very similar to that associated with several thousands of extrachromosomal copies of the viral genome. These data underline two essential requisites for hamster polyomavirus to become lymphomagenous: suppression of the late coding functions of the viral genome and expression of the viral oncogenes above a threshold level. 相似文献
12.
Z E Stoliarov 《Molekuliarnaia biologiia》1990,24(1):17-22
A model describing the mechanism of linear integration of cellular and viral DNA is proposed. The possible role of cellular DNA integration in the process of human carcinogenesis is considered. 相似文献
13.
Geoffrey J. Hayton Colin K. Pearson Jeremy R. Scaife Hamish M. Keir 《The Biochemical journal》1973,131(3):499-508
Newly synthesized (3)H-labelled DNA was extracted from baby hamster kidney cells (BHK-21/C13 cells) and was shown to possess single-stranded properties when examined by column chromatography on benzoylated naphthoylated DEAE-cellulose, hydroxyapatite and methylated albumin on kieselguhr, and by its affinity for nitrocellulose filters. Some of the newly synthesized DNA was shown to be of lower molecular weight than the bulk of the DNA when examined by alkaline sucrose-density-gradient centrifugation. The properties observed were not affected by treatment of the DNA with ribonuclease, Pronase or amylase. The effect of the size of the DNA on its observed properties was examined and is discussed. It is concluded that DNA synthesis in BHK-21/C13 cells proceeds according to the discontinuous-mechanism model in at least one of the strands. 相似文献
14.
Identification of temperature-sensitive DNA- mutants of Chinese hamster cells affected in cellular and viral DNA synthesis. 总被引:1,自引:1,他引:1
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We described a strategy which facilitates the identification of cell mutants which are restricted in DNA synthesis in a temperature-dependent manner. A collection of over 200 cell mutants temperature-sensitive for growth was isolated in established Chinese hamster cell lines (CHO and V79) by a variety of selective and nonselective techniques. Approximately 10% of these mutants were identified as ts DNA- based on differential inhibition of macromolecular synthesis at the restrictive temperature (39 degrees C) as assessed by incorporation of [3H]thymidine and [35S]methionine. Nine such mutants, selected for further study, demonstrated rapid shutoff of DNA replication at 39 degrees C. Infections with two classes of DNA viruses extensively dependent on host-cell functions for their replication were used to distinguish defects in DNA synthesis itself from those predominantly affecting other aspects of DNA replication. All cell mutants supported human adenovirus type 2 (Ad2) and mouse polyomavirus DNA synthesis at the permissive temperature. Five of the nine mutants (JB3-B, JB3-O, JB7-K, JB8-D, and JB11-J) restricted polyomavirus DNA replication upon transfection with viral sequences at 33 degrees C and subsequent shift to 39 degrees C either before or after the onset of viral DNA synthesis. Only one of these mutants (JB3-B) also restricted Ad2 DNA synthesis after virion infection under comparable conditions. No mutant was both restrictive for Ad2 and permissive for polyomavirus DNA synthesis at 39 degrees C. The differential effect of these cell mutants on viral DNA synthesis is expected to assist subsequent definition of the biochemical defect responsible. 相似文献
15.
Polyoma and cell DNA synthesis in mouse L cells temperature sensitive for the replication of cell DNA. 总被引:6,自引:1,他引:5
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Sheinin 《Journal of virology》1976,17(3):692-704
Polyoma (Py) virus multiplies, at 34 and 38.5 C, in wild-type (WT-4) and in ts A1S9 mouse L cells, which are temperature sensitive for growth and for DNA replication (R. Sheinin, 1976; L. H. Thompson et al., 1970). De novo synthesis of double-stranded, fully covalently closed Py DNA has been shown to proceed by semiconservative replication in WT-4 and ts A1S9 cells at the permissive and nonpermissive temperatures. Cell DNA is made late during infection, by both cell types and at both temperatures. Semiconservative replication of cell DNA proceeds in Py-infected WT-4 cells incubated at 34 or at 38.5 C and in Py-infected ts A1S9 cells incubated at 34 C. In virus-infected ts A1S9 cells incubated at 38.5 C, cell DNA synthesis appears to proceed almost entirely by a process analogous to repair replication. The inability of ts A1S9 cells to produce large-molecular-weight chromosomal DNA strands, at 38.5 C, by the normal mechanism is not overcome by Py infection. 相似文献
16.
Adeno-associated virus Rep78 protein and terminal repeats enhance integration of DNA sequences into the cellular genome. 总被引:10,自引:6,他引:4
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Two adeno-associated virus (AAV) elements are necessary for the integration of the AAV genome: Rep78/68 proteins and inverted terminal repeats (ITRs). To study the contribution of the Rep proteins and the ITRs in the process of integration, we have compared the integration efficiencies of three different plasmids containing a green fluorescent protein (GFP) expression cassette. In one plasmid, no viral sequences were present; a second plasmid contained AAV ITRs flanking the reporter gene (integration cassette), and a third plasmid consisted of an integration cassette plus a Rep78 expression cassette. One day after transfection of 293 cells, fluorescent cells were sorted by flow cytometry and plated at 1 cell per well. Two weeks after sorting, colonies were monitored for stable expression of GFP. Transfection with the GFP plasmid containing no viral sequences resulted in no stable fluorescent colonies. Transfection with the plasmid containing the integration cassette alone (GFP flanked by ITRs) produced stable fluorescent colonies at a frequency of 5.3% +/- 1.0% whereas transfection with the plasmid containing both the integration cassette and Rep78 expression cassette produced stable fluorescent colonies at a frequency of 47% +/- 7.5%. Southern blot analysis indicated that in the presence of Rep78, integration is targeted to the AAVSI site in more than 50% of the clones analyzed. Some clones also showed tandem arrays of the integrated GFP cassette. Both head-to-head and head-to-tail orientations were detected. These findings indicate that the presence of AAV ITRs and the Rep78 protein enhance the integration of DNA sequences into the cellular genome and that the integration cassette is targeted to AAVS1 in the presence of Rep78. 相似文献
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19.
Excision of viral DNA from host cell DNA after induction of simian virus 40-transformed hamster cells. 总被引:4,自引:2,他引:2
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Simian virus 40-transformed hamster cells were induced to produce infectious virus by treatment with mitomycin C or gamma-irradiation. A portion of the simian virus-40 DNA, which is integrated into the host cell genome in uninduced cells, was recovered in a pool of relatively low-molecular-weight DNA early after induction treatment in the absence of DNA replication. The data indicte that excision of the viral genome occurs subsequent to the induction stimulus. 相似文献
20.
The pathway by which semliki forest virus (SFV), a membrane-containing animal virus, enters BHK-21 cells was studied morphologically and biochemically. After attaching to the cell surface, the majority of viruses was rapidly trapped into coated pits, internalized by endocytosis in coated vesicles, and sequestered into intracellular vacuoles and lysosomes. Direct penetration of viruses through the plasma membrane was never observed. To assess the possible involvement of lysosomes in the release of the genome into the cytoplasm, the effect of five lysosomotropic agents, known to increase the lysosomal pH, was tested. All of these agents inhibited SFV infectivity and one, chloroquine (the agent studied in most detail), inhibited a very early step in the infection but had no effect on binding, endocytosis, or intracellular distribution of SFV. Thus, the inhibitory effect was concluded to be either on penetration of the nucelocapsid into the cytoplasm or on uncoating of the viral RNA. Possible mechanisms for the penetration of the genome into the cytoplasm were studied in vitro, using phospholipids-cholesterol liposomes and isolated SFV. When the pH was 6.0 or lower, efficient fusion of the viral membranes and the liposomal membranes occurred, resulting in the transfer of the nucleocapsid into the liposomes. Infection of cells could also be induced by brief low pH treatment of cells with bound SFV under conditions where the normal infection route was blocked. The results suggest that the penetration of the viral genome into the cytosol takes place intracellularly through fusion between the limiting membrane of intracellular vacuoles and the membrane of viruses contained within them. The low pH required for fusion together with the inhibitory effect of lysosomotropic agents implicate lysosomes, or other intracellular vacuoles with sufficiently low pH, as the main sites of penetration. 相似文献