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1.
Assignment of the T-antigen gene of simian virus 40 to human chromosome C-7 总被引:12,自引:16,他引:12
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C M Croce A J Girardi H Koprowski 《Proceedings of the National Academy of Sciences of the United States of America》1973,70(12):3617-3620
Hybrid cell clones between mouse cells deficient in thymidine kinase (EC 2.7.1.21) and two different human cell lines transformed by simian virus 40 (SV40) and deficient in hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) were examined for SV40 tumor (T) antigen(s). Concordant segregation of the gene(s) for SV40 T antigen and human chromosome C-7 was observed in these hybrids. The human chromosome C-7 which contains the gene(s) for SV40 T antigen is preferentially retained by the majority of the hybrid clones tested. When hybrid clones positive and negative for SV40 T antigen, derived from the fusion of SV40-transformed Lesch-Nyhan fibroblasts with mouse cells, were fused with CV-1 permissive cells, SV40-specific V antigen was observed only in the cultures derived from fusion of the hybrid clones positive for T antigen. This result indicates a linkage relationship between human chromosome C-7, SV40 T-antigen gene(s), and SV40 genome(s) integrated in the human transformed cells. 相似文献
2.
Genetics of type II glycogenosis: Assignment of the human gene for acid α-glucosidase to chromosome 17
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Gian G. D''''Ancona James Wurm Carlo M. Croce 《Proceedings of the National Academy of Sciences of the United States of America》1979,76(9):4526-4529
We have studied somatic cell hybrids between thymidine kinase (EC 2.7.1.75) deficient mouse cells and human diploid fibroblasts for the expression of human acid alpha-glucosidase (EC 3.2.1.20). A deficiency in this enzyme is associated with the type II glycogenosis or Pompe disease. All 30 somatic cell hybrids selected in hypoxanthine/aminopterin/thymidine medium expressed human acid alpha-glucosidase and galactokinase (EC 2.7.1.6) and retained human chromosome 17; counterselection of the same hybrids in medium containing 5-bromodeoxyuridine resulted in the growth of hybrids that concordantly lost the expression of human acid alpha-glucosidase and galactokinase as well as human chromosome 17. Hybrids between thymidine kinase-deficient mouse cells and fibroblasts from a patient with Pompe disease that contained human chromosome 17 were found not to express human acid alpha-glucosidase. Because we have already shown that hybrids between mouse peritoneal macrophages and GM54VA simian virus 40-transformed human cells selectively retain human chromosome 17 and lose all other human chromosomes, we tested 13 independent mouse macrophage x GM54VA hybrid clones, including two that retained human chromosome 17 and no other human chromosomes, for the expression of human acid alpha-glucosidase and galactokinase. All 13 hybrid clones were found to express these human enzymes. Thus, we conclude that the gene coding for human acid alpha-glucosidase is located on human chromosome 17. 相似文献
3.
Tumorigenicity of simian virus 40-transformed human cells and mouse--human hybrids in nude mice. 总被引:1,自引:0,他引:1
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H Koprowski C M Croce 《Proceedings of the National Academy of Sciences of the United States of America》1977,74(3):1142-1146
Four different human cell lines transformed by simian virus 40 (SV40) were tested for their tumorigenicity in athymic nude mice. Two of these lines, W18Va2 and GM52VA, were found to be tumorigenic when inoculated at a concentration of 1 x 10(7) cells per mouse. The other two cell lines, LN-SV and GM54VA, were found to induce very small tumors only after the injection of approximately 1 x 10(8) cells per mouse. Somatic cell hybrids between either LN-SV or GM54VA SV40-transformed human cells and normal mouse peritoneal macrophages, which have retained the human chromosomes carrying the SV40 genome, were found to be much more tumorigenic than the SV40-transformed human cell parents. These experiments suggest that the genetic background in which the human chromosomes carrying the SV40 genome are present plays a role in the modulation of the expiration of malignancy. 相似文献
4.
Assignment of gene(s) for cell transformation to human chromosome 7 carrying the simian virus 40 genome. 总被引:1,自引:5,他引:1
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C M Croce H Koprowski 《Proceedings of the National Academy of Sciences of the United States of America》1975,72(5):1658-1660
Somatic cell hybrid clones between either C57BL/6 or Balb/c mouse peritoneal macrophages and two different simian virus 40 (SV40)-transformed human cell lines deficient in hypoxanthine phosphoribosyltransferase (EC 2.4.2.8; IMP:pyrophosphate phosphoribosyltransferase) were obtained in hypoxanthine-aminopterin-thymidine selective medium. All the hybrid cell clones contained the human chromosome 7, which carries the SV40 genome, and were SV40 tumor (T)-antigen positive. No hybrid cell clones studied displayed the density-dependent inhibition of cell growth characteristic of normal cells; all clones had a high saturation density and gave origin to cell colonies when plated in soft agar. Since the expression of the transformed phenotype was always associated with the presence of the human chromosome 7, which carries the SV40 genome, it is concluded that this chromosome contains gene(s) [Tr gene(s)] coding for "transforming factor(s)." 相似文献
5.
Chromosome Assignment of the T-Antigen Gene of Simian Virus 40 in African Green Monkey Cells Transformed by Adeno 7-SV40 Hybrid
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Carlo M. Croce Kay Huebner Hilary Koprowski 《Proceedings of the National Academy of Sciences of the United States of America》1974,71(10):4116-4119
Somatic cell hybrids between mouse cells deficient in thymidine kinase [ATP:thymidine 5′-phosphotransferase (EC 2.7.1.75)] and two different monkey cell lines transformed by an adeno 7-SV40 hybrid have been produced using both a semiselective and a double selective procedure. Concordant segregation of the expression of SV40 T antigen with a specific monkey chromosome has been observed in all the mouse-monkey hybrid clones examined. Subcloning of three SV40 T antigen positive hybrid clones resulted in their segregation into SV40 T antigen-positive and negative subclones. Positive correlation between the SV40 T antigen and the same monkey chromosome has been observed in all the subclones examined. 相似文献
6.
Another chromosomal assignment for a simian virus 40 integration site in human cells. 总被引:8,自引:0,他引:8
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R Kucherlapati S P Hwang N Shimizu J K McDougall M R Botchan 《Proceedings of the National Academy of Sciences of the United States of America》1978,75(9):4460-4464
Somatic cell hybrids derived from fusion of GM637, a human cell line transformed by simian virus 40, and mouse B82 cells were examined for simian virus 40 T antigen, V antigen, and viral DNA. All hybrid cell lines that contained viral DNA were T-antigen positive. Cells that did not have viral DNA were T-antigen negative. We determined that there is a single viral insertion in these hybrid cells. Correlation of T-antigen expression and viral DNA with the partial complements of the human genome retained in the hybrids shwed that the inserted viral genome is in human chromosome 8. The integrated viral DNA is stable; free viral DNA found in GM637 does not insert at other potential sites in the human genome. 相似文献
7.
A F Miranda L E Babiss P B Fisher 《Proceedings of the National Academy of Sciences of the United States of America》1983,80(21):6581-6585
Molecular studies of the biochemical alterations involved in human myopathies have been restricted because of the finite life-span and slow growth rate of cultures derived from primary tissue. Because the tumor virus simian virus 40 (SV40) can alter both the growth properties and longevity of human cells, we have infected skeletal muscle cultures derived from four biopsies with a small-plaque variant of SV40 and analyzed the biological and biochemical properties of cloned myoblast derivatives. At early times after infection, myoblasts fused normally into multinucleated myotubes, and both unfused and fused cells contained SV40 tumor antigen (T antigen). After six to eight subcultures after infection, the ability of myoblasts to fuse diminished, and clonal cell lines were generated with increased growth rates and saturation densities. Transformed cultures also lost contact inhibition of growth and became anchorage independent. Unlike untransformed myoblasts, SV40-transformed clones did not undergo an increase in creatine kinase activity or a transition of creatine kinase isoenzymes from the BB form to the muscle-specific MM form. Analysis of the pattern of SV40 DNA integration by Southern blotting hybridization analysis in two cloned SV40-transformed myoblast cell lines (KJ-SV40 and PK-SV40) indicated that KJ-SV40 contained at least one site of SV40 DNA integration into chromosomal DNA and PK-SV40 contained at least three sites of SV40 DNA covalently linked to cellular DNA. Cell lysates and growth medium from PK-SV40 transformants contained infectious small-plaque variant SV40, whereas KJ-SV40 did not contain or produce detectable virus. These studies demonstrate that human myoblasts can be immortalized by SV40. This procedure may prove useful for generating large quantities of genetically deficient human cells for biochemical and molecular analysis. 相似文献
8.
N Howell R Sager 《Proceedings of the National Academy of Sciences of the United States of America》1980,77(5):2844-2847
This paper describes the rare cytoplasmic transmission of defective simian virus 40 (SV40) viral DNA from enucleated cells (i.e., cytoplasts) of the SV40-transformed mouse cell line SVT2 (chloramphenicol-resistant) into cybrid cells formed by fusion of these cytoplasts with BALB/c 3T3 cells (thymidine kinase-deficient). The cybrids were selected in medium containing 1% serum, bromodeoxyuridine, and chloramphenicol. They were identified by their 3T3 chromosome content, by the instability of tumor (T)-antigen expression, by their transformed phenotype, and by their drug resistance. The yield of rare cybrids was about 5 x 10(-7) 0.1% of the yield on medium with 10% serum. The presence of the SV40 genome was detected by the expression of SV40-specific T antigen and confirmed (unpublished data) by hybridization of viral DNA probes with restriction enzyme fragments of nuclear DNAs from cybrid clones. Restriction site mapping (unpublished data) showed that at least 1 kilobase of host flanking DNA on each side of the SV40 DNA was included in the transferred segment. The transforming DNA was not stably integrated initially, as judged by cellular heterogeneity in T-antigen expression. Stable T-antigen-positive and negative subclones were recovered in 10% serum; instability could be retained for at least 30 doublings during growth in 1% serum. The instability is interpreted as evidence of non-integration or unstable integration of the transferred DNA into the host genome. The cytoplasmic transfer is interpreted as evidence that chromosomal fragments or intact chromosomes can be transferred rarely through the cytoplasm in cybrid crosses. 相似文献
9.
J Horst C Weckler E Jacob C Aulehla-Scholz W Deppert 《Proceedings of the National Academy of Sciences of the United States of America》1982,79(15):4645-4649
The recombinant plasmid p102 based on pBR322 carrying approximately equal to 50% of the replicator proximal early region of simian virus 40 (SV40) DNA, including the viral origin of replication, has been constructed. It lacks a major part of the large tumor (T) antigen 3'-coding region, the T-antigen termination codon, and the polyadenylylation site. The plasmid was transferred together with the herpes simplex virus thymidine kinase (TK) gene as a selectable marker to mouse LTK- cells. TK+ cell clones were isolated and their high molecular weight DNAs were shown by DNA blotting and hybridization experiments to contain the SV40 DNA fragment from the recombinant. In some of these clones, heterogeneous expression of the SV40 DNA fragment could be detected by immunofluorescence while, in control experiments in which a plasmid containing the complete SV40 early DNA region was used, this extensive heterogeneity of T-antigen expression was not observed. RNA . DNA hybridization experiments showed that the SV40-specific RNA of those clones is polyadenylylated. The molecular weight of the T-antigen-related protein coded by p102 corresponded well to the expected coding capacity of the SV40 DNA fragment. Small tumor antigen was not expressed. 相似文献
10.
Simian virus 40-related antigens in three human meningiomas with defined chromosome loss. 总被引:5,自引:0,他引:5
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A F Weiss R Portmann H Fischer J Simon K D Zang 《Proceedings of the National Academy of Sciences of the United States of America》1975,72(2):609-613
Two out of seven meningiomas tested in early cell cultures by indirect immunofluorescence staining showed simian virus 40 (SV40)-related tumor (T) antigen. In one tumor 90% of the cells were positive. An additional SV40-related antigen (U) was found in 10% of cells of a third tumor. These findings indicate that the meningioma cells showing a positive reaction are transformed by a papova virus that has at least partly the same antigenic properties as SV40 virus. SV40-related viral capsid (V) antigen was absent in all the meningiomas tested. No virus infectious for African green monkey kidney (AGMK) cells could be isolated. The tumors positive for T and U antigens showed the chromosome aberration typical for human meningiomas, i.e., the loss of one chromosome, G-22. The T-antigen-positive tumors showed further hypodiploidization. Experiments to rescue virus from the T-antigen-positive tumors showed further hypodiploidization. Experiments to rescue virus from the T-antigen-positive meningioma cells were performed: fusion of cells pretreated with 8-azaguanine with cells premissive for SV40 led to a low percentage (0.01-0.05%) of V-antigen-positive nuclei in heterokaryon cultures. On the basis of these results, the possibility of a correlation between the meningioma, a relatively common intracranial tumor in man, and an SV40-related papova virus must be considered. It remains to be shown whether this virus is a causative agent for human meningiomas. 相似文献
11.
12.
Replication of simian virus 40 origin-containing DNA in vitro with purified proteins. 总被引:53,自引:15,他引:53
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C R Wobbe L Weissbach J A Borowiec F B Dean Y Murakami P Bullock J Hurwitz 《Proceedings of the National Academy of Sciences of the United States of America》1987,84(7):1834-1838
Simian virus 40 (SV40) DNA replication dependent on the SV40 origin of replication and the SV40 large tumor (T) antigen has been reconstituted in vitro with purified protein components isolated from HeLa cells. In addition to SV40 T antigen, these components included the DNA polymerase alpha-primase complex, topoisomerase I, and a fraction that contained a single-stranded DNA binding protein. The latter protein, which sediments at 5.1 S on glycerol gradients and copurifies with two major protein species of 72 and 76 kDa, was isolated solely by its ability to support SV40 DNA replication. The purified system retained the species-specific DNA polymerase alpha-primase requirement previously observed with crude fractions; the complex from HeLa cells supported SV40 replication, whereas that from calf thymus and mouse cells did not. DNA containing the polyomavirus origin of replication was replicated in a system containing polyomavirus T antigen, the HeLa single-stranded DNA binding protein-containing fraction, and DNA polymerase alpha-primase complex from mouse, but not HeLa, cells. While crude fractions yielded closed circular duplex DNA, none was detected with the purified system. Nevertheless, the addition of a crude fraction to the purified system yielded closed circular monomer products. 相似文献
13.
14.
15.
Epstein-Barr virus and human chromosomes: close association of the resident viral genome and the expression of the virus-determined nuclear antigen (EBNA) with the presence of chromosome 14 in human-mouse hybrid cells.
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K Yamamoto F Mizuno T Matsuo A Tanaka M Nonoyama T Osato 《Proceedings of the National Academy of Sciences of the United States of America》1978,75(10):5155-5159
Fourteen hybrid clones derived from the fused cultures of human lymphoblastoid FV5 cells and 5-bromodeoxyuridine-resistant mouse fibroblastic MCB2 cells grown in hypoxanthine/aminopterin/thymidine selective medium were examined for the presence of Epstein-Barr virus (EBV) DNA, the expression of the virus-determined nuclear antigen (EBNA), and the presence of human chromosomes, in the course of serial passage in vitro. Among the hybrid clones tested, 3 were positive for EBV DNA and EBNA, whereas the remaining 11 were totally negative. The chromosome investigations showed that human chromosome 14 was consistently involved in all three EBV genome-positive and EBNA-positive hybrid clones, but not in any negative clones. In 10 subclones isolated from 1 of the 3 positive clones, all of which contained only chromosome 14 of the human chromosomes, a concordant segregation of EBNA, EBA DNA, and chromosome 14 was evident. These findings suggest that the resident EBV genome is closely associated with chromosome 14 and the presence of this particular chromosome alone is sufficient for the maintenance and the expression of EBV genetic information in human lymphoblastoid cells. 相似文献
16.
Template supercoiling during ATP-dependent DNA helix tracking: studies with simian virus 40 large tumor antigen. 总被引:11,自引:3,他引:8
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L Yang C B Jessee K Lau H Zhang L F Liu 《Proceedings of the National Academy of Sciences of the United States of America》1989,86(16):6121-6125
Incubation of topologically relaxed plasmid DNA with simian virus 40 (SV40) large tumor antigen (T antigen), ATP, and eubacterial DNA topoisomerase I resulted in the formation of highly positively supercoiled DNA. Eukaryotic DNA topoisomerase I could not substitute for eubacterial DNA topoisomerase 1 in this reaction. Furthermore, the addition of eukaryotic topoisomerase I to a preincubated reaction mixture containing both T antigen and eubacterial topoisomerase I caused rapid relaxation of the positively supercoiled DNA. These results suggest that SV40 T antigen can introduce topoisomerase-relaxable supercoils into DNA in a reaction coupled to ATP hydrolysis. We interpret the observed T antigen supercoiling reaction in terms of a recently proposed twin-supercoiled-domain model that describes the mechanics of DNA helix-tracking processes. According to this model positive and negative supercoils are generated ahead of and behind the moving SV40 T antigen, respectively. The preferential relaxation of negative supercoils by eubacterial DNA topoisomerase I explains the accumulation of positive supercoils in the DNA template. The supercoiling assay using DNA conformation-specific eubacterial DNA topoisomerase I may be of general use for the detection of ATP-dependent DNA helix-tracking proteins. 相似文献
17.
Genetics of the connective tissue proteins: Assignment of the gene for human type I procollagen to chromosome 17 by analysis of cell hybrids and microcell hybrids 总被引:2,自引:0,他引:2
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Cholappadi V. Sundar Raj Robert L. Church Lawrence A. Klobutcher Frank H. Ruddle 《Proceedings of the National Academy of Sciences of the United States of America》1977,74(10):4444-4448
Somatic cell hybrids between mouse and human cell lines have been used to identify the specific chromosome that governs the synthesis of type I procollagen. Fourteen hybrid clones and subclones were derived independently from crosses between mouse parents [LM (thymidine kinase-negative) or A9 (hypoxanthine phosphoribosyltransferase-negative)] and human cells (human diploid lung fibroblasts WI-38 or diploid skin fibroblasts GM5, GM17, and GM9). The cultures were labeled with [(3)H]proline in modified Eagle's medium without serum. Radioactive procollagens were purified from the medium by the method of Church et al. [(1974) J. Mol. Biol. 86, 785-799]. DEAE-cellulose chromatography was used to separate collagen and type I and type III procollagen. Human type I procollagen was assayed by double immunodiffusion analysis with type I procollagen antibodies prepared by immunizing rabbits with purified human type I procollagen. These analyses combined with karyology and isozyme analyses of each hybrid line have produced evidence for the assignment of the gene for human type I procollagen to chromosome 17. A human microcell-mouse hybrid cell line containing only human chromosome 17 was positive for human type I procollagen, lending further support to the assignment of the human type I procollagen gene to chromosome 17. Finally, by using a hybrid line containing only the long arm of human chromosome 17 translocated onto a mouse chromosome, the type I procollagen gene can be assigned more specifically to the long arm of chromosome 17. 相似文献
18.
Immunologic selection against simian virus-40 transformed cells: Concomitant loss of viral antigens and early viral gene sequences
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Jürgen M. Kuster Peter T. Mora Myles Brown George Khoury 《Proceedings of the National Academy of Sciences of the United States of America》1977,74(11):4796-4800
A clonal line of highly oncogenic "spontaneously transformed" mouse cells (T AL/N clone 3) was transformed in tissue culture by simian virus 40 (SV40) and subsequently recloned. The clone of SV40-transformed cells (subclone 1) expressed SV40-specific T (nuclear) and transplantation antigens but was 100 times less tumorigenic than the parent T AL/N clone 3 cells. When large numbers of subclone 1 cells (10(4)-10(5)) were injected into syngeneic AL/N mice, tumors were produced. From the tumors, cell lines were established in culture, all of which were consistently negative for T antigen. Tumor lines tested were found not to contain SV40-specific transplantation antigen and had again become highly tumorigenic. The original subclone 1 cells contained about one copy of SV40 DNA per diploid amount of cell DNA, as well as RNA complementary to the early region of the SV40 genome. The T antigen-negative cells from tumor line 124 contained approximately 0.5 copy of SV40 DNA per diploid equivalent and did not synthesize any detectable virus-specific RNA. Reassociation kinetic analysis with restriction enzyme fragments of viral DNA demonstrated that the cells from tumor line 124 (and also the clones of this line) had lost DNA sequences predominantly from the early region of the SV40 genome. The results indicate that a set of stably integrated SV40 DNA sequences can be present in a cell without the expression of viral antigens. 相似文献
19.
Somatic cell hybrids between mouse peritoneal macrophages and simian-virus-40-transformed human cells: II. Presence of human chromosome 7 carrying simin virus 40 genome in cells of tumors induced by hybrid cells. 总被引:2,自引:2,他引:2
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C M Croce D Aden H Koprowski 《Proceedings of the National Academy of Sciences of the United States of America》1975,72(4):1397-1400
Cells derived from tumors induced in "nude" mice after injection of cells that were hybrids between mouse peritoneal macrophages and simian virus 40 (SV40)-transformed human cells were found to retain the human chromosome 7 carrying the SV40 genome, and indicate that the presence of human chromosome 7 carrying the SV40 genome is responsible for the expression of the tumorigenic phenotype in the hybrid cells. 相似文献
20.
L M Sompayrac K J Danna 《Proceedings of the National Academy of Sciences of the United States of America》1981,78(12):7575-7578
With standard protocols for DNA infection, only a small fraction (about 4%) of monkey cells exposed to purified DNA of simian virus 40 (SV40) exhibits signs of infection. We have devised a protocol by which we can extend the time of exposure of BSC-1 cells to DNA in the presence of low concentrations of DEAE-dextran. The efficiency of infection is proportional to the time of exposure. With an 8-hr exposure, we are reproducibly able to infect 25% of the cells, and we have been able to achieve levels of infection as high as 50% with a 16-hr exposure. The percentage of cells infected was measured either by scoring for nuclei positive for SV40 tumor antigen or by an infectious centers assay. We also report the use of ethidium bromide as a nonspecific nuclear counterstain in the immunofluorescence assay for SV40 tumor antigen. 相似文献