Ètude du mècanisme de l'induction chez des cellules de hamster syrien transformèes par le virus SV40 I. Propriètès d'une lignèe cellulaire clonale

Tournier, P; Cassingéna, R.; Wicker, R.; Coppey, Jacques; Suárez, H. G.

doi:10.1002/ijc.2910020207

Cited by 95 publications

(50 citation statements)

References 25 publications

(9 reference statements)

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“…The molecular and cytological events of the abortive infection in mouse kidney (MK) cells have been described (4,12,13). Under the conditions used in this study (10R PFU/ml; 370) a weak immunofluorescence reaction for T-antigen could be detected in 1-2% of the nuclei 7-8 hr after infection; later the relative number of positive nuclei (and the intensity of the immunofluorescence reaction) rapidly increased, reaching by 24 Under the conditions used (10P PFU/ml; 370) 1-5% of the nuclei contained T-antigen by [8][9] hr after infection; later the relative number of positive nuclei (and the intensity of the immunofluorescence reaction) rapidly increased, reaching 25-50% by 15 hr and 90-100% by [20][21][22][23][24] hr after infection. Time course of infection was similar in confluent BSC and Vero monkey kidney cell lines and also in confluent primary monkey kidney cell cultures.…”

Section: Resultsmentioning

confidence: 68%

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Two forms of simian-virus-40-specific T-antigen in abortive and lytic infection.

Ahmad-Zadeh¹,

Allet²,

Greenblatt³

et al. 1976

Proc. Natl. Acad. Sci. U.S.A.

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Simian-virus40-specific T-antigen was isolated by immunoprecipitation. From other studies we have proof that the T-antigen described in this work is coded by the viral DNA. The molecular weight estimated from electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels of T-antigen (1, 4,1) of a part of SV40 DNA ("early region") that comprises about 45% of total length (5, 6). Evidence discussed elsewhere suggested that early 19S mRNA directs synthesis of a single, rather large protein, the SV40-specific T-antigen, which is required to induce lytic and abortive infection, to initiate and maintain cell transformation, and probably also to induce primary tumors in animals (details in refs. 4 and 7).In the present work we isolated T-antigen by immunoprecipitation. The T-antigen isolated in this way is shown to be coded by the viral DNA. We compared the molecular properties of T-antigen in abortive and lytic infection. Our The results were the same whether crude viral lysates or highly purified viral preparations were used for infection. Virus (0.3 ml per dish) was adsorbed at 370 for 90 min; the cultures were then covered with 10 ml of medium containing 10% bovine serum; in most experiments primary mouse kidney cultures were covered with serum-free medium (4).Parallel cultures were mock-infected with 0.3 ml of medium and then treated as SV40-infected cultures.Cultures were labeled with [asS]methionine Ci/ mmol; The Radiochemical Centre, Amersham, England) by addition of 3 ml per dish of prewarmed (370) medium (E serum) which contained only 2 glg/ml of unlabeled methionine (instead of 30 .ug) and 50 ,Ci/ml of [a5S]methionine. At the end of the labeling period the cultures were washed twice with 5 ml of cold (20) isotonic phosphate buffer; then the cells were gently scraped off in 1 ml of the same phosphate buffer with a silicone policeman and centrifuged for 5 min at 500 X g.Preparation of "Soluble Extracts." The pellets were suspended at concentrations varying between 107 and 108 cells in 5 ml of extraction buffer (2.5 mM Tris-HCl, pH 7.4, 0.5 M LiCl, 1 mM EDTA), sonicated (MSE 100 W Ultrasonic Disintegrator) four times for 15 sec in an ice-water bath, and then immediately centrifuged for 30 min at 30,000 X g at 40. The supernatant was removed and is referred to as "soluble extract." In early experiments phenylmethylsulfonyl fluoride (0.1-0.3 mg/ml) and dithiothreitol (1 mM) were present in the extraction buffer.Immunoprecipitation. Most experiments were performed with sera (anti-T) obtained from Syrian hamsters bearing tumors induced by inoculation of an SV40-transformed hamster cell line (9). One preparation was a gift of Drs. E. and P. May, others were made in our laboratory. Hamster anti-T sera (batches 3 X 1888 and 4 X 21) were received from the National Cancer Institute (Bethesda, Md.). The anti-T sera used had specific complement fixing titers of 160-640. Control serum was a pool of 15 normal adult Syrian hamster

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Section: Resultsmentioning

confidence: 68%

“…Most experiments were performed with sera (anti-T) obtained from Syrian hamsters bearing tumors induced by inoculation of an SV40-transformed hamster cell line (9). One preparation was a gift of Drs.…”

Section: And 7)mentioning

confidence: 99%

Two forms of simian-virus-40-specific T-antigen in abortive and lytic infection.

Ahmad-Zadeh¹,

Allet²,

Greenblatt³

et al. 1976

Proc. Natl. Acad. Sci. U.S.A.

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“…clonal antibody, or 200 p1 of hybridoma culture supernatant, or 10 p1 of polyclonal anti-T serum obtained from tumor-bearing hamsters that had been injected with the SV40-transformed cell line TSV-5 clone 2 [16]. The samples containing monoclonal antibodies PAb 402 or 423 (subclass G1) were then supPurification of monoclonal antibodies [ 151 Hybridoma culture supernatant (200 ml) was adjusted with 1 M Mops/Na+, pH 8, to a final concentration of 0.1 M Mops and then centrifuged in a Sorvall GSA rotor for 5 min at 2000 rev./min.…”

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confidence: 99%

Binding sites for monoclonal antibodies and for mRNPs on SV40 large T‐antigen determined with a cleavage map

Schwyzer

Tai

Studer

et al. 1983

European Journal of Biochemistry

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Immune complexes of simian virus 40 large T-antigen with monoclonal papovavirus protein antibodies PAb 416, PAb 402, or PAb 423 were bound to protein-A-Sepharose and then cleaved into discrete fragments by limited tryptic proteolysis. PAb 402 protected a specific cleavage site, located approximately within amino acid residues 450 -500, from tryptic proteolysis; PAb 423 protected another site within residues 675 -699. As shown by immunoblotting, 12'I-labeled PAb 416 was bound to a 17-kDa N-terminal fragment of large T-antigen (amino acid residues 1 -130), and PAb 423 was bound to several overlapping fragments derived from the C terminus of large T-antigen. These monoclonal antibodies were then used as accessibility probes to study the interaction of mRNPs with cytoplasmic large T-antigen. Whereas small T-antigen and nuclear large T-antigen were fully immunoreactive, cytoplasmic large T-antigen reacted poorly with PAb 402 or polyclonal antibodies unless the mRNP moiety was removed by treatment with EDTA/RNase A. In contrast, mRNP/T-antigen complexes were fully immunoreactive with PAb 416 or PAb 423 and did not require treatment with EDTA/RNase A. The results suggest that the binding site of PAb 402 is blocked due to the interaction with mRNPs whereas the N-terminal binding site of PAb 416 and the C-terminal binding site of PAb 423 remain accessible to antibodies. For structural analysis, large T-antigen was bound as immune complex to protein-A -Sepharose and then cleaved into discrete fragments by limited tryptic proteolysis; cleavage occurred predominantly between arginine-130 and lysine-13 1 and at several sites in the C-terminal third of large T-antigen as shown by amino acid sequence analysis [12]. In the present report, we use this cleavage technique to determine the binding sites of monoclonal antibodies PAb 416, 402, and 423 directed against large T-antigen [13, 141. These monoclonal antibodies, as well as polyclonal antibodies, are then used as accessibility probes to study the interaction of large T-antigen with mRNPs. We show that some immunoreactive sites of large T-antigen are blocked as a result of mRNP binding and become accessible Abbreviations. T-antigen, tumor antigen; SV40, simian virus 40; mRNP, messenger ribonucleoprotein; NaDodSO,, sodium dodecyl sulfate; Mops, 4-morpholinepropanesulfonic acid; IgG, immunoglobulin G; PAb, papovavirus protein antibody.

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“…A fundamental feature of virus-transformed cells is the persistence of viral genetic information (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). In the cases which have been carefully studied to date, the persistent viral genes appear to be covalently linked to host genes (i.e., "integrated"), and expression of one or more integrated viral genes seems to be required for the maintenance of the transformed phenotype (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27).…”

mentioning

confidence: 99%

Integrated simian virus 40 sequences in transformed cell DNA: analysis using restriction endonucleases.

Ketner

Kelly²

1976

Proc. Natl. Acad. Sci. U.S.A.

187

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The DNAs from five independent simian virus 40 (SV40) transformants of the BALB/c 3T3 mouse cell line were digested with either the HpaII or the BamHI restriction endonuclease and the resulting fragments were fractionated by gel electrophoresis. The DNA fragments were denatured in situ in the gel and transferred to a membrane filter. Fragments containing viral DNA were detected by hybridization with high specific activity 32P-labeled SV40 complementary RNA (cRNA) synthesized in vitro. Each of the lines yielded a small number of fragments containing SV40 DNA and the fragments from each line were different. This observation shows that the structure of the integrated SV40 DNA and/or its location in the host DNA are different in each line.A fundamental feature of virus-transformed cells is the persistence of viral genetic information (1-12). In the cases which have been carefully studied to date, the persistent viral genes appear to be covalently linked to host genes (i.e., "integrated"), and expression of one or more integrated viral genes seems to be required for the maintenance of the transformed phenotype (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27). Little is known at present about the detailed structure of integrated viral DNA or about the molecular mechanisms involved in integration. This is largely due to the fact that viral DNA represents a very small fraction (about 10-6) of transformed cell DNA.In the past several years a good deal of information has accumulated about the structure and function of the genome of the papovavirus simian virus 40 (SV40). Much of this information has been acquired using site-specific bacterial restriction enzymes to dissect the viral DNA and construct detailed physical maps (28). In this paper we show that similar techniques can be used to study the structure of integrated SV40 DNA. Our approach is analogous to that previously employed by Botchan and McKenna (29) Cells and Cell DNAs. BALB/c 3T3 (A317) cells were obtained from G. Todaro and propagated on Eagle's minimum essential medium (MEM) with 20% fetal calf serum. A number of independent SV40-transformed cell lines were derived from these cells by a method similar to that of Todaro and Green (33). BALB/c 3T3 cells growing in 1.6 cm microwells (Linbro) were infected with SV40 (small plaque, strain 776) at a multiplicity of 0.5 PFU per cell after the cells had reached approximately 90% confluence. Twelve to 24 hr after infection the cells were trypsinized and the contents of each microwell were plated in a 100 mm petri dish. Thereafter, the cells were maintained on Eagle's minimum essential medium supplemented with 10% fetal calf serum (MEM-10) which was changed at weekly intervals. Colonies of transformed cells recognizable by their density and altered morphology were first visible at about 2 weeks after infection. The average number of transformed colonies per dish was about three. No transformed colonies were observed in five control dishes containing mock-infected cells. At 3-4 weeks after inf...

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Ètude du mècanisme de l'induction chez des cellules de hamster syrien transformèes par le virus SV40 I. Propriètès d'une lignèe cellulaire clonale

Cited by 95 publications

References 25 publications

Two forms of simian-virus-40-specific T-antigen in abortive and lytic infection.

Two forms of simian-virus-40-specific T-antigen in abortive and lytic infection.

Binding sites for monoclonal antibodies and for mRNPs on SV40 large T‐antigen determined with a cleavage map

Integrated simian virus 40 sequences in transformed cell DNA: analysis using restriction endonucleases.

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