Roles of the simian virus 40 tumor antigens in transformation of Chinese hamster lung cells: studies with simian virus 40 double mutants

We have constructed a dl8841tsA58 double mutant and a t+T-early-region deletion mutant and have used these mutants to study the roles of the simian virus 40 tumor antigens (T and t) in transformation. Our major conclusions are that (i) although the mutant tsA58 is not temperature sensitive for the maintenance of transformation, the d18841tsA58 double mutant is; (ii) small t antigen can provide at least one, but not all, of the functions required for the maintenance of transformation; and (iii) at least two different functions are required for the maintenance of simian virus 40 transformation.To examine the roles that the simian virus 40 (SV40) large T and small t antigens play in initiating and maintaining cellular transformation, we have constructed a double mutant that combines the temperature-sensitive large T antigen mutation of mutant tsA58 (19) with the small t antigen mutation of mutant d1884 (14). We present evidence here that the double mutant is temperature sensitive for both initiating and maintaining transformation but that the parental mutant, tsA58, is temperature sensitive only for initiating transformation. From these results we conclude that the SV40 small t antigen can perform at least one of the functions required for maintaining cellular transformation.The tsA mutants of SV40 were selected because of their lytic behavior, that is, because their replication is impaired at high temperature.Because their temperature-sensitive mutations map in the coding region for large T antigen, tsA mutants have been very useful for identifying and studying large T antigen functions required for the lytic growth of SV40 (20). In contrast, they have not been as useful for studying the involvement of large T antigen in SV40 transformation.As

supporting

confidence: 78%

A simian virus 40 dl884/tsA58 double mutant is temperature sensitive for abortive transformation

Sompayrac¹,

Danna²

1983

“…Both proteins influence viral transformation. Analysis of deletion mutants suggests that the small t antigen is required for the efficient initiation of transformation when growth is restricted soon after infection (5, 11,28,42) but plays a miniimal role in the subsequent maintenance of the transformed phenotype either in vitro or in vivo (22,38). Large T antigen, on the other hand, may play a continuingly active role in transformation.…”

mentioning

confidence: 99%

Separation of lytic and transforming functions of the simian virus 40 A region: two mutants which are temperature sensitive for lytic functions have opposite effects on transformation

Pintel¹,

Bouck²,

Mayorca³

1981

Thirty-six of 40 rat cell clones transformed to anchorage independence at low multiplicity of infection by simian virus 40 tsA58 were heat sensitive for continued expression of the transformed phenotype. tsA1499 is an 81-base-pair deletion at 21 map units which is like tsA58 in that it is also heat sensitive for lytic growth, belongs to the A complementation group, and produces rat cell transformants which contain a thermolabile T antigen. Unlike tsA58, however, tsA1499 generated rat cell transformants efficiently at the temperature at which it was lytically defective, and 10 of 17 clones transformed by tsA1499 were cold rather than heat sensitive for the continued maintenance of the transformed phenotype. The lytic and transforming activities of the A region thus appeared to function independently in mutant tsA1499.

“…Transformed cells. The N-and A-type tsA209, SV40-transformed rat cells and the tsA209 and tsA209 viable deletion double-mutant-transformed Chinese hamster lung (CHL) cells have been described previously (20,27,28).…”

Section: Methodsmentioning

confidence: 99%

“…The gels were then treated with 0.2 M NaOH-0.6 M NaCl for 45 to 60 min, neutalized with 1 M Tris-hydrochloride (pH 7.3)-0.6 M NaCl for 45 min, and equilibrated with 6x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate) for 30 to 45 min with gentle rotation. Blotting on nitroceliulose membrane filter paper (0.45 pm; type HAWP; Millipore Corp.) was performed downward (gel at the top) for approximately 3 h and upward for approximately 20 h, soaking the 3 MM paper bridge in 6x SSC. The nitrocellulose paper was rinsed for 15 min in 6x SSC and air dried.…”

Section: Methodsmentioning

confidence: 99%

Integration of the simian virus 40 genome into cellular DNA in temperature-sensitive (N) and temperature-insensitive (A) transformants of 3T3 rat and Chinese hamster lung cells

Chepelinsky¹,

Seif²,

Martin³

1980

Self Cite

phenol and once with chloroform-isoamyl alcohol (24: 1, vol/vol). The extracts were then dialyzed, incubated with 0.1 mg of RNase A (Boehringer) per ml, incubated again with proteinase K (0.1 mg/ml), reextracted, and dialyzed, as described by Botchan et al. (4). The molecular weight of the DNA was checked by agarose gel electrophoresis. Restriction enzymes, digestion, and agarose gels. Cellular DNA (25 ,Lg) was digested for 2 to 5 h with restriction enzymes by using the incubation media specified by New England Biolabs or Bethesda Research Laboratories, depending on the source of the enzyme. After incubation, 20 mM EDTA and 0.1 to 0.2 M NaCl (final concentrations) were added. The samples were extracted once with phenol and once with chloroform-isoamyl alcohol (24:1). Sodium ace-184