VIROLOGY 133, 341-353 (1984) Transformation by Purified Early Genes of Simian Virus 40 LONG-SHENG CHANG, MARY M. PATER, NANCY I. HUTCHINSON, AND GIAMPIERO DI MAYORCA’ Deportment of Microbidogy, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103 Received September 9, 1983; accepted Decewher 9, 198.3 A rapid soft-agar assay using baby hamster kidney (BHKn ~1.13) cells has been developed to establish the functional roles for the large T and small t antigens of SV40 in trans- formation. Plasmids expressing either large T or small t antigens of SV40 have also been constructed and these plasmids have been used separately or in combination for trans- formation. A large T clone, pD3-05, containing a deletion in the small t-specific coding region rO.5840.54 map units (mu)], transformed a low-background subclone of baby hamster kidney (BHKai ~1.13) cell line and Flll rat fibroblasts to anchorage independence at a low level (lo-20 and l%, respectively, of an early region clone from wild type [WT], pW2). A WT-derived small t clone, pW2-t, containing a deletion in the large T-specific coding region (0.373-0.169 mu), did not transform Flll cells, but transformed BHKzl cells at a very low level (about 2% of pW2). Another WT-derived small t clone, pW2-t/Bl, containing a larger deletion in the large T-specific coding region (0.512-0.169 mu), did not transform either BHKzl or Flll cells. However, cotransformation with pD3-05 clone and pW2-t or pW2-t/B1 clone increased the frequency of transformation to about the same level as that of pW2. The ability of the small t clones to enhance the transformation efficiency of the large T clone was not due to recombination between the two plasmids, since cotransformation with pD3-05 and a small t clone without the polyadenylation [poly(A)] signal sequence from WT, pW-t8, did not increase the frequency of transformation. When the frequency of transformation was determined by the focus assay using Flll cells, pD3-05 transformed as well as pW2 Also, cotransformation with pD3-05 and pW2-t/I31 did not increase the frequency of focus formation. Therefore, the small t antigen was not required for this morphological transformation. INTRODUCTION The early region of the simian virus 40 (SV40) is known to be required for malig- nant transformation. Two proteins, a 94,000-dalton large tumor (T) antigen and a 1’7,000-dalton small tumor (t) antigen, are encoded by this region (for references see Tooze, 1981). Both of these tumor an- tigens have been shown to be involved in transformation. However, their precise roles in initiation and maintenance of viral transformation have not been defined. Large T antigen has been shown to be involved in the initiation of stable trans- formation by SV40 since transformation ’ Author to whom requests for reprints should be addressed. by tsA mutants, which produce a temper- ature-sensitive (ts) large T antigen, fails to occur at the nonpermissive temperature (Kimura and Dulbecco, 1973; Tegtmeyer, 1975). Large T antigen also appears to be required continuously for the maintenance of transformation since tsA mutant- transformed clones of rat, hamster, rabbit, or mouse cells may lose various trans- formed characteristics when grown at the nonpermissive temperature (Tooze, 1981). We (Bouck et aL, 1978) and several other groups (Feunteun et aL, 1978; Sleigh et aL, 1978; Fluck and Benjamin, 1979; Frisque et aL, 1979; Martin et aL, 1979a, b; Pipas et ah, 1979; Seif and Martin, 1979a, b; Stein- berg and Pollack, 1979) previously reported that viable small t mutants of SV40 with deletions between 0.59 and 0.54 map units 341 0042-6822/84 $3.00 Copyright 0 IS&1 by Academic Press, Inc. All rights of reproduction in any form reserved