Polyacrylamide gel electrophoresis and tryptic peptide fingerprint analysis of the proteins made in a cell-free system derived from L-cells and immunoprecipitated with simian virus 40 (SV40) anti-T serum demonstrated that both SV40 large-T and small-t antigens are synthesized in vitro in response to mRNA isolated from productively infected CV1 cells. Sucrose density centrifugation in gradients containing 85% formamide showed that the mRNA's for both forms of T-antigen sediment at about 17.5S,

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... th the mRNA for small-t sedimenting marginally, but reproducibly, ahead of the mRNA for large-T. Hybridization experiments using restriction endonuclease fragments Hae III-E and Hind 11/111-B showed that all fractions active in the cell-free synthesis of both forms of Tantigen hybridized equally to both fragments. This suggests that the mRNA's for SV40 T-antigens are at least partly virus coded and that the bulk of the early SV40 mRNA contains sequence information from both ends of the early region. The data are consistent with the suggestion that the large-T mRNA is spliced. SV40 complementary RNA (the product of transcription of SV40 DNA using Escherichia coli RNA polymerase) was also translated in the L-cell system and gave two families of polypeptides which specifically immunoprecipitate with anti-T serum. One family (the small-t family) includes a polypeptide indistinguishable by gel electrophoresis and tryptic peptide fingerprinting from small-t isolated from cells. The other family (the 60K family) has a major component with molecular weight approximately 60,000 and includes other polypeptides with molecular weights ranging from approximately 14,000 to about 70,000. The 60K family has peptides in common with large-T but not with small-t. Together, the peptides of the small-t and 60K families account for virtually all of the methionine peptides of SV40 large-T. We conclude from these results (i) that small-t is probably entirely, and large-T at least predominantly, virus coded; (ii) that the small-t and 60K families represent the translation products of two different portions of the early region of SV40 DNA (approximately 0.65 to 0.55 map units and 0.54 to 0.17 map units); and (iii) that although most, if not all, of the large-T and small-t peptides are present in the cell-free product, some feature of sequence arrangement of SV40 complementary RNA prevents the translation of full-length large-T and results instead in the synthesis of fragments. We suggest that the absence of a splice in the complementary RNA is responsible for this result. Simian virus 40 (SV40) T-antigen (T-Ag) can be isolated from SV40-induced tumor cells, from SV40-transformed cells, and from productively infected cells, both early and late after infection (3, 35) . T-Ag has been implicated as an important control element in the initiation of viral DNA synthesis (5, 49), in the transcription of viral DNA (2, 51), and in the initiation and maintenance of transformation (4, 23, 24, 50). When extracted from productively infected cells or from SV40-transformed cells under conditions which minimize proteolytic degradation, at least two forms of T-Ag are detected (26, 33). These have apparent molecular weights in the range 90,000 (90K) to lOOK and 15K to 20K and are referred to as large-T and small-t, respectively. Fingerprinting data show that small-t contains a subset of the methionine tryptic peptides present in large-T, but that in addition small-t also contains two methionine peptides not present in large- T (26, 32, 33, 48). Presumably, therefore, large-T and small-t are coded for in part by a common DNA sequence. Early in the productive infection cycle, a 19S cytoplasmic polyadenylic acid [poly(A)]-containing SV40 RNA is detected. This hybridizes with the E strand of the region of SV40 DNA extending counter-clockwise from about 0.67 to 54 on May 9, 2020 by guest http://jvi.asm.org/ Downloaded from (Sigma) (pH 7.5), 150 mM NaCl, 5 mM EDTA (pH 7.0), 0.05% Nonidet P-40 (NET buffer), and 1 mg of bovine serum albumin per ml. A 10-,ld sample of either VOL. 28, 1978 on May 9, 2020 by guest