We analyzed the sequences involved in vivo in the initiation of simian virus 40 (SV40) late transcription occurring in the absence of both SV40 origin sequences and T antigen. The constituent elements of the SV40 late promoters have already been the subject of extensive studies. In vitro studies have resulted in the description of two putative domains of the late promoters. The first domain consists of an 11-base-pair (bp) sequence, 5'-GGTACCTAACC-3', located 25 nucleotides (nt) upstream of the SV40 major late initiation site (MLIS) (J. Brady, M. Radonovich, M. Vodkin, V. Natarajan, M. Thoren, G. Das, J. Janik, and N. P. Salzman, Cell 31:624-633, 1982 The major regulatory sequences of simian virus 40 (SV40), controlling both early and late transcription as well as viral DNA replication, are clustered within a 400-base-pair (bp) sequence of DNA. This region has been the subject of several investigations aimed at characterizing the component sequences of the early and late promoter elements. These studies have revealed this area to be a complex mosaic of cis-acting control elements, some of which are unique to either the early or late promoter, while others appear to be common to both. The early promoter is composed of at least three spatially distinct elements: (i) the TATA box; (ii) a G-C-rich region; and (iii) the enhancer element. The TATA box, which is contained within a 17-bp A-T-rich sequence, dictates the position of the major early initiation sites (at nucleotides [nt] 5230 to 5237, 21 to 28 bp downstream from the TATA box) used preferentially before viral DNA replication (3, 8, 17). The G-C-rich region contains six copies of the sequence 5'-PyPyCCGCCC-3' in two direct repeat sequences of 21 bp and a third, almost perfect repeat of 22 bp. This region is an essential element of the early promoter and is known to contain the binding sites for the cellular transcription factor Spl (12,15). A certain amount of evidence suggests that this element functions in a bidirectional manner (15,24). The third element, the enhancer, augments in vivo transcription from the early promoter (3,10,16,19,25,32,34). This element, which includes two 72-bp direct repeats and 20 bp upstream from these repeats (relative to the early start sites) is so called because it can stimulate in vivo transcription from heterologous * Corresponding author.promoters irrespective, to a large extent, of its position or orientation relative to the promoter.The component parts of the SV40 late promoter are less well understood. In vitro studies have resulted in the description of two putative domains of the late promoter. The first domain, defined by Brady et al. (7), consists of an 11-bp sequence, 5'-GGTACCTAACC-3', located 25 nucleotides upstream of the major SV40 late cap site. Base substitution within this transcriptional control sequence affects the in vitro transcriptional efficiency of the SV40 major late initiation site (MLIS). The second domain, described by Brady et al. (nt 75 to 95) (6) and by Hansen and Sharp (nt 72 to 114) (21), is l...

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Simian virus 40 (SV40) transformed V 11 F 1 clone 1 subclone 7 rat cells (subclone 7) do not synthesize normal-size large T antigen (Mr, 90,000); instead, they produce a 115,000 Mr super T antigen (115K super T antigen). This super T antigen is SV40 virus coded, and its synthesis results from rearrangement and amplification of integrated viral DNA sequences in subclone 7 (May et al., Nucleic Acids Res. 9:4111-4128, 1981). In this study the functional activities of 115K super T antigen were compared with the functional activities of SV40 large T antigen. Transfection experiments were performed with (i) cosmid SVE 5 Kb and plasmid pSVsT, both containing the super T antigen gene and (ii) plasmids pSV1 and pSV40, both containing the large T antigen gene. Transfection of pSVsT DNA or SVE 5 Kb DNA into secondary cultures of rat kidney cells induced the formation of transformed cell foci with an efficiency that was about 50% of the efficiency of pSV1 DNA or pSV40 DNA. Concomitant with the transforming activity, two other activities were also retained by super T antigen, namely, the ability to enhance the level of host cellular protein p53 and the capacity to bind to p53. In contrast, pSVsT and SVE 5 Kb DNAs were markedly deficient in the capacity to support tsA58 DNA replication in CV1-P cells at a nonpermissive temperature (41°C), as shown by cotransfection experiments. The yield of virus produced in these experiments was 400-fold less than the yield obtained in parallel experiments with pSV40 or pSV1. However, SVE 5 Kb and pSVsT have a functional SV40 replication origin, as shown by their efficient replication in COS 1 cells which provided functional large T antigen. Super T antigen also possesses a specific affinity for sequences of SV40 viral origin. Our results suggest that under certain conditions, evolutionary changes in T antigen take place and that these changes could be restricted to the phenotypic requirement of maintaining a structure that is able to induce cell transformation, to form a complex with p53, and to enhance the cellular level of p53. Therefore, there appears to be a close relationship among the activities of T antigen involved in transforming cells, in binding to p53, and in enhancing the p53 cellular level. Moreover, this set of activities appears to be separable from the replicative ability of T antigen, based on the observation that 115K super T antigen is markedly defective for initiating viral DNA synthesis. Many simian virus 40 (SV40)-transformed cell unusual forms of large T antigens. However, lines synthesize unusual forms of SV40 T antisome SV40-transformed cell lines express super gens that are structurally related toSV40 large T T antigens in the absence of any detectable antigen and are immunoprecipitated by antiamount of normal-size large T antigen (Mr, SV40 tumor serum and by antiserum or mono-90,000), suggesting that the super T antigens clonal antibodies specific for large T antigen. present in these cell lines are able to carry out These unusual antigens are either larger or som...

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We have previously cloned the gene encoding a 115,000-Mr super T antigen (115K super T antigen), an elongated form of the Simian virus 40 large T antigen, originating from the rat cell line V 11 Fl clone 1, subclone 7 (May et al., J. Virol. 45:901-913, 1983). DNA sequence analysis has shown that the 115K super T antigen gene contains notably an in-phase duplication of a sequence located in the region of tsA mutations. We have also shown that the 115K super T antigen gene is able to induce the formation of transformed foci in transfected rat cells. After rat cell cultures were transfected with the cloned gene encoding 115K super T antigen, we obtained a large number of transformants as reported in this paper. In these transformants, we detected a very high frequency of new T antigen variants, as shown by immunoprecipitation of the cell extracts with anti-simian virus 40 tumor serum followed by electrophoresis in sodium dodecyl sulfatepolyacrylamide gels. Based on these results and all of the data presently available, it appears likely that the input plasmid or cosmid DNAs containing the cloned gene were first subjected to recombination events that yield new variant T antigen genes before these recombinant genes become integrated. The new variant T antigens observed in the transformants were predominantly those comigrating with normal-size large T antigen. In fact, these latter variants appeared to be indistinguishable from wild-type large T antigen as judged by restriction mapping by Southern blotting of the total genomic DNA of the transformants. Models of intermolecular or intramolecular homologous recombination occurring between or within the input plasmid or input cosmid DNA molecules are proposed to account for the formation of such revertants.Large chromosomal rearrangements have been found in many living organisms, notably in eucaryotes. These changes in chromosome structure (recombinations) include rearrangements, amplifications, deletions, and translocations of DNA segments. Such recombinations are involved (i) in the genome evolutiort (11), (ii) in the rearrangements of mammalian antibody genes during lymphocyte development (36), (iii) in the changes, mediated by mobile elements, in gene order and expression (36), and (iv) in the genesis of B cell-derived tumors in mice and humans (19).Similarly, after infection by simian virus 40 (SV40) or its DNA transfection into cultured mammalian cells, the SV40 genome frequently undergoes mutatibns and recombihations in these cells (5,30,38,41,42,48,49).In SV40-transformed rat or mouse cell lines, rearrangements of viral and adjacent cellular sequences occur during or after integration (2,4,9,16,26,28,34). The altered templates encode either shortened forms (truncated T antigens [7]) or elongated forms (super T antigens [8, 23, 25]) of large T antigen. The templates for truncated T antigens are interrupted in the distal exon of the SV40 early region, whereas the templates for super T antigen frequently contain in-phase tandem duplications of the tsA region (23, 25...