Faculty Bibliography

Cells transformed by Polyoma virus (Py) can undergo a high rate of excision or amplification of integrated viral DNA sequences, and these phenomena require the presence of homology (i.e., repeats) within the viral insertion as well as a functional viral large T antigen (T-Ag). To determine whether the main role of large T-Ag in excision and amplification was replicative or recombination-promoting, we studied transformed rat cell lines containing tandem insertions of a ts-a Py molecule (encoding a thermolabile large T-Ag) with a deletion of the origin of viral DNA replication. Culturing of these cells at the temperature permissive for large T-Ag function did not result in any detectable excision or amplification of integrated Py sequences. We then introduced into origin-defective lines a recombinant plasmid containing the viral origin of replication and the gene coding for resistance to the antibiotic G418. All G418-resistant clones analyzed readily amplified the integrated plasmid molecules when grown under conditions permissive for large T-Ag function, showing that these cells produced viral large T-Ag capable of promoting amplification in trans of DNA sequences containing the Py origin. These observations strongly suggest that Polyoma large T antigen promotes excision or amplification of viral DNA by initiating replication at the integrated origin, providing a favorable substrate for subsequent recombination

We examined the transforming properties of polyomavirus DNA molecules which can produce a functional large T-antigen but which are cis defective for viral DNA replication. The inability of these molecules to replicate results from the deletion of sequences comprising the viral replication origin. We found that even in the presence of a functional large T-antigen, transformation of rat cells by these viral DNAs was greatly reduced when compared with replication-competent parental DNA, and cells transformed by origin-minus mutants generally contained the integrated viral DNA in a nontandem arrangement. Therefore, polyomavirus large T-antigen promotes the establishment of transformation and tandem integration by interacting with the viral origin of DNA replication. This indicates that viral DNA synthesis is directly involved in these processes

We have analyzed the regulation of transcription of integrated SV40 DNA and of five cellular genes during the cell cycle of two lines of SV40 transformed mouse 3T3 cells. These cells (ts SV3T3) are temperature sensitive for the expression of the transformed phenotype and at the nonpermissive temperature (39 degrees C) become arrested in G1 at low serum concentrations. SV40 specific RNAs are not detected either in the nuclear or in the cytoplasmic poly(A+)RNA of quiescent cells, suggesting control at the level of transcription. After serum stimulation, however, viral transcription increases and reaches its maximum during S-phase. The expression of a group of selected housekeeping genes has received parallel analysis to determine whether other cellular genes, beside the integrated SV40, are shut off in G1 arrested cells or are expressed in restricted periods of the cell cycle. We have found that, while the mRNAs for collagen, adenosinphosphoribosiltransferase (APRT) and the mouse major histocompatibility complex (H2) are present throughout the cell cycle, the genes coding for the multifunctional protein CAD and dehydrofolate reductase are cell-cycle regulated

We have studied the pattern of transcription of the early region of polyoma virus DNA after the onset of the late phase of lytic infection of mouse cells. Following initiation of viral DNA synthesis, the early/late switch is accompanied not only by efficient production of late mRNAs but also by the appearance of previously unidentified early-strand RNAs which have certain structural features in common with the classical early mRNAs. Stable poly(A)+RNAs have been identified by blot analysis and S1 nuclease mapping that are not detected early during infection or in polyoma virus-transformed cells. One group consists of transcripts whose 5' ends map 150-200 nucleotides upstream from the major early 5' ends (at positions 148 and 153 on the polyoma virus genome) but whose splicing pattern and poly(A) addition sites are indistinguishable from those of mRNAs produced early in infection. The 5' exons of these early region transcripts contain an open translational reading frame that extends from nucleotide positions 5,255 to 124 and is capable of encoding a basic protein of 53 amino acids. Transcription of these RNAs does not appear to be negatively regulated by large tumor antigen. A transcript of 1,800 nucleotides appears to map predominantly between 93 and 26 map units and does not contain sequences present in the early mRNA 5' exons. These data suggest that, after the onset of polyoma DNA replication, the activation of new early-strand promoters leads to the expression of previously untranscribed viral DNA sequences

The frequency of sister chromatid exchange (SCE) was determined in rat fibroblasts transformed by wild-type polyoma virus or by a mutant temperature sensitive for viral large tumor antigen function (ts-a). Elevated SCE frequencies were observed in two wild-type transformed cell lines growing at 37 degrees and in four ts-a-transformed lines upon growth at the permissive temperature for large viral tumor antigen (33 degrees). The increase in SCE frequency in ts-a-transformed cells at 33 degrees was reversed by growth at 39 degrees (nonpermissive for T-antigen function). An increase in SCE at 33 degrees was not observed in untransformed cells or in a ts-a-transformed cell line which makes a defective large viral tumor antigen. These results suggest that large viral tumor antigen can induce SCEs. Since large viral tumor antigen is also responsible for amplification of integrated viral DNA sequences (4), we tried to correlate this phenomenon with the increased SCE frequency. However, increasing SCE artificially by growing cells in the presence of 12-O-tetradecanoylphorbol-13-acetate did not result in amplification of integrated viral DNA in the absence of large viral tumor antigen function. Thus, there is no simple causal relationship between increased SCE and amplification

We have studied transcription of integrated viral DNA sequences in a variety of ts-a polyoma virus-transformed rat cells and cured revertants (which had undergone excision of variables amounts of integrated viral DNA) to characterize the structure of viral mRNA's produced in these lines under conditions in which integrated DNA is stable. Our results indicate that cells containing intact early region sequences, either in single-copy or tandem insertions, produce mRNA's indistinguishable from those observed early in lytic infections; sequences complementary to the polyoma late region were not transcribed from integrated viral DNA. Cured revertants no longer encoded full-length early mRNA's , but produced viral transcripts whose 3' ends mapped at an alternative early region polyadenylic acid attachment site at 99 map units or extended in to flanking host sequences. The phenotype of these revertant cells correlated with the abundance of these transcripts, suggesting that the transforming function(s) of polyoma virus controls the cellular phenotype in a dose-dependent manner. Unexpected results were obtained from studies of cells containing tandem repeats of defective viral DNA in which the polyadenylic acid attachment signal at 25.8 map units and surrounding sequences were deleted. In these cases, polyadenylated mRNA's were observed that contained sequences complementary to the early strand of the polyoma late region. These mRNA's (some larger than 8 kilobases) originated at the viral early promoter, extended into the late region, and continued into the early region of the contiguous repeat in the tandem. The multimeric mRNA's produced contained defective early regions in tandem with late region sequences. S1 analysis indicated that whereas the 5' early region sequences of readthrough transcripts were spliced in the usual manner, internal early region repeats were either unspliced or used only one of the small early region splices. When deletions in the viral readthrough transcripts were observed. This suggests that sequences nearby the AAUAAA sequence at 26 map units may control transcription termination of the polyoma early region