The DNA tumor virus, SV40, has two transcription units that run in opposite directions from a common control region. One is expressed early during lytic infection, the other late. An in vitro transcription system directs synthesis of both early and late RNA, initiating transcription at correct sites. Components of the in vitro system include RNA polymerase II and several additional factors, one of which, SP1, binds to a specific site in SV40 DNA, in a common upstream control region between the early and late startpoints. Our hope is to exploit the SV40 system as a model for understanding the influence of upstream control sequences on p romoter activity, with special emphasis on the complex interactions involved in activation of late viral transcription. This will involve further purification of the enzymes involved in transcriptional initiation, and detailed characterization of the multistep promoter selection and initiation reaction. Possible late-specific protein factors will be sought, using the smae approaches that led to discovery of Sp1. New mutants will be constructed, targeted to areas of interest, using effecient methods of site-directed in vitro mutagenesis, and their transcription will be measured in vitro and in vivo. It is anticipated multiple sequence elements will be involved in late transcription, and special attention will be given to the effects of spatial rearrangements. In addition, the role of the SV40 oncogene product, large T antigen, in induction of late transcription will be explored. Final aspects of the project include characterization ofthe interaction between the factor required for SV40 transcription and selected host-cell promoters, and an attempt to find other factors, which may operate by a mechanism akin to that of SP1, and that are involved in cell-type specific expression of immunoglobulin genes.
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