The genetic information of all higher organisms resides in their chromosomes; therefore, the molecular mechanisms by which chromosomes are replicated and transcribed are fundamental to our understanding of the proliferation and differentiation of all cells. Simian virus 40 chromosomes utilize cellular host processes to accomplish these functions and thus, are apropriate but relatively simple model systems for study. The properties of virally expressed proteins as they relate to the control of viral chromosome functions are also of interest. This proposal considers the question of how viral chromatin structure, specifically T-antigen: SV40 chromosome complexes, afffects the replication and expression of its genetic information. The use of immunoprecipitation techniques utilizing several monoclonal anti-T antibodies to purify T-containing mature, replicating and transcribing SV40 chromosomes, in vivo and in vitro replication and transcription assays to characterize function along with techniques such as electron microscopy, restriction endonuclease digestion, hybridization assays, sedimentation fractionation and gel electrophoresis to identify, characterize and quantitate T-containing DNA species will clarify the role of T-antigen with respect to in vivo SV40 chromosomal replication and transcription. Nuclease digestion and specific labeling studies of T-containing viral chromosomes will be used to determine whether chromatin phasing directs biological events at specific DNA sequences. In addition, the chemistry and functional properties of a highly reactive, aminesensitive ester site in SV40 T-antigen will be examined with respect to viral function. Similar sites recently described in several plasma proteins are not only critical for function of the native molecules, but are, upon activation, capable of covalent interaction with receptor molecules.
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