This work will focus on the proteins involved in the replication of adenovirus DNA. Specifically, DNA encoding these proteins will be moved onto bacterial plasmids which will allow expression of the viral DNA in bacteria. The proteins of interest are the E2b terminal protein, the E2b encoded polymerase and the 72K DBP from region E2a. These proteins will be synthesized as fusion products containing less than 15 amino acids, and in some cases only 1, from the Beta-galactosidase gene of E. coli or the cro gene of lambda. The microbially synthesized products will be purified and tested in vitro for their ability to function in adenovirus replication assays. Subsequently, specific modifications will be made in the primary structure of these proteins by altering the DNA encoding them. The effect of these alterations on the activity and properties of these proteins will then be tested. Conditional lethal mutations in the adenovirus terminal protein as well as additional mutations within the 120k polymerase gene will also be created in intact virus. These mutations will be characterized for their effect on viral DNA replication and on the virus growth cycle. We will also create antisera against these proteins by utilizing synthetic peptides as well as fragments of the proteins synthesized in bacteria as antigens. These sera will be used to study the synthesis and role of the replication proteins in the infected HeLa cell.
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