This work will focus on the proteins and genes involved in the replication of adenovirus DNA. The proteins of interest are the E2b terminal protein, the E2b encoded DNA polymerase and the 72K DBP from region E2a. The DNA encoding these proteins will be moved onto bacterial plasmids which will allow the synthesis of these proteins in bacteria. Once produced, the proteins will be isolated and tested in vitro for their ability to function in adenovirus replication assays. The proteins will also be introduced directly into infected HeLa cells by the method of protoplast fusion. Their ability to compliment mutants defective for DNA replication will be assessed. 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. We will also hook the E2b genes up to the SV40 early promotor and carry out transient expression studies with the purpose of defining the structure of the mRNA encoding the E2b proteins. These genes will then be introduced into HeLa cells on a plasmid containing the E.coli gpt gene, a selectable marker, for the purpose of obtaining E2b expresser cell lines. An attempt will also be made to determine if URF 7, the largest unidentified open reading frame in region E2b is actually expressed into a protein during the infectious cycle. Conditional lethal mutations and deletion mutations will also be made in the E2b genes. These mutations will be characterized for their effect on viral DNA replication and on the virus growth cycle.
|Rekosh, D; Lindenbaum, J; Brewster, J et al. (1985) Expression in Escherichia coli of a fusion protein product containing a region of the adenovirus DNA polymerase. Proc Natl Acad Sci U S A 82:2354-8|
|Smith, H C; Berezney, R; Brewster, J M et al. (1985) Properties of adenoviral DNA bound to the nuclear matrix. Biochemistry 24:1197-202|