Date of Award
Doctor of Philosophy (PhD)
Dr. Philip E. Branton
Human adenoviruses are known to transform rodent cells in culture and these cells are tumorgenic when injected into new born animals. It has been well established that the early region 1 (El) of human adenovirus type 5 is necessary and sufficient for oncogenic transformation. The El region is comprised of two transcription units known as E1A (0 to 4.5% of the genome) and E1B (4.5 to 11.2%), each of which produces multiple species of mRNAs and polypeptides. E1A is also required to activate the transcription of other viral early regions. In the present study anti-peptide sera were used to identify and characterize these viral proteins.
Anti-peptide sera specific for the amino- and carboxy-termini of E1A were raised and these two sera precipitated an identical set of four major polypeptides of 52, 50, 48.5, and 45K and two minor species of 37.5 and 35K. Studies using E1A mutant viruses also revealed that 52, 48.5, and 37.5K polypeptides are derived from the 1.1 kb mRNA, and the 50, 45, and 35K species from the 0.9 kb mRNA of E1A. These sera were also used to identify polypeptides that are associated with E1 proteins. A set of five cellular polypeptides consisting of >250K, 105K (doublet), 68K, and 65K species were found to co-precipitate with E1A proteins under various conditions and the nature of this association was investigated using the anti-peptide sera as well as an E1A-specific monoclonal activity.
Antisera against synthetic peptides corresponding to the both termini of E1B 58K were also raised and used to identfy 58K from wild-type and mutant-infected cells. It had previously been shown that protein kinase activity was associated with 58K. To ask if protein kinase activity was intrinsic to this viral protein several conventional methods were used to purify 58K and the results suggested that such activity may be intrinsic to this viral protein.
The anti-peptide sera were used to purify El proteins. A simple purification procedure using these sera and their corresponding synthetic peptides was developed and highly purified 58K and E1A proteins were obtained. Attempts were made to study protein kinase activity using these purified El proteins, however, the results were inconclusive and it was not possible to unequivocally determine if kinase activity was intrinsic to them.
Yee, Siu-Pok, "Characterization of Human Adenovirus type 5 Early Region 1 Proteins Using Anti-peptide Antibodies" (1985). Open Access Dissertations and Theses. Paper 1163.