The early region 1a oncogene of adenovirus 5 codes for proteins that can activate transcription of viral and cellular genes. This study describes the construction of three deletions and one point mutation that together span the entire coding region of the second exon of E1A. The exon-2 mutants were tested for their ability to activate transcription from the adenovirus early region 3 promoter (E3) in transient expression assays. Dl1116 (dl aa 205-221) did not affect transactivation of E3 in pKCAT-23. Sub1117 (dl exon-2 aa) and dl1115 (dl aa 188-204) were unable to activate transcription. Pm1131 (SER-219 to stop) had a reduced transactivating efficiency but was still able to stimulate transcription. These results define the 3' boundary of a transactivation domain on the E1A proteins as being between positions 188 and 204. Results obtained in our lab define the 5' boundary as being between 138-147 (Jelsma et al., 1988). The mutants that could not transactivate were tested for their ability to block wildtype E1A transactivation of the E3 promoter in assays similar to those described by Glenn and Ricciardi (1987). Dl1115 and sub1117 appeared to block transactivation by WT E1A. In transient expression assays, the fatty acid sodium butyrate was found to stimulate transcription of the CAT gene, when added to the medium of HeLa cells transfected with pKCAT-23. This suggests that sodium butyrate is transactivating the Ad 5 E3 promoter. / Thesis / Master of Science (MS)

The purpose of this study was to assess the effect of early region 1 functions of Ad5 on the ability of viruses containing a selectable marker in early region 3 (E3) to transform mammalian cells. To do this I have constructed and characterized five recombinant viruses containing the thymidine kinase (tk) gene from Herpes Simplex Virus type 1 (HSV1) inserted in E3. The biochemical transformation assay performed using these recombinant viruses allowed the separation of the selection process (incorporation and expression of tk) from the requirement for expression of E1 functions. The method of isolating the desired recombinant viruses was in vivo recombination following mixed infection of 293 cells. The parental viruses used were: hr 1 which expresses a truncated version of the E1a 243R product lacking amino acids 166 to 2437 pm975 which fails to express the E1a 289R product d1312 from which the majority of the E1a coding region has been deleted: hr6 which fails to express a wild type E1b 496R protein; and wild type Ad 5. Each coinfection was done with d1E1,3tk, a previously constructed recombinant virus (Haj-Ahmad and Graham, 1986). Using the resulting recombinant viruses, semipermissive tk- Rat 2 cells were infected and selected for conversion to the tk+ phenotype, as well as being assayed for viability post infection. Comparisons were made of tk transformation frequencies with and without correction for differential cell viability measured after infection with different viruses. Correction for differential cell viability greatly reduced the differences in transformation frequencies observed directly. However hr1tk remained able to induce tk+ transformation at a significantly greater frequency than hr6tk, pm975tk, or Ad5tk. The mutants d131,2tk and d1E1,3tk gave statistically indistinguishable results corresponding to an intermediate level of transformation, while hr6tk, pm975tk, and Ad5tk were grouped together as being least efficient at transformation. The infected Rat2 cell viability assays provide evidence of a correlation involving expression of the early region la (Ela) 289 residue product, efficient viral DNA replication, and cell death. Recombination frequencies obtained during the isolation of the recombinant viruses varied greatly depending on the combination of infecting parental viruses. The following factors appeared to affect recombination frequency: 1. the input ratio of the coinfecting viruses; 2. interference in the replication of d1E1,3tk relative to the other virus present; and 3. the presence of small numbers of mismatched base pairs (seven) near the left terminus of some of the viruses used in coinfection with d1E1,3tk. / Thesis / Master of Science (MS)