The E1 region of the group C adenoviruses is able to induce oncogenic transformation of rodent cells in culture and is also necessary for the efficient transcription of other viral genes. The proteins of the E1a transcription unit have been shown to play a pivotal role in both of these activities. In order to better understand the regions of the E1a proteins required for transformation and viral growth a program of random insertion mutagenesis was undertaken by D. S. Bautista to help identify important domains. The 39bp linker insertion oligonucleotides were designed to encode a 13 amino acid in frame insertion in one orientation or a closed reading frame insertion in the opposite orientation. As well the insertion mutants could be collapsed by digestion with BamHI to generate a 2 amino acid in frame insertion. Using this method all three types of mutants were generated at 18 different sites within the E1a coding sequences. The purpose of this project was to assay these E1a mutants for the ability to cooperate with EJb in the transformation of primary baby rat kidney cells using DNA-mediated transfection and also to 'rescue' the mutants into infectious virus and study the ability of the mutant virus to replicate on HeLa cells. Results showed that only closed reading frame mutations upstream of the unique region were completely negative for transformation. Conversely, 13aa or 2aa insertions outside of the unique region impaired but did not abolish transformation. However 8 of the 9 insertions in the unique region of the 289R protein of E1a were defective for transformation of BRK cells in E1a plus E1b DNA-mediated transformation assays. To determine whether the unique region played a direct role in the transformation process or if it had an indirect role such as the transactivation of E1b the transformation assay was carried out using selective media that allow~ growth of foci transformed by E1a alone. Results from this assay showed that the unique region mutants combined with E1b were able to transform with about the same efficiency as E1a alone. The transformation assay was also performed using the unique region mutants in an E1a only background cotransfected with the EJ-ras oncogene. Results from these experiments showed that the unique region mutants in an E1a only background could cooperate with ras in transformation as well as wild-type E1a. From these results it was concluded that the unique region does not play a direct role in transformation by E1 but is required for the efficient expression of E1b which results in wild type transforming frequencies. The actual role of E1b in transformation is unknown. The insertion mutants were also 'rescued' back into infectious virus to study their effect on the ability of the viruses to replicate. The results showed that only viruses in which the unique region was either eliminated or altered were defective for growth on HeLa cells. Transactivation assays carried out by D. Bautista showed results which were comparable to results of infectivity assays. Taken together the results suggest that only the unique region is required for transactivation and only the ability of E1a to transactivate is of importance for viral replication in HeLa cells. / Thesis / Master of Science (MS)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/23133 |
| Date | 10 1900 |
| Creators | McGrory, Joel |
| Contributors | Graham, Frank, Biology |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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