Using technology based upon genetic suppressor elements (GSEs), which the applicant has played a large role in developing, Dr. Gudkov wishes to identify cellular factors affecting either expression or function of the p53 tumor suppressor gene. Regulation of transcription of p53 has not received a lot of scientific scrutiny since elevated levels of p53 protein- associated with its growth inhibitory and apoptotic roles is primarily due to conformational changes leading to an increased stability. Using a p53 responsive promoter-Lac Z reporter construct in transgenic mice Dr. Gudkov has shown tissue and developmental stage-specific expression of the Lac Z reporter. This expression correlates directly with p53 mRNA expression. He has further demonstrated that widespread apoptosis in early embryos following DNA damage is correlated directly with p53 mRNA expression and its translation. The applicant intends to determine the regulation of this mRNA expression. Various parameters will be investigated, including the possibility of positive and negative transcriptional factors and mRNA stability. cDNA expression libraries (full length cDNAs and GSEs) with be used to screen for clones affecting p53 mRNA expression. Using the GSE strategy that was used successfully to identify a growth suppressor, p33(ING1), the applicant will screen for clones that induce phenotypes associated with p53 inactivation. In this case GSEs are isolated from retroviral libraries of randomly fragmented cDNA sequences identified through two-hybrid interaction trap procedures using p53 and p33(ING1) proteins as baits. The third major aim will be to attempt to place the position and role of these candidate proteins in the p53 pathway, via overexpression and suppression studies. The expression patterns of the candidate proteins and their chromosome map positions are included in these aims.
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