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Phelps, Stephanie Fonda

Microbiology and Molecular Genetics

2004

M.S.

Bacterial artificial chromosomes (BACs) can propagate up to 300 kilobases of genomic DNA and have become important tools to study the organization and expression of large eukaryotic genes. Elements that regulate gene expression, such as locus control regions, insulators, promoter and enhancer elements, mRNA splicing, transport, and translation signals may reside long distances from the gene or within the gene itself. In order to study how this variety of complex signals can effect developmental and tissue-specific gene expression, four BACs containing the human tumor suppressor gene TP53 were obtained. The BACs were mapped to chromosome 17p13.1 to determine their gene environment and positions relative to one another. The TP53 BACs were genetically modified by recA-mediated homologous recombination with sacB counterselection. Modifications included the placement of an IRES-hrGFP-pA marker in the 3' untranslated region of TP53 or enhanced green fluorescent protein (EGFP) on the vector backbone of the BAC. Transfection of these modified BACs and subsequent immunofluorescence and Western analysis allowed us to examine how p53 expression from BACs was regulated in response to damage, and to determine if p53 was able to transactivate p53-dependent genes. These studies will help establish conditions for using BACs to restore wild-type levels of gene expression and function, and define the minimal amount of 5' and 3' sequence surrounding the gene necessary for normal regulation.