BIOCHEMISTRY AND GENETICS OF MAMMALIAN/APRT

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We shall continue invistigating the mechanism of mutation at the APRT locus in Chinese hamster, mouse, and human cell lines. Analysis of mutants will be done by DNA sequencing, and comparative studies with the mutated proteins. Among mutagenic effects, we are interested in studying the effect of aneuploidizing agents, which have been reported to cause a high frequency of mutations at this locus. In vivo mutagenesis will be paralleled by studies with in vitro mutagenesis. Regulation of transcription and translation of 'house keeping' genes will be studied using APRT as a model. This will be done by a combination of deletion mapping in the promoter region, CAT assays with promoter segments of the APRT gene linked to the CAT gene, and by analysis of DNA binding sites on the gene. The effect of enhancer elements and viral LTRs on APRT expression will be examined. We shall analyze the effect of placing the APRT gene under hormonal control on transcription, translation, and nucleotide pools. Comparative analysis of phosphoribosyltransferases from E. coli, Drosophila melanogaster, and mammalian sources show extensive aminoacid homology. We shall sequence the yeast APRT gene, and compare the nucleotide and aminoacid sequences. Using the approach of comparative sequence analysis, in vitro mutagenesis, and monoclonal antibody interaction, we shall define the functional domains of the protein.