The regulation of gene expression is a central theme of molecular biology. It has now been found that eukaryotic cells utilize many mechanisms to attenuate messenger RNA levels (Darnell, 1982; Nevins, 1983; 1984). Among these mechanisms is polyadenylation- -a step required for synthesis and stabilization of active mRNA. It has already shown that there is a requirement for genetic sequences downstream of the poly(A) cleavage site (McDevitt et al., 1984; Gil and Proudfoot, 1984; Sadofsky and Alwine, 1984; McLauchlan et al., 1985; Hart et al., 1985a) in addition to the known, conserved AAUAAA sequence (Proudfoot and Brownlee, 1976). I have shown that this sequence may vary, that different sequences can be exchanged (Hart et al., 1985a), and that the sequence is recognized in an in vitro reaction (Hart et al., 1985b). I now plan to fractionate the nuclear extract that recognizes and processes the primary transcript, determine components involved in this process, and isolate intermediate complexes from the in vitro reaction. By determining specific interactions between isolate factors and known poly(A) site sequence mutations, the mechanism of utilization of a poly(A) site can be determined, and this will show us the steps available for regulation. Towards that end, I also plan to use deletion analysis to locate the sequences required for regulation of a specific poly(A) site (the dihydrofolate reductase (DHFR) gene, Leys and Kellems 1981; Kaufman and Sharp, 1983). With the regulatory signals and the poly(A) mechanism and components, I plan to identify regulatory factors for the DHFR poly(A) site. This knowledge will provide specific examples for a gene regulation step that may be crucial for tissue differentiation, development, and likely, carcinogenesis.
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