Polyadenylation of messenger RNA precursors is a critical step in the synthesis of mRNAs and an event that is regulated for some transcription units. Studies of expression of modular dihydrofolate reductase genes have suggested that inefficient polyadenylation can limit the synthesis of mRNA from a DNA segment and that the efficiency of polyadenylation at some sites is cell growth dependent. In the latter case, polyadenylation at some sites occurs more efficiently in growing cells as compared to resting cells. The possible growth dependent regulation of polyadenylation will be further studied by using recombinant DNA methods to construct tandem arrangements of a potential growth dependent site and a non-growth dependent site. The relative amount of mRNA polyadenylated at the two sites will be compared under different growth and physiological conditions. In addition, sequences specifying the polyadenylation reaction will be explored by constructing systematic deletions of sequences both 5' and 3' to the consensus AAUAAA sequence. The in vivo efficiency of these deletions will be examined by subcloning into the early region of a SV40 vector. The biochemical components responsible for cleavage and polyadenylation of mRNA precursors will be identified and purified by developing an in vitro system that processes exogenously added template RNA. Initially, this template RNA will encompass the L3 site in the late transcription unit of adenovirus 2. We have shown previously that nuclear precursor RNA can be cleaved and polyadenylated at the L3 site during incubation of isolated nuclei. This will be the starting point for development of a soluble system.
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