The objective of this research is to use the bovine growth hormone (bGH) and bovine prolactin (bPRL) genes to study three aspects of post-transcriptional gene regulation that influence the steady-- state levels of their corresponding messenger RNAs. (1) The bGH primary transcript is alternatively processed such that the last intron (intron D) is not removed. Apparently functional mRNAs containing intron D are found on polysomes in both pituitary and transfected tissue culture cells. Such mRNAs are predicted to code for an altered form of bGH in which the carboxy domains (108aa) are different from wild type. We will analyze nucleotide signal sequence in the downstream exon which influence this splicing event along with factors which interact with these sequences using site-directed mutagenesis, transfection systems and cell-free extract. Antisera generated against peptides contained within the carboxy domain will be used to study the variant form of growth hormone arising from this alternate splicing event. (2) The high efficiency of the bGH polyadenylation signal, coupled with its short effective sequence of only 52 nucleotides, renders it a good target for characterizing the essential elements required for efficient polyadenylation. A short downstream UGU sequence, which is known to be a critical component of this polyadenylation signal, and a strong stem structure which brackets the AAUAAA consensus signal will be targets for modification by in vitro mutagenesis. Mutants will be tested by an analytical procedure that allows accurate comparisons of polyadenylation efficiency both in cells and in cell-free extract. (3) A new approach for measuring the half-life of long-lived mRNAs that utilizes the inducible hsp70 promoter will be used to identify those features of bGH and bPRL mRNAs that contribute to their unusual stability. The availability of this assay will allow determination of hypothesized stability elements in these messages and the behavior of these sequences in the presence of known mRNA instability signals Collectively, characterization of these three post-transcriptional aspects of gene regulation will help to clarify the mechanisms where by the pituitary hormone messages reach such high steady- state level. Furthermore, they will provide a better understanding of intrinsic signals in mRNA that influence message steady state levels.
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