The major goal of this research is to determine the degree to which mRNA stability is dependent upon tissue specific factors, the intrinsic nucleotide structure of the mRNA, and the flanking regions contained in the genomic sequence coding for the message. To approach these studies we will use DNA-mediated gene transfer techniques to introduce the bovine growth hormone (bGH) gene into a variety of cell types and then examine the stability of bGH mRNA transcripts made in these cells. Both pituitary and non-pituitary cells will be used as recipient cells for bGH gene transfer, including a stable cell-line of virally transformed bovine pituitary cells. The bGH gene is particularly well-suited for such studies since it codes for an unusually stable mRNA, its expression is regulated by both glucocorticoid and thyroid hormones and the GH gene is contained in a relatively small restriction fragment (2kb) which lacks apparent internal Pol III promoters. These studies should provide basic information on the role of mRNA stability in the regulation of gene expression. The availability of this transformation system will also allow us to study several other potentially important control mechanisms for regulating GH gene expression. We have described a highly conserved sequence of nucleotides in the 5'-flanking region of the bGH gene which is 38 nt in length and shows greater than 90% homology with similar regions of the rat and human GH genes. The function of this sequence, which could potentially be involved in hormonal regulation of this gene, will be examined by in vitro site-directed mutagenesis.
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