The goals of this project are to identify cis-acting regulatory sequences flanking the GH1, CSHP1, CSH1, GH2 and CSH2 genes in the human growth hormone (hGH) cluster and to determine their role in regulating tissue specific transcription of these genes. To accomplish these goals a series of recombinant plasmids will be constructed by fusing the bacterial gene, chloramphenicol acetyltransferase (CAT), to varying amounts of DNA lying 5' to each of the genes in the hGH gene cluster. Human term and first trimester placental as well as rat anterior pituitary cells will be transfected with these recombinant plasmids by Ca2PO4 precipitation. Positive or negative regulatory regions will be identified by comparative analysis of assayed levels of CAT activity. Specific bases that are essential for these regulatory functions will be identified using plasmid constructs that are modified by Bal 31 exonuclease or oligonucleotide directed site mutagenesis. Tissue specificity will be analyzed by comparing the CAT activity of analogous plasmid constructions corresponding to each gene in each cell type. The importance of spacial arrangements on the ability of 5' sequences to regulate the promoter sequences for these genes will be evaluated by assaying CAT activity from a variety of plasmids in which the normal distances separating these regions have been altered. The information obtained from these studies will contribute to our understanding of the tissue specific transcriptional, regulation of this complex of growth promoting genes which will provide insight into the regulation of other eukaryotic multigene families.
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