We are attempting to gain insight into the biologic and biochemical events that determine and regulate tissue-specific gene expression and the alterations that accompany or follow neoplastic transformation. Four biologic systems exemplifying differential gene expression are under study. Alpha 2u globulins are a gene family manifesting tissue-specific gene expression which ceases upon neoplastic transformation. Alternate processing sites within intron six have been detected. The 7 kb gene has seven exons, a (GT)27 repeat sequence flanked by enhancer sequences as well as a 5' glucocorticoid receptor consensus sequence. Members of this gene family have been inserted into transgenic mice and transfected into various cell lines to study the relationship between gene structure, development, and tissue-specific gene expression. Cell-free transcriptional and binding studies are attempting to identify tissue-specific transcriptional regulators. Enhancers and tissue specific transacting proteins as determinants of tissue-specific immunoglobulin gene expression are being investigated. Deletions and exchange of putative regulatory regions followed by transfection into various cell types may identify regulatory regions. Translational inhibitors may determine the degree to which proteins modulate gene expression. Dopamine agonist regulation of transcription of selected members of the kallikrein gene family is being examined. cDNAs of expressed kallikrein genes have been isolated from a lambda GT10 pituitary library. In vitro nuclear transcription and tissue levels of kallikrein mRNA will be evaluated. Experiments are continuing to isolate cDNAs to the alpha and beta subunits of the hormone inducible Na/K ATPase. Transfected fibroblasts contain two Na/K ATPases, one of which has low affinity for and is resistant to ouabain. (V)
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