Control of eukaryotic gene expression ts exerted at developmental and homeostatic levels through proteins whose interactions within the nucleus dictate the transcriptional activities of most cellular genes. Steroid receptors may represent the best characterized subset of this family primarily because the recent molecular cloning of their respective genes has yielded tremendous structural and functional insight. The intent of this renewal application is to utilize receptor cDNAs, previously recovered and characterized, to study the evolution, structure, function, and biology of this receptor protein, and to evaluate its role in tissue resistant diseases. Murine and porcine receptor cDNAs will be obtained and their sequences compared with currently existing avian and human receptor cDNAs. Cloning of the human receptor natural gene will provide an overall view of its organization, allow us to evaluate its regulatory elements, and will form the basis for identifying genetic defects in resistant cells. Transient transfection of receptor cDNA (both intact and mutant) into eukaryotic recipient cells will be utilized to evaluate the peptide's requirements for function (DNA- and steroid-binding). Transcriptional evaluation will be made by employing natural and cloned target gene promoters as reporter genes for transcriptional activity (CaBP, osteocalcin, MMTV). Peptides produced via prokaryotic hosts will be utilized to define precisely the nature of gene elements conferring D- sensitivity. The tertiary structure of important peptide domains will be evaluated employing zinc. Experiments regarding the cell biology of receptor will employ developmental, ontogenic, and homeostatic models. Key elements which trigger receptor gene expression will be ascertained. Autologous hormonal upregulation of receptor mRNA will be probed at the transcriptional level, and the receptor gene promoter scanned for receptor recognition sites. The relationship between this and other regulatory elements will be evaluated. Expression of receptor cDNA will be employed to evaluate the absolute receptor requirements for certain bioresponses and to evaluate the effect of receptor concentration on biologic activity. Cell lines derived from patients with the human D-resistant syndromes will be critically evaluated at the nucleic acid level for genetically-determined receptor abnormalities. Both DNA (polymorphisms) and RNA (size) will be screened. Direct assessment of this syndrome will be achieved by the molecular cloning of receptors with specific functional defects. Intact receptor cDNA will be employed in complementation studies to ascertain whether dysfunctional receptors represent exclusive defects. Our long term goals are to understand 1) the regulation of gene expression 2) receptor regulatory biology, and 3) the genetic basis for D-resistant syndromes. We believe that these goals can be met most efficiently utilizing current molecular biologic approaches.
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