Steroid receptors mediate the action of their hormonal ligands by interacting with genomic DNA in a sequence specific manner to cause subsequent enhancement or suppression of hormone-sensitive gene transcription. However, steroid receptors are a class of labile proteins of low abundance, whose physical characteristics, functional properties, and biosynthesis have yielded reluctantly to investigation. The purpose of this application is to develop cloned nucleic acid probes for a steroid receptor mRNA which will then be exploited in detecting receptor mRNA, evaluating its cellular regulation, and isolating cloned genomic DNA containing the natural receptor gene. The achievement of these goals will enhance significantly our knowledge of receptor biology, and open a new avenue in receptor research. More importantly, however, they mesh with our long term goal of understanding structure-function relationships for a polypeptide known to be a eukaryotic gene regulating protein. We propose to develop cDNA libraries from mRNA derived from receptor-containing cells of chick and human intestine, as well as from human fibroblasts by cloning into a phage expression vector Lambdagtll, followed by amplification of the library in E. coli. Libraries (1-10 million) recombinants/ug cDNA) will then be screened for expression of a fusion protein (Beta-galactosidase-receptor) via comparison of pure receptor/fusion protein tryptic peptide maps or receptor amino acid sequence/cloned DNA base sequence, as well as through hybridization arrested translation. Receptor cDNA will then be exploited in studies designed to examine the presence, abundance, and physical characteristics of receptor mRNA and to evaluate principles potentially involved in its cellular regulation. This cDNA will also be utilized in a hybridization selection protocol to enrich for receptor mRNA, and a full-length cDNA prepared and cloned in pBR322. Finally, the cloned probes will be utilized to screen appropriate genomic DNA libraries and to isolate recombinant DNA carrying the natural receptor gene. The development of these probes will permit experiments designed to enhance our understanding of the role and action of steroid hormone receptors as regulatory elements of transcription. Most important, however, their use will permit us to begin evaluation of the genetic basis for human tissue-resistance which exists for virtually all steroid hormones.
| Mangelsdorf, D J; Pike, J W; Haussler, M R (1987) Avian and mammalian receptors for 1,25-dihydroxyvitamin D3: in vitro translation to characterize size and hormone-dependent regulation. Proc Natl Acad Sci U S A 84:354-8 |
