The overall objective of the proposed research is to gain insight into the structure, function, and expression of human steroid 5alpha-reductase and to understand how the gene is altered in subjects with steroid 5alpha- reductase deficiency. Over the last year we have succeeded in isolating a cDNA to the rat steroid 5alpha-reductase. This cDNA now makes possible an analysis of the molecular genetics of the human steroid 5alpha-reductase. Our initial goal in this new project will be the isolation of a cDNA clone to the human steroid 5alpha-reductase mRNA by cross-hybridization with a rat cDNA probe. The human steroid 5alpha-reductase cDNA will be characterized by DNA sequence analysis and by expression in mammalian cells. Our second objective is to isolate and characterize the normal steroid 5alpha-reductase gene from a genomic DNA library. The exon-intron structure of the gene will be determined and compared to the sequence of the protein. Intron DNA sequences will be used to derive a series of oligonucleotide primers that can be used to amplify individual exons of the gene. Our third goal is to characterize several key mutations in the gene that are found in male pseudohermaphrodites with steroid 5alpha-reductase deficiency. These mutations affect the enzyme's Michaelis-Mentin constants for substrate or cofactor, and their study will define important structural domains in the protein. The identification of mutations will be accomplished using the polymerase chain reaction followed by sequence analysis of the amplified DNA. A fourth goal is to expand the structure- function information derived from the naturally occurring mutations by creating and expressing a limited number of site-directed mutations in the steroid 5alpha-reductase cDNA. We will focus on identifying amino acids that form the cofactor and substrate binding domains of the protein. The final objective is to gain insight into the transcriptional regulation of steroid 5alpha-reductase mRNA expression. We will concentrate on defining general transcription signals and androgen-responsive elements in the promoter of this highly regulated gene.
| Russell, D W; Berman, D M; Bryant, J T et al. (1994) The molecular genetics of steroid 5 alpha-reductases. Recent Prog Horm Res 49:275-84 |
| Wigley, W C; Prihoda, J S; Mowszowicz, I et al. (1994) Natural mutagenesis study of the human steroid 5 alpha-reductase 2 isozyme. Biochemistry 33:1265-70 |
| Russell, D W; Wilson, J D (1994) Steroid 5 alpha-reductase: two genes/two enzymes. Annu Rev Biochem 63:25-61 |
| Thigpen, A E; Silver, R I; Guileyardo, J M et al. (1993) Tissue distribution and ontogeny of steroid 5 alpha-reductase isozyme expression. J Clin Invest 92:903-10 |
| Berman, D M; Russell, D W (1993) Cell-type-specific expression of rat steroid 5 alpha-reductase isozymes. Proc Natl Acad Sci U S A 90:9359-63 |
| Thigpen, A E; Cala, K M; Russell, D W (1993) Characterization of Chinese hamster ovary cell lines expressing human steroid 5 alpha-reductase isozymes. J Biol Chem 268:17404-12 |
| Davis, D L; Russell, D W (1993) Unusual length polymorphism in human steroid 5 alpha-reductase type 2 gene (SRD5A2). Hum Mol Genet 2:820 |
| Hirsch, K S; Jones, C D; Audia, J E et al. (1993) LY191704: a selective, nonsteroidal inhibitor of human steroid 5 alpha-reductase type 1. Proc Natl Acad Sci U S A 90:5277-81 |
| Wilson, J D; Griffin, J E; Russell, D W (1993) Steroid 5 alpha-reductase 2 deficiency. Endocr Rev 14:577-93 |
| Jenkins, E P; Andersson, S; Imperato-McGinley, J et al. (1992) Genetic and pharmacological evidence for more than one human steroid 5 alpha-reductase. J Clin Invest 89:293-300 |
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