Using cDNA clones prepared in previous studies in this laboratory in the modified Okayama-Berg cloning vector, it is proposed to construct and express chimeric P-450 steroid hyroxylases in COS 1 cells of monkey kidney origin in order to examine the role of specific P-450 sequences in steroid hydroxylase activities. In particular, chimeric proteins will be constructed between P-45017 alpha and P450C21 and between P-45017 alpha and P-450scc. The P-450 domains to be examined in these studies include the Ozols tridecapeptide and the heme-binding region. It will be tested whether either of these regions of P-450C21 can impart 21- hydroxylase activity to P-45017 alpha. In addition it will be tested whether either of these regions of P-450scc can impart cholesterol side chain cleavage activity to P-45017 alpha and whether either of these regions of P-45017 alpha can impart 17 alpha-hydroxylase/17, 20-lyase activity to P-450scc. These chimeric proteins will be prepared from their specific cDNA inserts utilizing the two-primer mutagenesis procedure of Zoller and Smith. The resultant chimeric cDNA molecules will be expressed in COS 1 cells using the same procedure previously utilized by this laboratory to express P-45017 alpha, P-450scc and adrenodoxin cDNAs. Also it is proposed to carry out site-specific mutagenesis of specific amino acid residues of anrenodoxin by this procedure. Finally, the two-primer mutagenesis procedure will be utilized to investigate the amino acid sequence requirements for insertion of P-45017 alpha into the endoplasmic reticulum and P- 450scc and adrenodoxin into their correct submitochondrial compartments. These studies represent the first investiggation of expression and structure-function relationships of P-450 and adrenodoxin using mammaliam cells. The results of these studies will provide information on specific steroid hydroxylase structure- function relationships as well as general aspects of P-450 structure vs. function. In addtion they will provide new insights into the biogenesis of functional steroid hydroxylase complexes.
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