Cloning and Characterization of a Novel Member of the Cytochrome P450 Subfamily IVA in Rat Prostate

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Abstract
To isolate cDNAs for forms of cytochrome P450 from rat prostate, a λ gt11 cDNA library from this tissue was screened with a mixture of oligonucleotide probes directed against the conserved heme binding region of different P450 isozymes. A cDNA clone (PP1) encoding a part of a novel form of cytochrome P450 was isolated and the deduced amino acid sequence showed 76% identity with cytochrome P450 IVA1, indicating that PP1 is a member of the same subfamily. Northern blot analysis of total RNA from prostates of untreated rats revealed that two mRNAs of approximately 2.8 and 2.2 kb hybridize to PP1. The level of mRNA was induced fivefold above the level in intact animals by androgen treatment of castrated rats. Analysis of poly(A)+RNA levels in different tissues on Northern blots showed high constitutive expression of PP1 in the kidney, but no signal was detectable with RNA from liver; a weak signal was detected in the retina. Subsequent screening of a rat kidney cDNA library led to the isolation of the full-length clone KP1, which differs from Ppl only in three nucleotide positions. KP1 is 1,957 bp long and contains a 1,527-bp-long open reading frame encoding a protein of 508 amino acids. In situ hybridization of rat kidney sections with PP1 showed that this P450 form is expressed in the outer stripe of the outer medulla, indicating its localization in the proximal tubules.