Cloning and Characterization of the Bovine Gene for Steroid 21-Hydroxylase (P-450c21)

Abstract

Steroid 21-hydroxylase activity is required for the synthesis of both cortisol and aldosterone. Using two manually synthesized oligonucleotide probes, we screened a bovine genomic DNA library and identified a phage, λE11, carrying the gene for the steroid 21-hydroxylase, P-450_C21. The identity of λE11 was initially proven by initiating dideoxy sequencing from the two oligonucleotides directly on the full-length, uncleaved phage template. Hybridization of total bovine genomic DNA to λE11 restriction fragments indicates the presence of repetitive sequences in or near the P-450_C21 gene. Northern blots indicate that the mature mRNA exists in two principal forms of about 2.2 and 2.4 kb in length. Southern blots indicate there are two copies of the gene in the bovine genome. Sequence analysis of a 1141-bp Eco RI fragment of the gene shows three complete exons and a portion of a fourth exon, correctly determining the amino acid sequence of 148 amino acids of this important enzyme. This cloned 1141-bp fragment cross-hybridizes to human genomic DNA, indicating it should be a useful probe for studying the human P-450C21 gene in patients having impaired 21-hydroxylase activity.