The structure and characterization of type I P-450(15) alpha gene as major steroid 15 alpha-hydroxylase and its comparison with type II P-450(15) alpha gene.
The structures of genes 15 alpha OH-1 and 15 alpha OH-2, within the mouse steroid 15 alpha-hydroxylase (P-450(15) alpha) family were determined. Genes 15 alpha OH-1 and -2 encoded mouse Type I and II P-450(15) alpha, respectively (Squires, E.J., and Negishi, M. (1988) J. Biol. Chem. 263, 4166-4171). The two genes, which spanned approximately 8 kilobase pairs of total length, showed nearly identical structures and were divided into nine exons at the same positions. A high nucleotide sequence homology (greater than 96%) indicated that 15 alpha OH-1 and -2 were duplicated within 5 million years. The two major transcription start sites were located at 14 and 24 base pairs (bp) upstream from the initiation Met in both genes. No tissue-specific difference in 15 alpha OH-1 and -2 transcriptional start sites was found in mouse liver and kidney. Both 15 alpha OH-1 and -2 had TATA and CAAT boxes at 30 and 100 bp, respectively, upstream from their major transcription start sites. A glucocorticoid regulatory element was present at 336 bp upstream from the start site in both genes. In addition to these motifs, 15 alpha OH-2 had an SV40 enhancer core sequence immediately downstream from its CAAT box. There were only 11 substitutions between Type I and II P-450(15) alpha in their 494-amino acid residues. Type I cDNA-transfected cell homogenates had testosterone 15 alpha-hydroxylase activity at approximately 9 pmol/min/mg protein. Type I also catalyzed progesterone and androstenedione 15 alpha-hydroxylase activities. Type II, on the other hand, exhibited little activity toward these steroids. The results indicated that Type I was the major steroid 15 alpha-hydroxylase. The differential 15 alpha OH-1 expression, therefore, determined the sexual dimorphism of the tissue-specific 15 alpha-hydroxylase activity in mice.
Lindberg
R
R
Pharmacogenetics Section, National Institute of Environmental Health Sciences, Triangle Park, North Carolina 27709.
Burkhart
B
B
Ichikawa
T
T
Negishi
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D000072467:Cytochrome P450 Family 2; D004247:DNA / Q000235:genetics; D005786:Gene Expression Regulation; D051379:Mice; D008969:Molecular Sequence Data; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D013250:Steroid Hydroxylases / Q000235:genetics*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014158:Transcription, Genetic; D014162:Transfection
D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C460223:Cyp2a4 protein, mouse; D000072467:Cytochrome P450 Family 2; C050356:steroid 15-alpha-hydroxylase