We characterized the structural genes of sex-specific mouse P450s: the male-specific steroid 16alpha-hydroxylase (P450 16alpha) and the female-specific steroid 15alpha-hydroxylase (P450 15alpha). The nucleotide and deduced amino acid sequences indicate that these sex-specific steroid hydroxylases have evolved as the new members within the different P450 subfamilies. Our site-directed mutagenesis studies show that P450 15alpha-dependent 15alpha-hydroxylase activity critically depends on the residue at position 209. Moreover, the cytochrome's spin equilibrium is altered by the amino acid substitutions at this position in P450 15alpha, indicating that the 209 resides close to the 6th ligand of the heme protein. We confirmed, by nuclear run-on assay and the transgenic mouse, that the sex-specific P450 gene expressions were regulated by growth hormone transcriptionally. On the other hand, the P450 induction by exogenous chemicals and metals, including pyrazole and CoCl2, are regulated post-transcriptionally, but pre-translationally. We used in vitro transcription, DNase 1 footprinting, and gel retardation to identify two proximal cis-acting transcription elements (SDI and CTE) in the 5'-flanking region of P450 16alpha gene. Moreover, SDI is the strong one and specific for this gene and, therefore, may be involved in the male-specific gene transcription. Also, our transient transfections showed the presence of positive and negative regulatory elements (PRE and NRE) in the far upstream of the P450 16alpha gene. In addition, preliminary results suggested that these are growth hormone-response elements. It appeared, therefore, that PRE, NRE and SDI together provide P450 16alpha gene with the hormone-dependent, male-specific transcription. These results are extremely important to understand the sex-and tissue-dependent toxicity and carcinogenicity of endogenous hormones and exogenous chemicals and also their polymorphism.
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