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Publisher Summary Vitamin D 3 is hydroxylated to 25-hydroxyvitamin D 3 by a vitamin D 3 25-hydroxylase (25-hydroxylase) located in liver mitochondria or endoplasmic reticulum. 25-hydroxyvitamin D 3 is then transported to the kidney through the bloodstream. In kidney mitochondria, it is hydroxylated at the 1 α -position by a 25-hydroxyvitamin D 3 1 α -hydroxylase to 1 α ,25-dihydroxyvitamin D 3 in calcium-depleted animals or hydroxylated at the 24 R -position to 24 R ,25-dihydroxyvitamin D 3 by a 25-hydroxyvitamin D 3 24-hydroxylase (24-hydroxylase) in animals that are calcium or 1,25-dihydroxyvitamin D 3 repleted. Both 25- and 24-hydroxylases are purified and their cDNA clones are isolated. These hydroxylases constitute novel families in the P450 super family, that is, CYP27 for 25-hydroxylase and CYP24 for 24-hydroxylase. This chapter discusses the molecular cloning of these hydroxylases. Screening of cDNA libraries is performed by nucleic acid hybridization or by an immunological detection method. Construction of cDNA libraries, isolation of cDNA clones, and nucleotide sequencing are performed according to established methods. |
