Abstract
Over 90% of cases of congenital adrenal hyperplasia are due to disordered 21-hydroxylation, which converts progesterone and 17-hydroxyprogestone (17-OHP) to DOC and 11-deoxycortisol. 21-hydroxylase is a single 49000 dalton microsomal cytochrome P450, termed P450C21, which represents <0.1% of adrenocortical protein. We prepared polyadenylated messenger RNA (mRNA) from demedulated bovine adrenals and used this mRNA as template for the synthesis of double stranded (DS) complementary DNA (cDNA). The 3' ends of the DScDNA were extended with deoxycytosine using terminal deoxynucleotidyl transferase; this "tailed" DScDNA was inserted into the Pst I site of pBR322 similarly tailed with deoxyguanosine. Transformation of E. coli 294 yielded >60,000 clones. Based on a partial porcine amino acid sequence, we used the manual phosphoramidite method to synthesize a family of 32 15-base oligonucleotides and used these to prime the synthesis of a specific cDNA probe. Clones hybridizing to the 15mer-primed cDNA were then probed with a different family of 64 17-base oligonucleotides we synthesized based on a different portion of the amino acid sequence. Of 10,000 clones containing cDNA inserts longer than 800 bases, 5 hybridize efficiently with both probes, indicating they carry bovine P450C21. cDNA sequences. These DNA clones will permit the determination of the structure of bovine P450C21 as well as human P450C21 and its pathogenic variants.
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Chung, BC., Matteson, K., Morin, J. et al. CLONING OF cDNA ENCODING BOVINE 21-HYDROXYLASE. Pediatr Res 18 (Suppl 4), 166 (1984). https://doi.org/10.1203/00006450-198404001-00438
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DOI: https://doi.org/10.1203/00006450-198404001-00438