Conversion of
cholesterol to
pregnenolone is mediated by the
cholesterol side-chain cleavage (SCC)
enzyme, P450scc. Deficient SCC activity causes congenital lipoid adrenal
hyperplasia (also known as
20,22 desmolase deficiency), a potentially lethal defect in the synthesis of all
steroid hormones. To probe for possible genetic defects causing this disease we synthesized four
oligodeoxyribonucleotides containing 63 to 72 bases corresponding to portions of the bovine
complementary DNA (
cDNA) sequence for P450scc. The bovine
oligonucleotides were labeled and used directly to probe Southern blots of normal human genomic
DNA, revealing a pattern indicating there is a single P450scc gene in the human genome. Hybridization to Northern blots of normal human and bovine adrenal
messenger RNA indicates that P450scc
messenger RNA is about 2.0 kilobases long in both species. Hybridizations of the
oligonucleotides to genomic
DNA from three unrelated patients with SCC deficiency did not detect a deletion in the human P450scc gene. The bovine sequence
oligonucleotides were then used to isolate a human P450scc
cDNA clone. The isolated P450scc
cDNA fragment contains 818 bases encoding 239
amino acids of the
protein, the translation termination signal, and 98 bases of the
3' untranslated region. The sequence of this carboxy-terminal half of the human P450scc
protein is 72% homologous with the bovine sequence and contains an additional
amino acid not found in bovine P450scc; the human and bovine nucleotide sequences are 81% homologous. Repetition of the genomic
DNA blotting studies with the
cDNA probe gave the same results obtained with the bovine-sequence
oligonucleotide probes, confirming that SCC deficiency is not due to a deletion in the regions of the P450scc hybridizing with the probes. Long, chemically synthesized heterologous sequence
oligonucleotides containing unknown numbers of base mismatches with human sequences may thus be used to study human genes so that access to a
cDNA is not necessary for such studies.