RESUMO

In this study we report the cloning and sequencing of a cDNA for cholesterol side chain cleavage cytochrome P450scc from Syrian hamster adrenal glands. Isolation of P450scc mRNA was carried out with degenerate primer PCR together with 5' and 3' RACE protocol. The full-length cDNA comprises an open reading frame of 1563 bp encoding a polypeptide of 520 amino acid residues. The predicted protein sequence exhibits well-preserved heme- and steroid-binding domains and shares 89% amino acid sequence identity with rat and mouse enzymes. Transient transfection of HEK-293 cells with the cloned cDNA leads to the formation of pregnenolone from 25-hydroxycholesterol. Northern blot analysis showed expression of mRNAs for P450scc in the major steroidogenic tissues, namely, the adrenal cortex, testis, and ovary. In addition, tissue distribution analysis using the coupled reaction of RT-PCR and Southern blotting revealed that the mRNA of the enzyme is also expressed in various nonendocrine tissues, including the epididymis, Harderian gland, and lungs. The relative abundance of specific transcripts at these novel sites suggests that P450scc could potentially play an important role in regulating local steroid hormone synthesis.

Assuntos

Glândulas Suprarrenais/enzimologia , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Clonagem Molecular , DNA Complementar/genética , Expressão Gênica , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , Linhagem Celular , Enzima de Clivagem da Cadeia Lateral do Colesterol/química , Cricetinae , Embrião de Mamíferos , Humanos , Rim , Mesocricetus/genética , Dados de Sequência Molecular , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , RNA Mensageiro/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção