Vilchis F, Chávez B, Larrea F, Timossi C, Montiel F
Department of Reproductive Biology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Vasco de Quiroga No. 15, México DF 14000, Mexico.
Gen Comp Endocrinol. 2002 May;126(3):279-86. doi: 10.1016/s0016-6480(02)00003-5.
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.
在本研究中，我们报道了从叙利亚仓鼠肾上腺克隆并测序胆固醇侧链裂解细胞色素P450scc的cDNA。采用简并引物PCR结合5'和3' RACE方案分离P450scc mRNA。全长cDNA包含一个1563 bp的开放阅读框，编码一个由520个氨基酸残基组成的多肽。预测的蛋白质序列显示出保存良好的血红素和类固醇结合结构域，与大鼠和小鼠的酶具有89%的氨基酸序列同一性。用克隆的cDNA瞬时转染HEK-293细胞可导致从25-羟基胆固醇形成孕烯醇酮。Northern印迹分析显示P450scc的mRNA在主要的类固醇生成组织，即肾上腺皮质、睾丸和卵巢中表达。此外，使用RT-PCR和Southern印迹的偶联反应进行的组织分布分析表明，该酶的mRNA也在各种非内分泌组织中表达，包括附睾、哈德氏腺和肺。这些新位点上特定转录本的相对丰度表明，P450scc可能在调节局部类固醇激素合成中发挥重要作用。
