White P C, New M I, Dupont B
Proc Natl Acad Sci U S A. 1984 Apr;81(7):1986-90. doi: 10.1073/pnas.81.7.1986.
We isolated a cDNA clone encoding a bovine adrenal cytochrome P-450 specific for steroid 21-hydroxylation (P-450C21). Serum from rabbits immunized with purified P-450C21 precipitated a single protein from the products of an in vitro translation reaction using bovine adrenal mRNA. This protein migrated with P-450C21 on NaDodSO4/polyacrylamide gel electrophoresis. After sucrose gradient sedimentation, mRNA encoding P-450C21 was found in the 19S fraction. This fraction was reverse transcribed into double-stranded cDNA and inserted into the Pst I site of pBR322 by the dC X dG tailing procedure. Escherichia coli cells transformed with recombinant plasmids were screened with an in situ immunoassay using anti-P-450C21 serum and 125I-labeled staphylococcal protein A. Two colonies consistently bound anti-P-450C21 serum. They were identified as carrying the same plasmid by restriction mapping. This plasmid, pC21a, contains an insert of 520 base pairs. It hybridizes with mRNA encoding P-450C21. The peptide encoded by the insert in pC21a is highly homologous to two peptides isolated from porcine P-450C21 and shows limited homology to the P-450 induced by phenobarbital in rat liver. This clone may be useful in studying the molecular genetics of human congenital adrenal hyperplasia due to 21-hydroxylase deficiency.
我们分离出了一个编码牛肾上腺甾体21-羟化特异性细胞色素P-450（P-450C21）的cDNA克隆。用纯化的P-450C21免疫的兔血清从使用牛肾上腺mRNA的体外翻译反应产物中沉淀出一种单一蛋白质。该蛋白质在十二烷基硫酸钠/聚丙烯酰胺凝胶电泳上与P-450C21一起迁移。经过蔗糖梯度沉降后，在19S组分中发现了编码P-450C21的mRNA。该组分被逆转录成双链cDNA，并通过dC×dG加尾法插入到pBR322的Pst I位点。用抗P-450C21血清和125I标记的葡萄球菌蛋白A进行原位免疫测定，筛选用重组质粒转化的大肠杆菌细胞。有两个菌落始终结合抗P-450C21血清。通过限制性图谱分析确定它们携带相同的质粒。这个质粒，pC21a，含有一个520个碱基对的插入片段。它与编码P-450C21的mRNA杂交。pC21a中插入片段编码的肽与从猪P-450C21中分离出的两种肽高度同源，与大鼠肝脏中由苯巴比妥诱导的P-450显示出有限的同源性。该克隆可能有助于研究由于21-羟化酶缺乏引起的人类先天性肾上腺增生的分子遗传学。
