Oaks M K, Raff H
Immunology and Endocrine Research Laboratories, St Luke's Medical Center, Milwaukee, WI 53215, USA.
J Steroid Biochem Mol Biol. 1995 Sep;54(5-6):193-9. doi: 10.1016/0960-0760(95)00143-n.
The adrenocortical enzymes of the steroidogenic late pathway in the rat are aldosterone synthase (P450aldo), which catalyzes the production of aldosterone, and 11 beta-hydroxylase (P45011 beta), which catalyzes the production of corticosterone throughout the cortex. These two enzymes are highly homologous and are encoded by the genes CYP11B2 and CYP11B1, respectively. The purpose of the present study is to describe the development of two sets of primers and the reverse transcription-polymerase chain reaction (RT-PCR) conditions that are capable of discriminating between rat P450aldo and P45011 beta mRNAs. The P450aldo primer set did not amplify full length cDNA P45011 beta plasmid and the P45011 beta primer set did not amplify full length cDNA P450aldo plasmid indicating minimal crosstalk. The fidelity of the PCR primers and method was further established by sequencing the PCR products and demonstration of virtual identity with the published sequences of P450aldo and P45011 beta. RT-PCR of mRNA from adrenal capsules (zona glomerulosa) and subcapsules (zona reticularis/fasciculata) from rats demonstrated no effect of sodium diet on the expression of P45011 beta mRNA but an approximately 8-fold greater expresison in P450aldo mRNA on low vs high sodium intake. Similar results were found when single hemicapsules were subjected to RT-PCR, demonstrating the sensitivity of the method. We conclude that the two sets of PCR primers and the RT-PCR method described are capable of evaluating the expression of the highly homologous mRNAs for P450aldo and P45011 beta with great precision and sensitivity.
大鼠类固醇生成晚期途径中的肾上腺皮质酶是醛固酮合酶（P450aldo），它催化醛固酮的生成；还有11β - 羟化酶（P45011β），它在整个皮质中催化皮质酮的生成。这两种酶高度同源，分别由CYP11B2和CYP11B1基因编码。本研究的目的是描述两组引物的开发以及逆转录 - 聚合酶链反应（RT-PCR）条件，这些引物和条件能够区分大鼠P450aldo和P45011β mRNA。P450aldo引物组未扩增出全长cDNA P45011β质粒，P45011β引物组也未扩增出全长cDNA P450aldo质粒，表明交叉反应最小。通过对PCR产物进行测序并证明与已发表的P450aldo和P45011β序列具有实质同一性，进一步确定了PCR引物和方法的保真度。对大鼠肾上腺被膜（球状带）和被膜下组织（网状带/束状带）的mRNA进行RT-PCR分析表明，钠饮食对P45011β mRNA的表达没有影响，但低钠摄入时P450aldo mRNA的表达比高钠摄入时高约8倍。对单个半肾上腺进行RT-PCR时也发现了类似结果，证明了该方法的敏感性。我们得出结论，所描述的两组PCR引物和RT-PCR方法能够高精度和高灵敏度地评估P450aldo和P45011β高度同源mRNA的表达。
