SELECTIVE DETECTION OF MESSENGER-RNA FORMS ENCODING THE MAJOR PHENOBARBITAL INDUCIBLE CYTOCHROMES-P450 AND OTHER MEMBERS OF THE P450IIB FAMILY BY THE RNASE-A PROTECTION ASSAY

The identification of P450 mRNAs in a tissue poses the problem that members of the same P450 gene family share a high sequence homology. Studies based on oligomer probes rely on a probe covering only a few base pairs. In contrast in our study on the expression of the P450IIB gene family we used in vitro-generated antisense transcripts, covering several hundred base pairs, of the hypervariable and constant regions of the P450IIB1 and P450IIB2 cDNA, in the RNAse A protection assay of mRNA isolated from various tissues. RNAse A concentrations were adjusted to a level where this enzyme still yielded distinct fragments for a defined P450IIB1 antisense/P450IIB2 sense heteroduplex, which contained 24 scattered mismatches within a stretch of 285 nucleotides. In contrast nuclease S1 was not useful for the detection of mismatches within this heteroduplex. With this highly sensitive RNAse A protection assay we were able to distinguish between the expression of P450IIB1 and the expression of P450IIB2 in several organs. Our results strongly support earlier studies on the tissue specific expression of these enzymes, which had used oligomer probes (Omiecinski, C. J., 1986, Nucleic Acids Res. 14, 1525-1539). Moreover we detected the constitutive hepatic expression of a P450IIB gene which was distinct from P450IIB1 and IIB2. In addition we identified a P450IIB mRNA which was expressed at high levels in the preputial gland but not in the liver or any other organ tested. © 1990.