cDNA cloning and sequence and cDNA-directed expression of human P450 IIB1: identification of a normal and two variant cDNAs derived from the CYP2B locus on chromosome 19 and differential expression of the IIB mRNAs in human liver

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Abstract
A cDNA designated hIIB1, representing the entire coding sequence of a P450 in the IIB gene family, was isolated from a human liver .lambda.gt11 library by using the rat IIB1 cDNA as a probe. The hIIB1 protein, deduced from the cDNA sequence, contained 491 amino acids, had a calculated molecular weight of 56,286, and displayed 76% amino acid similarity with the rat IIB1 protein. Expression of the cDNA, using the vaccinia virus system, yielded a P450 that had a reduced CO-binding spectrum with an absorption maximum of 452 nm. The expressed human enzyme was able to catalyze the deethylation of 7-ethoxycoumarin. Total RNA from 13 livers was probed for levels of hIIB mRNA. Two livers had high levels, four contained moderate levels, and eight contained very low, or no detectable, mRNA. These data suggest either that defective hIIB1 genes exist in humans or that the hIIB1 gene is regulated and variably induced in our liver specimens. To search for mutant mRNA transcripts, libraries were constructed from livers expressing low levels of hIIB1 mRNA. A cDNA, designated hIIB2, was isolated that was identical with the hIIB1 cDNA except for the presence of an unusual alteration of the DNA near its 5'' end corresponding to the putative exon 4. This alteration was caused by a deletion of 29 bp and an insertion of 44 bp of nonhomologous DNA. This sequence replacement occurs at the junction of the third and fourth exons as predicted from the structure of the rat IIB1 gene, suggesting that a faulty splice might have given rise to the variant hIIb2 transcript. Due to the presence of an in-frame termination codon in the inserted DNA, this variant transcript can only produce a prematurely terminated protein. A third cDNA, designated hIIB3, was identified in two separate libraries that displayed 95% nucleotide and 93% cDNA-deduced amino acid sequence similarities to hIIB1. This transcript was found to possess a C .fwdarw. T change that resulted in a termination codon. The IIB genes (CYP2B locus) were localized to human chromosome 19 using the somatic cell hybrid mapping strategy. High-frequency restriction fragment length polymorphisms were detected in both BamHI and BglII digests.