The human cytochrome P450 3A locus. Gene evolution by capture of downstream exons

Abstract

Using a bacterial artificial chromosome (BAC) clone, we have mapped the human cytochrome P450 3A (CYP3A) locus containing the genes encoding for CYP3A4, CYP3A5 and CYP3A7. The genes lie in a head-to-tail orientation in the order of 3A4, 3A7 and 3A5. In both intergenic regions (3A4–3A7 and 3A7–3A5), we have detected several additional cytochrome P450 3A exons, forming two CYP3A pseudogenes. These pseudogenes have the same orientation as the CYP3A genes. To our surprise, a 3A7 mRNA species has been detected in which the exons 2 and 13 of one of the pseudogenes (the one that is downstream of 3A7) are spliced after the 3A7 terminal exon. This results in an mRNA molecule that consists of the 13 3A7 exons and two additional exons at the 3′ end. The additional two exons originating from the pseudogene are in an altered reading frame and consequently have the capability to code a completely different amino acid sequence than the canonical CYP3A exons 2 and 13. These findings may represent a generalized evolutionary process with genes having the potential to capture neighboring sequences and use them as functional exons.

Introduction

The cytochrome P450 3As (CYP3As) are heme-containing monooxygenases involved in the metabolism of various foreign compounds (Nelson et al., 1996). Four CYP3A genes have been described in human, CYP3A4, CYP3A5, CYP3A7 (Nelson et al., 1996) and the recently discovered CYP3A43 (Domanski et al., 2001). CYP3A4 is the most abundant cytochrome P450 in adult liver, CYP3A7 is mainly expressed in fetal liver, whereas CYP3A5 is expressed in kidney and intestine and shows a polymorphic expression in liver (Guengerich, 1999, Kivisto et al., 1996, Schuetz et al., 1992). CYP3A43 is generally expressed at low levels, with the highest expression being detected in liver, kidney, prosate and pancreas (Domanski et al., 2001). In spite of the different expression pattern, the CYP3A enzymes are characterized by a high structural similarity. The protein sequences show a significant identity, and the encoding genes have a well-conserved exon–intron structure as all consist of 13 exons (Hashimoto et al., 1993, Nelson et al., 1996). In line with the high structural similarity, the 3A4, 3A5 and 3A7 enzymes have overlapping specificities (Guengerich, 1999). The human CYP3A genes are thought to be localized in a cluster on chromosome 7q21–q22.1 (Brooks et al., 1988, Spurr et al., 1989). In addition to the four CYP3A genes, a putative CYP3A5P pseudogene has also been reported (Schuetz and Guzelian, 1995).

Gene duplication events are generally considered to be essential in the evolution of gene families (Gogarten and Olendzenski, 1999). However, gene duplications may also result in the generation of non-functional copies of genes, i.e. pseudogenes. By definition, pseudogenes are genomic sequences that exhibit a high similarity to one or more paralogous genes. Pseudogenes are considered non-functional as either these result in no production of protein at all, or, if expressed, the encoded protein lacks the potential to function as the normal one (Mighell et al., 2000). The characterization of the genomic structure of various cytochrome P450 gene clusters revealed that gene duplication events have in several cases led to the formation of pseudogenes (Nelson et al., 1996).

Here, we report the characterization of a bacterial artificial chromosome (BAC) clone spanning the human CYP3A locus. The clone encompasses the genes encoding for CYP3A4, CYP3A7 and CYP3A5, but not that of CYP3A43. In addition to the three functional CYP3A genes, several duplicated CYP3A exonic sequences, forming two distinct CYP3A pseudogenes, were found in the intergenic regions. Interestingly, a 3A7 variant mRNA has been detected having two exons of one of the pseudogenes spliced with the 3A7 terminal exon. Moreover, these two pseudogene exons are in an altered reading frame and consequently have the potential for coding an amino acid sequence that is completely different to that of the canonical CYP3As.