Introduction

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The human cytochrome P450 (CYP¹) 2A gene subfamily was previously found to comprise three genes, CYP2A6, CYP2A7, and CYP2A13, as well as two identical copies of a CYP2A7 pseudogene, CYP2A7PT and CYP2A7PC (or CYP2A7P1), which contain putative CYP2A coding sequences corresponding to exons 1 through 5 (Fernandez-Salguero et al., 1995). Both CYP2A6 and CYP2A13 are active toward many carcinogens and other toxicants, such as the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (Fernandez-Salguero and Gonzalez, 1995; Su et al., 2000) and the herbicide 2,6-dichlorobenzonitrile (Ding et al., 1996; Su et al., 2000), whereas CYP2A7 is not functional (Yamano et al., 1990; Ding et al., 1995). CYP2A6 is also the major coumarin 7-hydroxylase and nicotine C-oxidase in the liver (Fernandez-Salguero and Gonzalez, 1995; Messina et al., 1997; Yamazaki et al., 1999). Genetic polymorphisms in the CYP2A6 gene have been identified (Fernandez-Salguero et al., 1995; Nunoya et al., 1999; Oscarson et al., 1999), and intensive efforts are being made to determine the role of CYP2A6 gene mutations in individual susceptibility to disease and environmental toxicity (Pianezza et al., 1998; Kamataki et al., 1999; Kitagawa et al., 1999; Miyamoto et al., 1999). However, the genotyping methods used in at least some of these studies have been found to be unreliable (Oscarson et al., 1998; Pianezza et al., 1998; Sabol and Hamer, 1999), partly because of the presence of multiple CYP2A genes with high sequence homology.

The CYP2A genes are located in the CYP2A-2B-2F gene cluster (Hoffman et al., 1995) on human chromosome 19, where two mutant CYP2G genes, CYP2GP1 and CYP2GP2, were also found (Sheng et al., 2000). In mammalian animals, there appears to be only a single CYP2G gene, named CYP2G1, which is expressed only in the olfactory mucosa, and is active toward sex steroid hormones as well as odorants and other xenobiotics (cf. Hua et al., 1997; Gu et al., 1998). However, the two human CYP2G genes both have loss-of-function mutations: CYP2GP1 contains a single nucleotide deletion in exon 2 and a 2.4-kbp deletion between exons 3 and 7, whereas CYP2GP2 contains two nonsense mutations in exons 1 and 3, respectively (Sheng et al., 2000). In the current study, in an effort to identify additional, potentially functional CYP2G-related genes, we have isolated a new CYP2A gene fragment (named CYP2A7P2), which is linked to CYP2GP1 in an outward opposite orientation. The deduced partial amino acid sequence of CYP2A7P2 is 81 to 87% identical to corresponding sequences (exons 6-9) in the other human CYP2As. An analysis of CYP2A sequence alignment suggests that CYP2A7P2 may be derived from the same ancestral gene that gave rise to CYP2A7P1.

Materials and Methods

An EMBL3 genomic library, which was prepared with genomic DNA derived from the leukocytes of an adult female (CLONTECH, Palo Alto, CA), was screened by plaque hybridization with a ³²P-labeled E7-8 probe (about 0.2 kbp). The probe was obtained previously by PCR from human genomic DNA and contained parts of exons 7 and 8 and the entire intron 7 of a putative human CYP2G gene (Sheng and Ding, 1996). A total of 1.2 × 10⁶ phages were screened at a density of 200 plaque-forming units/cm². A single positive clone was isolated. The cloned DNA fragment (named S34) was released by digestion with SalI and inserted into pCR-Script vector (Stratagene, La Jolla, CA) to give pS34 plasmid. The latter was further digested with BamHI, EcoRI, HindIII, or Sau3AI to obtain various subclones. Nucleic acid sequences were determined initially from the subclones using vector primers, which revealed both CYP2A- and CYP2GP1-like sequences. Subsequently, pS34 was sequenced directly using primers designed according to CYP2GP1, CYP2A6, and CYP2A13 sequences, and by primer walking.

A bacteriophage P1 genomic library constructed with DNA from the foreskin fibroblasts of a Caucasian male was also screened (by Genome Systems, St Louis, MO) using the E7-8 probe. Only one positive clone, named P1G1, was identified; the recombinant DNA was isolated using a modified alkaline lysis procedure provided by Genome Systems. The isolated P1G1 DNA, containing an 85-kbp insert, was digested with PstI or BstEII for Southern blot detection of CYP2-related fragments. DNA fragments were resolved by pulsed-field agarose gel electrophoresis with use of a Hoefer PC500 Switchback pulse controller (Hoefer Pharmacia Biotech, San Francisco, CA), transferred to a Hybond-N nylon membrane (Amersham, Arlington Heights, IL), and analyzed by hybridization with ³²P-labeled oligonucleotide probes derived from CYP2A, CYP2B, or CYP2G coding sequences (Yamano et al., 1989; Sheng et al., 2000; Su et al., 2000). Positive fragments were gel-purified and subcloned into pCR-Script vector. The P1 phage DNA and subcloned fragments were analyzed by sequencing with numerous primers derived from CYP2A6, CYP2A7P1, CYP2A7P2, CYP2A13, CYP2B6, CYP2B7, CYP2F1, CYP2GP1, and CYP2GP2 to detect the presence of these genes and to identify potential CYP2A7P2 exons 1 to 5 sequences [see Nelson et al. (1996) for sources of P450 sequences]. DNA sequence analysis was performed with an automated DNA sequencer from Applied Biosystems, model 373A (Foster City, CA), at the Molecular Genetics Core Facility of the Wadsworth Center.

	Results and Discussion

Top Abstract Introduction Results and Discussion References

As shown in Fig. 1, pS34 contained a 15-kbp insert comprising exons 7 through 9 of CYP2GP1 (Sheng et al., 2000) and exons 6 through 9 of a previously unidentified CYP2A gene (CYP2A7P2); the two fragments were arranged in outward opposite directions with about 8 kbp of intervening sequence. The P1G1 clone contained a full-length CYP2GP1 gene (with the 2.4-kbp deletion between exons 3 and 7) and exons 6 through 9 of CYP2A7P2. The distance between CYP2A7P2 and CYP2GP1 was initially estimated using long distance PCR (data not shown), and was confirmed by the length and partial sequence of a PstI subclone of P1G1 (P1G1-SUBI; Fig. 1). All exon sequences were determined at least twice and from both orientations. CYP2A7P2 introns and over 50% of CYP2GP1 introns were sequenced at least once. Intron sizes were confirmed by PCR with adjacent exon primers.

View larger version (8K): [in this window] [in a new window]   Fig. 1.   Schematic representation of the structure of CYP2A7P2 with close linkage to CYP2GP1. The linked CYP2A7P2 and CYP2GP1 sequences were identified in two different genomic clones, S34 and P1G1, which were isolated from two independent genomic libraries. Putative exons (solid box for CYP2GP1 and open box for CYP2A7P2) were identified on the basis of sequence homology to known CYP2A and CYP2G genes, respectively, and are numbered on top. Solid lines indicate introns and other noncoding sequences. The restriction sites used for subcloning are also indicated. The cloned insert in S34 is about 15 kbp, and that in P1G1 is approximately 85 kbp.

Available sequence upstream of CYP2A7P2 exon 6 in the S34 clone, about 1.8 kbp, was also determined, but sequence with homology to known CYP2A exons 1 through 5 was not detected. In the P1G1 clone, the sequence upstream of CYP2A7P2 exon 6 was at least 4 kbp (Fig. 1). However, CYP2A-like exons 1 through 5 were not identified by sequence analysis of a subcloned 9-kbp BstEII fragment (P1G1-SUBII; Fig. 1) containing CYP2A7P2, with about 4 kbp upstream of exon 6, or by direct sequencing and PCR analyses of the P1G1 clone with numerous primers derived from known CYP2A exons 1 through 5. Since the sizes of intron 5 in CYP2A6 and CYP2A13 are 875 bp and 1719 bp, respectively (Fernandez-Salguero et al., 1995), these results suggest that the putative exons 1 through 5 of CYP2A7P2 have been translocated elsewhere or deleted.

The nucleotide sequence of putative CYP2A7P2 coding regions (exons 6-9) and partial intron sequences and intron sizes are shown in Fig. 2. Exon-intron junctions are predicted according to alignment with other human CYP2A sequences. CYP2A7P2 intron sizes are very similar to those reported for CYP2A6 and CYP2A13 (Fernandez-Salguero et al., 1995). The deduced amino acid sequence (exons 6-9) of CYP2A7P2 is 81.1 to 86.6% identical to corresponding sequences in the other human CYP2As (Fig. 3 and Table 1). Interestingly, the putative coding sequence (exons 1-5) of CYP2A7P1 is 84.1 to 87.7% identical to those of the known human CYP2A proteins (Table 1). In contrast, the sequence identities among CYP2A6, CYP2A7, and CYP2A13 are greater than 90.5%. Thus, it appears that CYP2A7P1 and CYP2A7P2 may be derived from the same ancestral gene, which was corrupted possibly by the insertion at intron 5 of a large genomic fragment translocated from another part of the genome.

View larger version (68K): [in this window] [in a new window]   Fig. 2.   Nucleotide sequence of human CYP2A7P2. The putative exon sequences and exon-intron junctions of CYP2A7P2 were derived from the S34 human genomic clone. Identical sequences were also found in the P1G1 clone. Exon sequences are shown in upper case whereas partial intron and 3'-flanking sequences are shown in lower case with the size of the introns indicated in parentheses. Coding nucleotides are numbered to the right of each line. The dotted lines represent abbreviated sequences. The sequence in brackets is from the CYP2A6 gene, which is complementary to the R4 reverse primer sequence used for genotyping CYP2A6 alleles (Fernandez-Salguero et al., 1995). The putative translation termination codon is underlined, whereas the conserved polyadenylation signal is double-underlined. Sequences corresponding to putative exons 1 through 5 of a CYP2A gene were not detected in either clone. -, a gap introduced in the R4 sequence to obtain optimal alignment. This sequence has been assigned GenBank accession nos. AF296253 through AF296256.

View larger version (105K): [in this window] [in a new window]   Fig. 3.   Comparison of deduced amino acid sequences of different human CYP2A genes and alleles. The alignment was done using the CLUSTAL program in PC/GENE (Oxford Molecular, Beaverton, OR). -, gaps introduced to obtain optimal alignment. Amino acids are numbered to the right of each line; conserved sequences [perfectly conserved (*) and well conserved (·)] are indicated underneath the amino acid. Arrows highlight a nonsense mutation (x) and two single nucleotide deletions (d) in CYP2A7P1. CYP2A6*1 and CYP2A7*1 sequences were based on Yamano et al. (1990); CYP2A6*3 (or CYP2A6v2), CYP2A7*2, and CYP2A7P1 sequences were based on Fernandez-Salguero et al. (1995); CYP2A13 sequence was from Su et al. (2000). For CYP2A7P1 sequence, gaps were introduced at the two single nucleotide deletions in exons 3 and 4, respectively, to maintain the original CYP2A-like reading frame. CYP2A7P2 was deduced from the coding nucleotide sequence from Fig. 2.

Nonsense mutations and frame-shift deletions are present in the putative coding sequences of CYP2A7P1 (Fernandez-Salguero et al., 1995), consistent with its designation as a pseudogene; however, similar loss-of-function mutations were not found in the putative CYP2A7P2 coding sequence. Nevertheless, the deduced amino acid sequence of CYP2A7P2 contained a deletion of a conserved Glu residue at position 330 and an unusually cysteine-rich segment at the carboxyl terminus, which may significantly alter the structure of the putative protein (Fig. 3). It is not known whether a second copy of CYP2A7P2 exists; however, the two copies of CYP2A7P1, CYP2A7PT and CYP2A7PC (Fernandez-Salguero et al., 1995), may have been generated by gene duplication after separation of CYP2A7P1 from CYP2A7P2.

The identification of CYP2A7P2 should facilitate future efforts to study genomic polymorphisms in other human CYP2A genes. However, the presence of CYP2A7P2 should not affect the results of recent CYP2A6 genotyping experiments (e.g., Pianezza et al., 1998) using the primers designed in a previous study (Fernandez-Salguero et al., 1995). The 3'-primer (R4 in Fernandez-Salguero et al., 1995) used in the first round of PCR has three nucleotide differences from the corresponding sequence in CYP2A7P2, one of them at the 3'-terminus, as well as a single nucleotide gap (Fig. 2). Furthermore, nonspecific products generated from CYP2A7P2 are unlikely to be reamplified by the nested PCR with primers derived from exon 3 of CYP2A6 (Fernandez-Salguero et al., 1995). More recent CYP2A6 genotyping protocols (Oscarson et al., 1998; Kitagawa et al., 1999) use primers derived from the 5'-half of the gene and thus would not detect the CYP2A7P2 sequence.

A full-length CYP2B7 gene, but not the functional CYP2B6 gene, was also detected in the P1G1 clone by using Southern blot analysis with oligonucleotide probes and by genomic PCR with primers derived from CYP2B6 and CYP2B7 cDNAs (data not shown). Sequence analysis of the P1G1 clone and a BstEII subclone containing most of the CYP2B7 gene revealed that the coding region was 100% identical to the reported CYP2B7 cDNA sequence, with a nonsense mutation in exon 7 of the gene sequence, as was previously found in a cDNA clone (Yamano et al., 1989). This result confirms that CYP2B7 is likely a pseudogene. Furthermore, the identification of CYP2B7 on P1G1 clone maps CYP2GP1 and CYP2A7P2 to the middle of the CYP2A-2B-2F gene cluster (Hoffman et al., 1995), close to CYP2B7. The precise distance and orientation of the CYP2B7 gene with regard to the CYP2GP1 and CYP2A7P2 genes are not yet known; attempts to map this physical distance were unsuccessful using long-distance PCR. However, the distance between the CYP2B7 gene and the CYP2GP1-CYP2A7P2 fragment may be less than 30 kbp since the entire insert in P1G1 was about 85 kbp, the length of the CYP2B7 gene is estimated to be about 25 kbp (based on the size of the highly homologous CYP2B6 gene, GenBank accession no. AC023172), and the combined length of the characterized sequences in the CYP2GP1-CYP2A7P2 region was at least 30 kbp (Fig. 1 and Sheng et al., 2000). This conclusion is consistent with the lack of detection of other CYP2 genes known to be near CYP2B7 on chromosome 19 (but at least about 35 kbp away from CYP2B7) in the P1G1 clone, including CYP2A6, CYP2A7, CYP2A7P1, CYP2A13, CYP2B6, and CYP2F1 (data not shown). CYP2GP2 was not detected in the P1G1 clone, either. It is not known whether CYP2B7P, which was reported to be about 20 kbp from CYP2B7 (Fernandez-Salguero et al., 1995), is present in the P1G1 clone because details of the structure of this pseudogene are not available. Finally, although a functional human CYP2G gene or allele (Sheng et al., 2000) has yet to be identified, the present finding that CYP2GP1 is located in the middle of the CYP2A-2B-2F gene cluster lends further support to the proposed evolutionary relationship among these four CYP2 subfamilies, which share the highest amino acid sequence similarities and are believed to have evolved from a single ancestral CYP gene (Hoffman et al., 1995).