Intergenic Transcripts Containing a Novel Human Cytochrome P450 2C Exon 1 Spliced to Sequences from the CYP2C9 Gene Susan C. Warner, Csaba Finta, and Peter G. Zaphiropoulos Center for Nutrition and Toxicology, Department of Biosciences at NOVUM, Karolinska Institute, Huddinge, Sweden The cytochrome P450 2C (CYP2C) gene locus was found to include a novel exon 1 sequence with high similarity to the canonical exon 1 of CYP2C18. Rapid ampli®cation of cDNA ends (RACE) and PCR ampli®cations of human liver cDNA revealed the presence of several intergenic species containing the CYP2C18 exon 1±like sequence spliced to different combinations of exonic and intronic sequences from the CYP2C9 gene. One splice variant was found to have an open reading frame starting at the canonical translation initiation codon of the CYP2C18 exon 1± like sequence. Another variant consisted of the nine typical CYP2C9 exons spliced after the CYP2C18 exon 1±like sequence through a segment of CYP2C9 59 ¯anking sequences. Moreover, analysis of bacterial arti®cial chromosome (BAC) clones revealed that the CYP2C18 exon 1±like sequence was located in the intergenic region between the Downloaded from https://academic.oup.com/mbe/article/18/10/1841/1060609 by guest on 23 September 2021 CYP2C19 and CYP2C9 genes. The ®nding that a solitary exon is spliced with sequences from a neighboring gene may be interpreted as representing a general evolutionary mechanism aimed at using the full expression potential of a cell's genomic informational content. Introduction The cytochrome P450 (CYP) superfamily of pro- There is increasing evidence that pre-mRNA splic- teins are heme-containing enzymes that play important ing cannot be explained by the simple mechanism of the roles in the synthesis and degradation pathways of var- spliceosome scanning 59 to 39, removing introns, and ious biomolecules, as well as being involved in the me- joining exons in the order they are found in the primary tabolism and detoxi®cation of numerous xenobiotics transcript. The existence of low-abundant, nontypical (Nelson et al. 1996; Chang and Kam 1999). In all known mRNA molecules has been documented in numerous cases, genes of the same subgroup (CYP subfamily) are studies (reviewed in Finta and Zaphiropoulos 2000c). genomically arranged in clusters, although individual RNA species containing scrambled exons, where con- clusters belonging to the same CYP family may be lo- sensus splice sites have been used to join exons in an cated on different chromosomes (Nelson et al. 1996). A order different from that of the genomic DNA (Nigro et total of around 50 genes belonging to the cytochrome al. 1991; Cocquerelle et al. 1992; Caldas et al. 1998; P450 superfamily have been identi®ed in humans (Nel- Chao et al. 1998; Marcucci et al. 1998; Crawford et al. son 1999). The human CYP2C subfamily is composed 1999; Surono et al. 1999; Takahara et al. 2000), and of four members containing nine exons with well-con- exon repetition in the form of tandem repeats of exons served intron/exon boundaries (Finta and Zaphiropoulos which are not the result of gene duplications (Caudevilla 2000a). The members of the CYP2C subfamily have et al. 1998; Akopian et al. 1999; Frantz et al. 1999; Finta been mapped to a cluster on chromosome 10q24 with and Zaphiropoulos 2000a) are examples of phenomena that indicate that pre-mRNA splicing is indeed a highly the order CYP2C18, CYP2C19, CYP2C9, CYP2C8 complex process. In addition, evidence of the existence (Gray et al. 1995). of RNA molecules possessing properties of circular Pre-mRNAs, the primary transcripts in eukaryotic molecules has been presented (Capel et al. 1993; Coc- cells, undergo a number of posttranscriptional modi®- querelle et al. 1993; Pasman, Been, and Garcia-Blanco cations, including the excision of noncoding intronic se- 1996; Li and Lytton 1999). Furthermore, nontypical quences by large protein/RNA complexes called splice- splicing events may result in the joining of canonical osomes (Smith and ValcaÂrcel 2000). Conserved se- exons with sequences, both exonic and intronic, from quences necessary for the identi®cation of exon/intron pseudogenes or neighboring genes belonging to the boundaries include two intronic dinucleotides, in most same gene family (Benson, Nguyen, and Maas 1995; cases GT and AG, located, respectively, at the 59 and 39 Zaphiropoulos 1999; Finta and Zaphiropoulos 2000b), splice sites (Burset, Seledtsov, and Solovyev 2000; as well as unrelated nearby sequences (Shimamura et al. Smith and ValcaÂrcel 2000). A branch site sequence con- 1998; Rogalla et al. 2000). taining a conserved A nucleotide located close to the 39 The present study was directed at investigating splice site is also required for exon/intron recognition splice variants within the human CYP2C subfamily, spe- (Smith and ValcaÂrcel 2000). ci®cally species encompassing a novel CYP2C18 exon 1±like sequence. Moreover, with the use of six indepen- dent bacterial arti®cial chromosome (BAC) clones, the Key words: pseudogene, alternative promoter, pre-mRNA splic- gene locus containing the CYP2C18 exon 1±like se- ing, gene evolution, Homo sapiens, cytochrome P450. quence was characterized. Address for correspondence and reprints: Center for Nutrition and Toxicology, Department of Biosciences at NOVUM, Karolinska Insti- Materials and Methods tute, SE-141 57 Huddinge, Sweden. E-mail: cDNA Synthesis [email protected]. Mol. Biol. Evol. 18(10):1841±1848. 2001 cDNA was synthesized from human liver total q 2001 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038 RNA using random hexamers or oligo dT primers. A 1841 1842 Warner et al. Table 1 Primers Used in the RACE and PCR Ampli®cations RACE and PCR primers (cDNA) 2C18 exon 1±like, forward ................. 59-TACAGTTGTCTTACTAAGAAGG 2C18 exon 1±like, forward (nested).......... 59-GCGTCGACTCTGGAAGAGGGAGGCTCCTATT 2C9 exon 9, reverse....................... 59-GCAGCCAGGCCATCTGCT 2C9 exon 9, reverse (nested) ............... 59-GCAAATCCATTGACAACTGGAGTG PCR primers (genomic DNA) 2C9 exon 1, forward ...................... 59-AGGCTCCAACCAAGTACA 2C9 exon 1, reverse....................... 59-ATTTGCTGATGTCCTTAATAC 2C9 exon 1, reverse (nested) ............... 59-GAGAATCCATTGAAGCCTT 2C9 exon 5, reverse....................... 59-GAAGTAATCAATGATAGGAGA 2C9 exon 9, forward ...................... 59-CTCTGGTTGACCCAAAGA 2C9 exon 9, reverse....................... 59-CAGCCAGGCCATCTGCT Downloaded from https://academic.oup.com/mbe/article/18/10/1841/1060609 by guest on 23 September 2021 2C18 exon 1±like, forward ................. 59-TACAGTTGTCTTACTAAGAAG 2C18 exon 1±like, forward (nested).......... 59-AAGAGGGAGGCTCCTATT 2C18 exon 1±like, reverse.................. 59-CAATAATCAGGAGAGGAGTA 2C19 exon 1, forward ..................... 59-ATGTTTGCTTCTCCTTTCAA 2C19 exon 1, reverse...................... 59-GGTTAAGGATTTGCTGACA 2C19 exon 9, forward ..................... 59-CTGTTTTTATTCCTGACCTT 2C19 exon 9, reverse...................... 59-GGAATGAAGCACAGCTGA 20-ml reaction mixture containing M-MLV Reverse athon cDNA Ampli®cation Kit (Clontech). The nested Transcriptase buffer (Promega), 1 mM dNTPs, 20 U gene-speci®c primer (table 1) was designed to include a RNasin (Promega), 30 pmol random hexamer primer SalI site to be used for subsequent cloning together with (Promega) or 10 pmol oligo dT primer (Promega), 10 the NotI site in the nested universal primer. Ampli®ca- mg RNA (Clontech), and 200 U M-MLV Reverse Tran- tions were carried out in a reaction mixture containing scriptase RNase H (2) Point Mutant (Promega) was in- Expand buffer 1, 350 mM dNTPs, 90 pmol gene-speci®c cubated at 428C for 1.5 h. primer (table 1), 10 pmol of the universal primer, and 3.5 U of Expand enzyme. Denaturation was at 948C for Isolation of BAC DNA 1 min, annealing at 608C (initial ampli®cation) or 708C (nested ampli®cation) for 1 min, and extension at 728C BAC clone RP11-400G3 was obtained from BAC- for 1±3 min (14 s/cycle). PAC Resources. BAC clones CIT-342L6, RP11-622P11, CIT-2061B9, CIT-2339N22, and CIT-3052K18 were supplied by Research Genetics. BAC DNA for PCR Intergenic PCR analysis of the six BAC clones for the presence of For the set of experiments performed to identify CYP2C19 exon 9, CYP2C9 exon 1, and the CYP2C18 intergenic products containing the CYP2C18 exon 1± exon 1±like sequence was isolated using the JetQuick like sequence, 1 ml human liver cDNA synthesized as Plasmid Miniprep kit (Genomed), but with phenol/chlo- described above was the template in all cases. The re- roform extractions and an ethanol precipitation instead action mixture for ampli®cations using Taq DNA poly- of column puri®cation. BAC DNA for the Southern blot merase (Promega) contained Taq buffer, 2 mM MgCl2, was obtained using the Concert High Purity Plasmid 200 mM dNTPs, 90 pmol of each primer (table 1), and Maxiprep System (Life Technologies) with an isopro- 5UTaq polymerase. With Expand polymerase, the re- panol precipitation included before column loading. action mixture was as described above. For the Perkin Elmer 480 thermal cycler, denaturation was at 948C for PCR Ampli®cations 1 min, annealing at the appropriate temperature (1±48C PCR ampli®cations were performed using either below the calculated melting temperature) for 1 min, and the Perkin Elmer 480 thermal cycler or the MJ Research extension at 728C for 1±3 min (14 s/cycle). When using PTC-200 DNA Engine. All reactions were carried out the MJ Research PTC-200 DNA Engine, ampli®cations for 30 cycles, unless stated otherwise, in a total volume were performed with 10 s at 928C, 30 s at the appro- of 50 ml using the hot start method. For the nested rapid priate annealing temperature, and 2 min at 728C. ampli®cation of cDNA ends (RACE) and intergenic PCR ampli®cations, 1 ml of the initial ampli®cation Genomic PCR products was used. Gene-speci®c primers (table 1) were synthesized by Cybergene AB (Huddinge, Sweden). The BAC clones were characterized by PCR using Taq DNA polymerase essentially as described above. Denaturation was at 928C for 10 s, annealing at the ap- 39 RACE Ampli®cations propriate temperature for 20 s, and extension at 728C The RACE reactions were performed on 5 ml Mar- for either 40 s or 1±3 min (14 s/cycle).