Evidence for Genomic Duplication of the Glutathione Transferase A3 Gene in Genus Rattus

Nasser Fotouhi-Ardakani, Robyn L. Schecter and Gerald Batist1,

Center for Translational Research in Cancer, Division of Experimental Medicine, Sir Mortimer B. Davis–Jewish General Hospital, McGill University, Montreal, Quebec, Canada

The glutathione transferases (GSTs) are a multigene family of detoxifying enzymes that are present in all eukaryotes and some bacteria. This group of enzymes has evolved to protect cells from toxic endogenous and xenobiotic electrophilic compounds. There are three distinct superfamilies of enzymes with glutathione transferase activity, namely, the cytosolic, membrane-bound, and metallo forms (Arca, Hardisson, and Suarez 1990Citation ). The natural evolution of GSTs has occurred by both convergent and divergent pathways (Pemble and Taylor 1992Citation ; Ji et al. 1995Citation ). The evolutionary pressure driving the diversification of GSTs is provided by the reactive oxygen species which are generated in cells exposed to an aerobic environment. The cytosolic enzymes are the best characterized examples of divergent evolution.

GSTs are extremely diversified and are grouped into a number of classes, designated alpha, mu, pi, sigma, theta, zeta, etc. (Board et al. 1997Citation ), which originate from an ancient group of proteins. Each class is composed of several subunits, of which the biologically active forms are either homo- or heterodimeric proteins. A common feature of the GSTs is their ability to bind glutathione; another property is their ability to recognize and detoxify compounds with diverse chemical structures. The evolution of the GSTs in which relatively low binding affinities are offset by broad substrate specificities constitutes an energy-efficient response to toxin exposure. Although substrate specificities are somewhat overlapping, the enzymes are critical to eukaryotic organisms in that they display unique activities toward a variety of harmful compounds (Board et al. 1997Citation ).

The three-dimensional structures of GSTs have a conserved chain fold of the glutathione-binding domain (Wilce and Parker 1994Citation ). The second domain is more variable and is principally involved with the electrophilic substrate. The structural differences contribute to the differential substrate selectivity seen between isozymes (Mannervik 1985Citation ). The generation of isozymes with novel substrate specificities has been attributed to gene conversion and exon shuffling (Mannervik 1985Citation ).

Although the evolutionary relationships of the various isoenzyme classes are not entirely certain, analysis of both the sequences and the exon-intron boundaries of several GST genes provides some insight. For example, the theta class protein is thought to be the evolutionary precursor for genes encoding alpha, mu, and pi proteins (Pemble and Taylor 1992Citation ), and the sigma class enzymes diverged from an ancestral precursor prior to the divergence of the alpha/mu/pi precursor (Ji et al. 1995Citation ). The relatedness of one subunit to another is determined primarily on the basis of sequence comparisons. However, there are no established criteria for the extent of sequence similarity necessary for a subunit to be considered a member of one class over another.

The isolation and structural organization of two overlapped phage clones of the glutathione transferase A3 gene of the Rattus sp. (rGSTA3 gene) was recently reported by our laboratory (Fotouhi-Ardakani and Batist 1999Citation ). During the course of analysis of the exon/intron splicing junctions of the rGSTA3 gene, we detected the presence of some nucleotide sequences identical to the 5'-flanking region of the rGSTA5 gene (Pulford and Hayes 1996Citation ). In order to confirm and extend this result and to elucidate the molecular evolution of the GST multigene family, the sequencing of the entire region of intron 1 through intron 2 of the rGSTA3 subunit gene was performed in this study.

We identified a nucleotide fragment of 690 bp in the 3' region of intron 1 of the rGSTA3 subunit gene which is similar to a segment of the 5'-flanking region (nucleotides -483 to +207) of the rGSTA5 gene (fig. 1A ). This region of the rGSTA5 with a high degree of nucleotide sequence identity to the 3' end of intron 1 of the rGSTA3 consists of a transcription start site, a TATA box, and two putative enhancer elements, namely hepatocyte nuclear factor-5 (HNF-5 at -392 bp), a liver- specific consensus binding site, and an antioxidant-responsive element (ARE) located at -421 bp (fig. 1A ). The sequence analysis revealed that HNF-5 and ARE consensus elements in intron 1 of the rGSTA3 clones were modified by one and two bases, respectively (fig. 1B ).



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Fig. 1.—A, The schematic diagram of the 5' region of the rGSTA5 and A3 genes. The regions of the rGSTA3 clone (analyzed in this study) that contain sequences identical to the segments of the rGSTA5 are located between two dotted lines. Open boxes represent untranslated exons, closed boxes represent coding exons, +1 indicates transcription start sites, ATG indicates translation start sites, and the positions of the primers used in PCR analysis are marked with arrowheads. The modified structure of the rGSTA5 gene was obtained from Pulford and Hayes (1996). B, Nucleotide sequence of the rGSTA3 subunit gene spanning the 3' region of intron 1 through a portion of intron 2. The nucleotide sequences of the identical regions in the rGSTA3 and A5 subunits are aligned, and dashed lines in A3 show nucleotides which are identical to those in rGSTA5. The gaps (*) were made to generate maximum alignments. The respective coding exons are shown in capital letters. The nucleotide sequences of the rGSTA5 gene were obtained from Pulford and Hayes (1996). +1 indicates the transcription start site of rGSTA5, ATG indicates start codons, and intron/exon splicing junctions are indicated by curved arrows. The positions of the primers used in PCR analysis are underlined. Dideoxy sequencing reactions (Sanger, Nicklen, and Coulson 1977) were performed using T7 Sequenase, version 2.0 (Amersham). The BLAST (basic local alignment search tool) program of the GenBank database (Altschul et al. 1997) was employed for construction of the sequence alignments

 
Similarly, the 5' end of intron 2 of the rGSTA3 clone has a stretch of 102 bp identical to the nucleotide sequences located in the 5' end of intron 1 of the rGSTA5 clone (fig. 1A and B ), forming the identical regions of 900 bp in length.

To verify that our observation was a true finding and not the result of cloning artifact, polymerase chain reaction (PCR) analysis was employed using the genomic DNAs from a rat mammary carcinoma cell line (MatB) and rat liver tissue, respectively. PCR reactions were performed as described previously (Fotouhi-Ardakani and Batist 1999Citation ) using primers 1–3 (fig. 1 ), where primer 1 was designed to be specific only to rGSTA3. The PCR products were analyzed on a 1% agarose gel (fig. 2A ) and then sequenced directly following the ds DNA cycle sequencing protocol (Gibco-BRL). PCR analysis (fig. 2A ) and sequencing of the products showed that the 3' region of intron 1 of rGSTA3 with high identity to 5'-flanking region of rGSTA5 is actually present in cells and tissues.



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Fig. 2.—A, PCR analysis of the 3' region of intron 1 of the rGSTA3 subunit gene. Agarose gel electrophoresis shows the PCR products from MatB cell line and rat liver tissue, in addition to a cloned phage. The PCR fragments were isolated from the gel and sequenced directly. M = molecular size marker in kb ({lambda} DNA cleaved with HindIII). Lanes 1 and 4, a rGSTA3 phage clone, {lambda}BF7 (Fotouhi-Ardakani and Batist 1999); lanes 2 and 5, MatB cells; lanes 3 and 6, rat liver tissue. Oligonucleotide primers used in the PCR analysis were primer 1 (5'-gaggggcacctgagtagttcttagc-3'), primer 2 (5'-ccagtttagtgtatccatcgg-3'), and primer 3 (5'-ggcttccccggcatggcagca-3'). The positions of these primers are indicated in figure 1 . B, Lack of functional activity of the rGSTA3 intronic sequences. The CAT expression plasmids, carrying the 3' region of intron 1 of the rGSTA3 gene (A) or its homologous rGSTA5 promoter region (-647 to +192; see fig. 1B ), were used to transfect HepG2 cells. The CAT assays were performed as described previously (Fotouhi-Ardakani and Batist 1999). pRSV-CAT and pCAT-Basic plasmids were used as positive and negative controls, respectively. The transfection efficiency was normalized by cotransfection with the plasmid containing the ß-galactosidase gene. These experiments were repeated at least three times in triplicate

 
In order to determine whether this region of the rGSTA3 clone with 95% identity to the regulatory region of the rGSTA5 clone has been altered to nonfunctionality or evolved a new function, we tested the rGSTA3 intronic sequence (fig. 1B ) and its homologous rGSTA5 promoter region in the same transient transfection experiments. The rGSTA5 sequence was included in the experiment as a control. The transcriptional activity of the rGSTA5 promoter was previously demonstrated by Pulford and Hayes (1996)Citation . The transfection and chloramphenicol acetyltransferase (CAT) assay conditions were as described elsewhere (Fotouhi-Ardakani and Batist 1999Citation ). Briefly, the PCR-generated DNA fragments of the respective regions of both subunit genes were inserted into the pCAT-Basic reporter plasmids. These constructs were then transiently transfected into the HepG2 (human hepatoma) cell line. The proteins were extracted 36 h after the transfection, and then the CAT assay analysis was performed (fig. 2B ). The transfection of the HepG2 cell line with this region of the rGSTA3 gene showed that it lacks the capability of modulating transcriptional activity, and, indeed, these intron sequences are nonfunctional under normal conditions (fig. 2B ).

An important force in genome evolution is gene duplication. Recently, in an empirical study, it was suggested that approximately half of all duplicated genes become functionally divergent and novel, while the remaining half become pseudogenes (Nadeau and Sankoff 1997Citation ). The ongoing large-scale sequencing of eukaryotic genomes is an excellent tool for providing, among other things, direct information about gene duplication events. In this paper, we report a remarkable sequence identity between rGSTA3 and A5 subunit genes which extends beyond their coding regions. Genomic sequences of the 711-bp regions upstream of their start codons contain a total of 38 individual nucleotide modifications that account for a 5% difference between the two subunits. In addition, there are a total of 48 base pair differences in the coding regions of the rGSTA3 and rGSTA5 subunit genes (Pulford and Hayes 1996Citation ; Fotouhi-Ardakani and Batist 1999Citation ). These differences in sequence between rGSTA3 and A5 will translate into structural differences and explain the differences in their substrate specificities. For example, the rGSTA5 homodimer was reported to exhibit 120- to 150-fold higher aflatoxin B1 exo-8, 9-epoxide conjugating activity than the rGSTA3 homodimer (Buetler et al. 1996Citation ). It is well known that the rGSTA3 enzyme has very high specific activity toward alkylating agents such as melphalan (Bolton, Colvin, and Hilton 1991Citation ). However, there is no report in the literature of such activity for rGSTA5. The relationship between the coding exons and a high conservation of nucleotide sequence in the intervening sequences reflects a gene duplication event during the evolution of the rat genome. This duplication event presumably led to mutations with ultimate diversification and functional benefits. The striking 95% identity between the promoter region of the rGSTA5 gene and the intronic region of the rGSTA3 gene suggests that this duplication event occurred relatively recently. Assuming the spontaneous mutation rate of 10-9/bp/year for vertebrates (Ohno 1985Citation ), we estimate that the duplication event of this region of rGSTA3 took place approximately 53 MYA.

In humans, it has been well documented that the GST genes occur in class-specific clusters on different chromosomes. Although there are less data available for other species, there are probably similar arrangements in other mammals. For example, mouse GST (mGST) alpha and pi genes are clustered on chromosomes 9 and 1, respectively. In rats, GST Yc genes (GSTA3 and GSTA5) are clustered on chromosome 9 (Yamada et al. 1992Citation ). This strongly supports the observation that the evolution of the GSTs has involved multiple gene duplication events.

In summary, we report evidence for a gene duplication within the alpha class of the GSTs in rats. These data suggest that the rGSTA3 subunit evolved as a duplication of the rGSTA5 subunit gene, increasing the range of catalytic activity within this class and affording further protection to deleterious agents.



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Fig. 1 (Continued)

 

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This work was supported by a grant from the National Cancer Institute of Canada. We wish to thank Dr. Marco Di Fruscro for critical reading of this manuscript. The nucleotide sequence reported in this study is deposited in the DDBJ/EMBL/GenBank with the accession number AF146746.


    Footnotes
 
Simon Easteal,

1 Keywords: class alpha GST gene duplication Rattus sp. genomics. Back

2 Address for correspondence and reprints: Gerald Batist, Center for Translational Research in Cancer, Sir Mortimer B. Davis–Jewish General Hospital, Montreal, QC, Canada. E-mail: gbatist{at}onc.jgh.mcgill.ca Back


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Accepted for publication October 13, 1999.