Center for Translational Research in Cancer, Division of Experimental Medicine, Sir Mortimer B. DavisJewish 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 1990
). The natural evolution of GSTs has occurred by both convergent and divergent pathways (Pemble and Taylor 1992
; Ji et al. 1995
). 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. 1997
), 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. 1997
).
The three-dimensional structures of GSTs have a conserved chain fold of the glutathione-binding domain (Wilce and Parker 1994
). 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 1985
). The generation of isozymes with novel substrate specificities has been attributed to gene conversion and exon shuffling (Mannervik 1985
).
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 1992
), and the sigma class enzymes diverged from an ancestral precursor prior to the divergence of the alpha/mu/pi precursor (Ji et al. 1995
). 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 1999
). 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 1996
). 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 ).
|
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 1999
) using primers 13 (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.
|
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 1997
). 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 1996
; Fotouhi-Ardakani and Batist 1999
). 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. 1996
). It is well known that the rGSTA3 enzyme has very high specific activity toward alkylating agents such as melphalan (Bolton, Colvin, and Hilton 1991
). 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 1985
), 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. 1992
). 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.
|
![]() |
Acknowledgements |
---|
![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
1 Keywords: class alpha GST
gene duplication
Rattus sp.
genomics.
2 Address for correspondence and reprints: Gerald Batist, Center for Translational Research in Cancer, Sir Mortimer B. DavisJewish General Hospital, Montreal, QC, Canada. E-mail: gbatist{at}onc.jgh.mcgill.ca
![]() |
literature cited |
---|
![]() ![]() ![]() |
---|
Altschul, S. F., T. L. Madden, A. A. Schäffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:33893402.
Arca, P., C. Hardisson, and J. E. Suarez. 1990. Purification of a glutathione S-transferase that mediates fosfomycin resistance in bacteria. Antimicrob. Agents Chemother. 34:844848.[ISI][Medline]
Board, P. G., R. T. Baker, G. Chelvanayagam, and L. S. Jermiin. 1997. Zeta, a novel class of glutathione transferases in a range of species from plants to humans. Biochem. J. 328:929935.[ISI][Medline]
Bolton, M. G., O. M. Colvin, and J. Hilton. 1991. Specificity of isozymes of murine hepatic glutathione S-transferase for the conjugation of glutathione with L-phenylalanine mustard. Cancer Res. 51:24102415.[Abstract]
Buetler, T. M., T. K. Bammler, J. D. Hayes, and D. L. Eaton. 1996. Oltipraz-mediated changes in aflatoxin B1 biotransformation in rat liver: implications for human chemointervention. Cancer Res. 56:23062313.[Abstract]
Fotouhi-Ardakani, N., and G. Batist. 1999. Genomic cloning and characterization of the rat glutathione S-transferase A3 subunit gene. Biochem. J. 339:685693.[ISI][Medline]
Ji, X., E. C. Von Rosenvinge, W. W. Johnson, S. I. Tomarev, J. Piatigorsky, R. N. Armstrong, and G. L. Gilliland. 1995. Three-dimensional structure, catalytic properties, and evolution of a sigma class glutathione transferase from squid, a progenitor of the lens S-crystallins of cephalopods. Biochemistry 34:53175328.
Mannervik, B. 1985. The isoenzymes of glutathione transferase. Adv. Enzymol. Relat. Areas Mol. Biol. 57:357417.[Medline]
Nadeau, J. H., and D. Sankoff. 1997. Comparable rates of gene loss and functional divergence after genome duplications early in vertebrate evolution. Genetics 147:12591266.
Ohno, S. 1985. Dispensable genes. Trends Genet. 1:160164.[ISI]
Pemble, S. E., and J. B. Taylor. 1992. An evolutionary perspective on glutathione transferases inferred from class-theta glutathione transferase cDNA sequences. Biochem. J. 287:957963.[ISI][Medline]
Pulford, D. J., and J. D. Haye. 1996. Characterization of the rat glutathione S-transferase Yc2 subunit gene, GSTA5: identification of a putative antioxidant-responsive element in the 5'-flanking region of rat GSTA5 that may mediate chemoprotection against aflatoxin B1. Biochem. J. 318:7584.[ISI][Medline]
Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:54635467.
Wilce, M. C. J., and M. W. Parker. 1994. Structure and function of glutathione S-transferases. Biochim. Biophys. Acta. 1205:118.[ISI][Medline]
Yamada, T., Y. Muramatsu, M. Yasue, T. Agui, J. Yamada, and K. Matsumoto. 1992. Chromosomal assignments of genes for rat glutathione S-transferase Ya (GSTA1) and Yc subunits (GSTA2). Cytogenet. Cell Genet. 61:125127.[ISI][Medline]