- Email: [email protected]
Structural Organization of the Human Microsomal GlutathioneS-Transferase Gene (GST12)
GENOMICS
36, 100–103 (1996) 0429
ARTICLE NO.
Structural Organization of the Human Microsomal Glutathione S-Transferase Gene (GST12) MICHAEL J. KELNER,*,1 MIRIAM N. STOKELY,* NICOLE E. STOVALL,*
AND
MARK A. MONTOYA*
*Department of Pathology, University of California, San Diego, 200 West Arbor Drive, San Diego, California 92103-8320 Received February 1, 1996; accepted June 18, 1996
The primary structure of the human microsomal glutathione S-transferase gene (GST12) was determined by genomic cloning. The gene structure of GST12 spans 12.8 kb and consists of four exons and three introns. The coding sequence resides on exons 2, 3, and 4. Sequencing of the exons revealed two nucleotide differences compared to a previous report of the cDNA sequence. The substitutions, however, were silent, as they did not alter amino acid composition or restriction enzyme sites. All introns commenced with nucleotides GTAA at the 5* boundary and ended with nucleotides AG at the 3* boundary, in agreement with the proposed consensus sequence for intron spliced donor and acceptance sites. The presence of an in-phase stop codon and an upstream false start codon in the 5*-untranslated region was confirmed. Although it was previously predicted that there existed another start codon in-phase and within 50 bp of this stop codon, coding for a second mini-cistron, we could not identify another start codon for greater than 200 bp prior to the stop codon. Thus, initiation is suppressed at the first or false start codon due to either the closeness of the stop codon or the suboptimal context of the codon. q 1996 Academic Press, Inc.
INTRODUCTION
The glutathione S-transferases (EC 2.5.1.18) are a family of enzymes catalyzing the conjugation of glutathione to a wide variety of lipophilic electrophiles that include many environmental mutagens and carcinogens (Morgenstern and DePierre, 1985). The enzymes exist in many different cytosolic isozymes as well as a microsomal form (GST12)2. The microsomal form (monomer Mr 17,300) resembles cytosolic enzymes in displaying broad substrate specificity and some inhibiSequence data from this article have been deposited with GenBank under Accession Nos. U46494–U46499. 1 To whom correspondence should be addressed. Telephone: (619) 543-5976. Fax: (619) 543-3730. E-mail: [email protected] sd.edu. 2 The human microsomal glutathione transferase gene was designated GST12 in 1989 at the Tenth International Workshop on Human Gene Mapping.
0888-7543/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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tion characteristics (Morgenstern and DePierre, 1985). The microsomal form, however, is immunologically distinct (Morgenstern et al., 1982) and has an amino acid sequence (Morgenstern et al., 1985) and a cDNA sequence (DeJong et al., 1988) unrelated to the cytosolic glutathione transferases. The microsomal enzyme exists functionally as a trimer of a 154-amino-acid subunit (Anderson et al., 1994; Hebert et al., 1995), whereas the cytosolic forms exist as homo- or heterodimers. The microsomal enzyme is the predominant leukotriene C4 membrane-bound binding site (Metters et al., 1993). A variety of environmental toxins are substrates for microsomal glutathione transferase including benzo(a)pyrene-4,5 oxide (Morgenstern et al., 1988), carbon tetrachloride (Reiter and Burk, 1988), and 1,2-dibromo-3-chloropropane (Miller et al., 1986). Recent evidence also indicates that renal toxicity of some halogenated chemicals is attributable to glutathione Sconjugate formation, then metabolism of these glutathione S-conjugates to corresponding cysteine S-conjugates, and bioactivation of the cysteine S-conjugates to nephrotoxins (Anders et al., 1988). Evidence also indicates that microsomal glutathione S-transferase is responsible for scavenging lipid soluble radicals (Mosialou and Morgenstern, 1989). The microsomal glutathione transferase is activated in vitro by a variety of methods including exposure to electrophiles, thiodisulfide interchange, protein-dimer formation, proteolysis, oxidation, and removal of an unidentified endogenous microsomal inhibitor (Morgenstern et al., 1979; Boyer et al., 1986; Aniya and Anders, 1989a,b, 1992; Lundqvist and Morgenstern, 1992). The role of promoter induction for regulation of cytosolic glutathione transferases is well documented. The role of induction for microsomal glutathione transferase has been studied, but only to a limited extent. Most inducers of cytosolic glutathione transferases have no effect on the microsomal activity, but some exceptions are noted in the literature. There is a mild, but consistent, increase in microsomal glutathione transferase activity in mice treated with 2(3)-ter-butyl-4-hydroxyanisole (Morgenstern and Dock, 1982). A more recent study noted that isoniazid, B-nap-
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thoflavone, clofibrate, and isosafrole produced moder- ferase was located in this P1 clone. A SacI/SacI 16.5-kb ate increases in microsomal glutathione transferase subclone was isolated and subcloned into pBluescript activity and mRNA content (Waxman et al., 1992), KS(0) at the SacI site. Sequencing confirmed that the suggesting that induction could have a role in regula- clone contained a region corresponding to the initial 5*tion of enzyme activity. untranslated sequence region and contained the entire The gene for GST12 is located on human chromo- gene. Restriction enzyme mapping and nucleotide sesome 12 (DeJong et al., 1990), but the structural orga- quencing revealed that the regions corresponding to nization of the gene is unknown. To determine the the initial 5* and the terminal 3* ends of the mRNA relevance of induction in regulation of microsomal glu- spanned 12.8 kb (Fig. 1). Characterization of the tathione transferase, we isolated a clone containing GST12 gene revealed four exons and three introns (Fig. the entire human gene. This will allow the construc- 1). The nucleotide sequences of all exons and their tion of promoter – reporter gene constructs for use in flanking regions were determined. The coding sequence xenobiotic gene induction studies. resides on exons 2, 3, and 4. The sequencing of the exons revealed two nucleotide differences compared to a previous report of a cDNA sequence (DeJong et al., MATERIALS AND METHODS 1988). The C residue in the third position for Arg 73 Materials and Radiochemicals. [a-35S]dATP was purchased from in the cDNA sequence (DeJong et al., 1988) was noted Amersham Life Science Corp. (Arlington Heights, IL). Restriction to be a G (silent mutation) in the genomic sequence enzymes were from Boehringer-Mannheim (Indianapolis, IN). The (exon 3). There was also an A r G change in the 3*pBluescript II KS(0) plasmid was from Stratagene Corp. (La Jolla, untranslated region (nucleotide 560) in exon 4 of the CA). Sea Plaque GTG agarose was from FMA BioProducts (Rockland, ME). Oligonucleotides were provided by the UCSD Cancer Center genomic clone. These substitutions did not result in a change in restriction enzyme sites. All introns started Core Facility. with nucleotides GTAA at the 5* boundary and ended Isolation of P1 clone. A probe corresponding to the last 409 nucleotides of human microsome glutathione transferase cDNA (DeJong with nucleotides AG at the 3* boundary (Table 1). This et al., 1988) was generated from a pCD human cDNA library (Dr. agrees with the proposed consensus sequence for intron Paul Berg, Stanford, CA) using PCR amplification. The sequences of spliced donor and acceptance sites, indicating that all the oligonucleotides used for PCR screening were CAT GGC GTA CAG GTT GCT GAA AAG TA corresponding to nucleotide 502 of the splicing sites between exons and introns of the human GST12 gene are functional (Mount, 1982). cDNA and GCATTC TTT AAA TTC TTT ATT TGA TG corresponding to nucleotide 885 of the cDNA (DeJong et al., 1988). The entire probe The nucleotide sequences of the 5*-flanking region was sequenced to confirm nucleotide identity. A human genomic fore(U46494), the 3*-flanking region (U46499), all four exskin fibroblast bacteriophage P1 library (Genome Systems, St. Louis, MO) was screened by PCR using these oligonucleotides. Three clones ons, the entire first intron, and the initial 5* and final 3* were identified as positive by PCR amplification. The PCR amplifica- regions of the last two introns are available (U46495– tion product from one of the clones, 2625, was sequenced to confirm U46498) (Fig. 1). In agreement with a previous report that the product corresponded to nucleotides 502 to 885 of the GST12 on partial cDNA sequence (DeJong et al., 1988), an incDNA sequence. phase stop codon and an out-of-phase false start codon PCR conditions. PCR parameters were 1 cycle at 957C for 30 s and were present 5* to the authentic ATG start codon. A 727C for 1 min; 45 cycles at 957C for 30 s, 497C for 30 s, and 727C for 30 s; and 1 cycle at 727C for 5 min. The reaction mix contained dNTPs at second in-phase start codon that was previously predicted to exist (De Jong et al., 1988) was not present. 200 mM KCl at 75 mM, magnesium chloride at 1.5 mM, pH 8.8. DNA sequence analysis. All plasmids were isolated and purified Regions approximately 3 kb upstream and 1.5 kb downusing Qiagen-tip 100 cartridges (Chatsworth, CA). Nucleotide sestream from the first and fourth exons, respectively, quence analysis was performed by Sanger’s dideoxynucleotide chain- were sequenced (Fig. 1). A DNA database search using termination method (Sanger et al., 1977, 1980) with sequencing the BLAST algorithms failed to find any homology to grade Taq DNA polymerase and the fmol thermocycling DNA sequencing kit by Promega Corp. (Madison, WI). A sequence labeling these two flanking regions or to any of the other regions presented (Fig. 1). reaction was performed using the Direct Incorporation SEQ protocol with 7deazaGTP to minimize band compression often associated with the high G / C content in genomic DNA. Oligonucleotide primers complementary to defined human genomic DNA sequences were also used to perform some of the sequencing reactions. Oligonucleotide sequences were determined on both strands of the DNA inserts. To ensure accuracy of the sequencing data, the gene was sequenced in each direction in overlapping fragments, and autoradiograms containing sequence letters were proofread after data entry.
RESULTS
Three clones were isolated from the human genomic P1 library. One clone, 2625, with a 75-kb insert, was chosen for further studies. Sequencing of the insert obtained from PCR amplification of this clone confirmed that the gene for microsomal glutathione trans-
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DISCUSSION
The human microsomal glutathione transferase gene (GST12) spans 12.8 kb and consists of four exons (Fig. 1). With the exception of the 3* end of exon 1, all exon– intron junctions follow the general consensus sequence for exon–intron junctions. Two nucleotide differences, compared to a partial cDNA sequence previously reported, were noted in the exons. These two changes were silent, as they did not result in amino acid changes nor did they alter restriction enzyme sites. The presence of an in-phase stop codon and an upstream false start codon (from the authentic start codon) in the 5*-untranslated region was confirmed. The
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KELNER ET AL.
FIG. 1. Structure of the human microsomal glutathione transferase (GST12) gene. Black boxes indicate exons of the gene. Numbers below the exons refer to the corresponding nucleotides in the mRNA sequence previously reported (DeJong et al., 1988). Regions sequenced, and their corresponding GenBank accession numbers, are noted above the map.
presence of the in-phase stop codon eliminates the possibility of an encoded microsomal leader sequence, so cotranslation insertion must be operative using internal sequence signals. The presence of the in-phase stop codon prior to the authentic start codon and the lack of a second in-phase start codon more than 300 bases upstream of the mRNA site are intriguing. According to the scanning model for eukaryotic translation initiation, the presence of an upstream start codon that codes for a heptadecapeptide that terminates past the authentic start codon should depress translation initiation at the authentic AUG (Kozak, 1987a, 1989). Ribosomes, however, may reinitiate at a downstream AUG provided a termination codon exists 5* in close proximity to the upstream codon and is functionally active (Kozak, 1987b). The in-phase stop codon is strategically located to alleviate the inhibition expected by the heptadecapeptide open reading frame if it is functionally active and terminates for another upstream reading frame. Such an arrangement would allow for translation initiation at the downstream authentic start codon (Kozak, 1987b). This arrangement, whereby the upstream inphase TGA terminates a second mini-cistron, requires the presence of another in-phase start codon in the
early 5*-untranslated region of mRNA. This arrangement would allow for translation and initiation at the downstream authentic start codon. This arrangement, however, does require the presence of another in-phase start codon in the early 5* region of the mRNA. As the initial cDNA clone was known not to be full length (DeJong et al., 1988) but to be missing approximately 40 nucleotides of the initial 5* region due to the presence of an internal EcoRI restriction enzyme site, it was presumed that another in-phase AUG was present in this unrecovered region. However, we could not identify another start codon for greater than 200 bases prior to the internal EcoRI site. Thus, there is no second mini-cistron present, and initiation should be depressed at the authentic start codon per the scanning model for eukaryotic translation initiation. There are two mechanisms that may explain why the first start codon is suppressed. It is possible that the closeness of the stop codon to the first false start codon (TGA A ATG) suppresses initiation at the false start codon. The second possibility is that the first AUG codon occurs in a suboptimal context, as it is lacking the critical G position at 4/ (Kozak, 1986, 1987a, 1995; Cavener and Ray, 1991). The gene for microsomal glutathione transferase
TABLE 1 Nucleotide Sequences of the Exon/Intron Junctions of Microsomal Glutathione Transferase Exon-3* 1 2 3
CAAGTT AGAAAG ACGGAG
Consensus sequence
AG
5*-Intron-3* GTAAGT GTAAGA GTAAAC A GT-A G
779 bp Ç3000 bp Ç6100 bp
5*-Exon TTTAAG GGATAG CCACAG C -AG T
ATTCCA GGTTTT AGCCCA
2 3 4
NNN
Note. Over 200 bp of the 5*- and 3*-flanking regions surrounding each exon was sequenced. Six nucleotides on both sides of the boundaries are provided.
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MICROSOMAL GLUTATHIONE TRANSFERASE (GST12)
(GST12) was previously assigned to human chromosome 12 (DeJong et al., 1990). Several regions of the gene both upstream and downstream were sequenced (Fig. 1). Despite recent publication of a detailed map of chromosome 12 (Krauter et al., 1995), a search of worldwide data banks failed to find homology. Thus, at this time, we cannot identify the neighboring genes. ACKNOWLEDGMENT This work was supported by NIH Grants ES04989 and CA52310.
REFERENCES Anders, M. W., Lash, L., Dekant, W., Elfarra, A. A., and Dohn, D. R. (1988). Biosynthesis and biotransformation of glutathione Sconjugates to toxic metabolites. Crit. Rev. Toxicol. 18: 311–314. Anderson, C., Weinander, R., Lundqvist, G., DePierre, J. W., and Morgenstern, R. (1994). Functional and structural membrane topology of rat liver microsomal glutathione transferase. Biochem. Biophys. Acta 1204: 298–304. Aniya, Y., and Anders, M. W. (1989a). Regulation of rat liver microsomal glutathione S-transferase activity by thiol/disulfide exchange. Arch. Biochem. Biophys. 270: 330–334. Aniya, Y., and Anders, M. W. (1989b). Activation of rat liver microsomal glutathione S-transferase by reduced oxygen species. J. Biol. Chem. 264: 1998–2002. Aniya, Y., and Anders, M. W. (1992). Activation of rat liver microsomal glutathione S-transferase by hydrogen peroxide: Role for protein-dimer formation. Arch. Biochem. Biophys. 296: 611–616. Boyer, T. D., Vessey, D. A., and Kempner, E. (1986). Radiation inactivation of microsomal glutathione S-transferase. J. Biol. Chem. 261: 16963–16968. Cavener, D. R., and Ray, S. C. (1991). Eukaryotic start and stop translation sites. Nucleic Acids Res. 19: 3185–3192. DeJong, J. L., Morgenstern, R., Jornvall, H., DePierre, J. W., and Tu, C-P. D. (1988). Gene expression of rat and human microsomal glutathione S-transferases. J. Biol. Chem. 263: 8430–8436. DeJong, J. L., Mohandas, T., and Tu, C-P. D. (1990). The gene for the microsomal glutathione S-transferase is on human chromosome 12. Genomics 6: 379–382. Hargus, S. J., Fitzsimmons, M. E., Aniya, Y., and Anders, M. W. (1991). Stereochemistry of the microsomal glutathione Stransferase catalyzed addition of glutathione to chlorotrifluoroethene. Biochemistry 30: 717–721. Hebert, H., Schmidt-Krey, I., and Morgenstern, R. (1995). The projection structure of microsomal glutathione transferase. EMBO J. 14: 3864–3869. Kozak, M. (1986). Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44: 283–292. Kozak, M. (1987a). An analysis of 5*-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15: 8125–8132. Kozak, M. (1987b). Effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosomes. Mol. Cell. Biol. 7: 3438– 3445. Kozak, M. (1989). The scanning model for translation: An update. J. Cell Biol. 108: 229–241. Kozak, M. (1995). Adherence to the first-AUG rule when a second
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AUG codon follows closely upon the first. Proc. Natl. Acad. Sci. USA 92: 2662–2666. Krauter, K., Montgomery, K., Voon, S-J., LeBlanc-Straceski, J., Renault, B., Marondel, I., Herdman, V., Cupelli, L., Banks, A., Lieman, J., Menninger, J., Bray-Ward, P., Nadkarni, P., Weissenbach, J., Le Paslier, D., Rigault, R., Chumakov, I., Cohen, D., Miller, P., Ward, D., and Kucherlapati, R. (1995). A second-generation YAC contig map of human chromosome 12. Nature 377(Suppl): 321– 333. Lundqvist, G., and Morgenstern, R. (1992). Mechanism of activation of rat microsomal glutathione transferase by noradrenaline and xanthine oxidase. Biochem. Pharmacol. 43: 1725–1728. Metters, K. M., Sawyer, N., and Nicholson, D. W. (1993). Microsomal glutathione S-transferase is the predominant leukotriene C4 binding site in cellular membranes. J. Biol. Chem. 269: 12816–12823. Miller, G. E., Brabec, M. J., and Kulkarni, A. P. (1986). Mutagen activation of 1,2-dibromo-3-chloropropane by cytosolic glutathione S-transferases and microsomal enzymes. J. Toxicol. Environ. Health 19: 503–518. Morgenstern, R., DePierre, J. W., and Ernster, L. (1979). Activation of microsomal glutathione S-transferase activity by sulfhydryl reagents. Biochem. Biophys. Res. Commun. 87: 657–663. Morgenstern, R., and Dock, L. (1982). A comparison of induction of microsomal glutathione S-transferase activity in the liver of the mouse and rat by dietary 2(3)-ter-butyl-4-hydroxyanisole (BHA). Acta Chem. Scand. B36: 255–256. Morgenstern, R., Guthenberg, C., and DePierre, J. W. (1982). Microsomal glutathione transferase: Purification, initial characterization and demonstration that it is not identical to the cytosolic glutathione S-transferases A, B, and C. Eur. J. Biochem. 124: 591– 597. Morgenstern, R., and DePierre, J. W. (1985). Microsomal glutathione transferase. Rev. Biochem. Toxicol. 7: 67–103. Morgenstern, R., DePierre, J. W., and Jornvall, H. (1985). Microsomal glutathione transferase. Primary structure. J. Biol. Chem. 260: 13976–13983. Morgenstern, R., Lundqvist, G., Hancock, V., and DePierre, J. W. (1988). Studies on the activity and activation of rat liver microsomal glutathione transferase, in particular with a substrate analogue series. J. Biol. Chem. 263: 6671–6675. Mosialou, E., and Morgenstern, R. (1989). Activity of rat liver microsomal glutathione toward products of lipid peroxidation and studies of the effect of inhibitors on glutathione-dependent protection against lipid peroxidation. Arch. Biochem. Biophys. 275: 289–294. Mount, S. M. (1982). A catalog of splice junction sequences. Nucleic Acids Res. 10: 459–472. Reiter, R., and Burk, R. F. (1988). Formation of glutathione adducts of carbon tetrachloride metabolites in a rat liver microsomal incubation system. Biochem. Pharmacol. 37: 327–331. Sanger, F., Nicklen, S., and Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463–5467. Sanger, F., Coulson, A. R., Barrell, B. G., Smith, A. J. M., and Roe, B. A. (1980). Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J. Mol. Biol. 143: 161–178. Waxman, D. J., Sundseth, S. S., Srivastava, P. K., and Lapenson, D. P. (1992). Gene-specific oligonucleotide probes for a, u, p, and microsomal rat glutathione S-transferases: Analysis of liver transferase expression and its modulation by hepatic enzyme inducers and platinum anticancer drugs. Cancer Res. 52: 5797–5802. Weber, G. F., and Waxman, D. J. (1993). Denitrosation of the anticancer drug 1,3-Bis(2-chloroethyl)-1-nitrosourea catalyzed by microsomal glutathione S-transferase and cytochrome P450 monooxygenases. Arch. Biochem. Biophys. 307: 369–378.
gnma
AP: Genomics
36, 100–103 (1996) 0429
ARTICLE NO.
Structural Organization of the Human Microsomal Glutathione S-Transferase Gene (GST12) MICHAEL J. KELNER,*,1 MIRIAM N. STOKELY,* NICOLE E. STOVALL,*
AND
MARK A. MONTOYA*
*Department of Pathology, University of California, San Diego, 200 West Arbor Drive, San Diego, California 92103-8320 Received February 1, 1996; accepted June 18, 1996
The primary structure of the human microsomal glutathione S-transferase gene (GST12) was determined by genomic cloning. The gene structure of GST12 spans 12.8 kb and consists of four exons and three introns. The coding sequence resides on exons 2, 3, and 4. Sequencing of the exons revealed two nucleotide differences compared to a previous report of the cDNA sequence. The substitutions, however, were silent, as they did not alter amino acid composition or restriction enzyme sites. All introns commenced with nucleotides GTAA at the 5* boundary and ended with nucleotides AG at the 3* boundary, in agreement with the proposed consensus sequence for intron spliced donor and acceptance sites. The presence of an in-phase stop codon and an upstream false start codon in the 5*-untranslated region was confirmed. Although it was previously predicted that there existed another start codon in-phase and within 50 bp of this stop codon, coding for a second mini-cistron, we could not identify another start codon for greater than 200 bp prior to the stop codon. Thus, initiation is suppressed at the first or false start codon due to either the closeness of the stop codon or the suboptimal context of the codon. q 1996 Academic Press, Inc.
INTRODUCTION
The glutathione S-transferases (EC 2.5.1.18) are a family of enzymes catalyzing the conjugation of glutathione to a wide variety of lipophilic electrophiles that include many environmental mutagens and carcinogens (Morgenstern and DePierre, 1985). The enzymes exist in many different cytosolic isozymes as well as a microsomal form (GST12)2. The microsomal form (monomer Mr 17,300) resembles cytosolic enzymes in displaying broad substrate specificity and some inhibiSequence data from this article have been deposited with GenBank under Accession Nos. U46494–U46499. 1 To whom correspondence should be addressed. Telephone: (619) 543-5976. Fax: (619) 543-3730. E-mail: [email protected] sd.edu. 2 The human microsomal glutathione transferase gene was designated GST12 in 1989 at the Tenth International Workshop on Human Gene Mapping.
0888-7543/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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tion characteristics (Morgenstern and DePierre, 1985). The microsomal form, however, is immunologically distinct (Morgenstern et al., 1982) and has an amino acid sequence (Morgenstern et al., 1985) and a cDNA sequence (DeJong et al., 1988) unrelated to the cytosolic glutathione transferases. The microsomal enzyme exists functionally as a trimer of a 154-amino-acid subunit (Anderson et al., 1994; Hebert et al., 1995), whereas the cytosolic forms exist as homo- or heterodimers. The microsomal enzyme is the predominant leukotriene C4 membrane-bound binding site (Metters et al., 1993). A variety of environmental toxins are substrates for microsomal glutathione transferase including benzo(a)pyrene-4,5 oxide (Morgenstern et al., 1988), carbon tetrachloride (Reiter and Burk, 1988), and 1,2-dibromo-3-chloropropane (Miller et al., 1986). Recent evidence also indicates that renal toxicity of some halogenated chemicals is attributable to glutathione Sconjugate formation, then metabolism of these glutathione S-conjugates to corresponding cysteine S-conjugates, and bioactivation of the cysteine S-conjugates to nephrotoxins (Anders et al., 1988). Evidence also indicates that microsomal glutathione S-transferase is responsible for scavenging lipid soluble radicals (Mosialou and Morgenstern, 1989). The microsomal glutathione transferase is activated in vitro by a variety of methods including exposure to electrophiles, thiodisulfide interchange, protein-dimer formation, proteolysis, oxidation, and removal of an unidentified endogenous microsomal inhibitor (Morgenstern et al., 1979; Boyer et al., 1986; Aniya and Anders, 1989a,b, 1992; Lundqvist and Morgenstern, 1992). The role of promoter induction for regulation of cytosolic glutathione transferases is well documented. The role of induction for microsomal glutathione transferase has been studied, but only to a limited extent. Most inducers of cytosolic glutathione transferases have no effect on the microsomal activity, but some exceptions are noted in the literature. There is a mild, but consistent, increase in microsomal glutathione transferase activity in mice treated with 2(3)-ter-butyl-4-hydroxyanisole (Morgenstern and Dock, 1982). A more recent study noted that isoniazid, B-nap-
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thoflavone, clofibrate, and isosafrole produced moder- ferase was located in this P1 clone. A SacI/SacI 16.5-kb ate increases in microsomal glutathione transferase subclone was isolated and subcloned into pBluescript activity and mRNA content (Waxman et al., 1992), KS(0) at the SacI site. Sequencing confirmed that the suggesting that induction could have a role in regula- clone contained a region corresponding to the initial 5*tion of enzyme activity. untranslated sequence region and contained the entire The gene for GST12 is located on human chromo- gene. Restriction enzyme mapping and nucleotide sesome 12 (DeJong et al., 1990), but the structural orga- quencing revealed that the regions corresponding to nization of the gene is unknown. To determine the the initial 5* and the terminal 3* ends of the mRNA relevance of induction in regulation of microsomal glu- spanned 12.8 kb (Fig. 1). Characterization of the tathione transferase, we isolated a clone containing GST12 gene revealed four exons and three introns (Fig. the entire human gene. This will allow the construc- 1). The nucleotide sequences of all exons and their tion of promoter – reporter gene constructs for use in flanking regions were determined. The coding sequence xenobiotic gene induction studies. resides on exons 2, 3, and 4. The sequencing of the exons revealed two nucleotide differences compared to a previous report of a cDNA sequence (DeJong et al., MATERIALS AND METHODS 1988). The C residue in the third position for Arg 73 Materials and Radiochemicals. [a-35S]dATP was purchased from in the cDNA sequence (DeJong et al., 1988) was noted Amersham Life Science Corp. (Arlington Heights, IL). Restriction to be a G (silent mutation) in the genomic sequence enzymes were from Boehringer-Mannheim (Indianapolis, IN). The (exon 3). There was also an A r G change in the 3*pBluescript II KS(0) plasmid was from Stratagene Corp. (La Jolla, untranslated region (nucleotide 560) in exon 4 of the CA). Sea Plaque GTG agarose was from FMA BioProducts (Rockland, ME). Oligonucleotides were provided by the UCSD Cancer Center genomic clone. These substitutions did not result in a change in restriction enzyme sites. All introns started Core Facility. with nucleotides GTAA at the 5* boundary and ended Isolation of P1 clone. A probe corresponding to the last 409 nucleotides of human microsome glutathione transferase cDNA (DeJong with nucleotides AG at the 3* boundary (Table 1). This et al., 1988) was generated from a pCD human cDNA library (Dr. agrees with the proposed consensus sequence for intron Paul Berg, Stanford, CA) using PCR amplification. The sequences of spliced donor and acceptance sites, indicating that all the oligonucleotides used for PCR screening were CAT GGC GTA CAG GTT GCT GAA AAG TA corresponding to nucleotide 502 of the splicing sites between exons and introns of the human GST12 gene are functional (Mount, 1982). cDNA and GCATTC TTT AAA TTC TTT ATT TGA TG corresponding to nucleotide 885 of the cDNA (DeJong et al., 1988). The entire probe The nucleotide sequences of the 5*-flanking region was sequenced to confirm nucleotide identity. A human genomic fore(U46494), the 3*-flanking region (U46499), all four exskin fibroblast bacteriophage P1 library (Genome Systems, St. Louis, MO) was screened by PCR using these oligonucleotides. Three clones ons, the entire first intron, and the initial 5* and final 3* were identified as positive by PCR amplification. The PCR amplifica- regions of the last two introns are available (U46495– tion product from one of the clones, 2625, was sequenced to confirm U46498) (Fig. 1). In agreement with a previous report that the product corresponded to nucleotides 502 to 885 of the GST12 on partial cDNA sequence (DeJong et al., 1988), an incDNA sequence. phase stop codon and an out-of-phase false start codon PCR conditions. PCR parameters were 1 cycle at 957C for 30 s and were present 5* to the authentic ATG start codon. A 727C for 1 min; 45 cycles at 957C for 30 s, 497C for 30 s, and 727C for 30 s; and 1 cycle at 727C for 5 min. The reaction mix contained dNTPs at second in-phase start codon that was previously predicted to exist (De Jong et al., 1988) was not present. 200 mM KCl at 75 mM, magnesium chloride at 1.5 mM, pH 8.8. DNA sequence analysis. All plasmids were isolated and purified Regions approximately 3 kb upstream and 1.5 kb downusing Qiagen-tip 100 cartridges (Chatsworth, CA). Nucleotide sestream from the first and fourth exons, respectively, quence analysis was performed by Sanger’s dideoxynucleotide chain- were sequenced (Fig. 1). A DNA database search using termination method (Sanger et al., 1977, 1980) with sequencing the BLAST algorithms failed to find any homology to grade Taq DNA polymerase and the fmol thermocycling DNA sequencing kit by Promega Corp. (Madison, WI). A sequence labeling these two flanking regions or to any of the other regions presented (Fig. 1). reaction was performed using the Direct Incorporation SEQ protocol with 7deazaGTP to minimize band compression often associated with the high G / C content in genomic DNA. Oligonucleotide primers complementary to defined human genomic DNA sequences were also used to perform some of the sequencing reactions. Oligonucleotide sequences were determined on both strands of the DNA inserts. To ensure accuracy of the sequencing data, the gene was sequenced in each direction in overlapping fragments, and autoradiograms containing sequence letters were proofread after data entry.
RESULTS
Three clones were isolated from the human genomic P1 library. One clone, 2625, with a 75-kb insert, was chosen for further studies. Sequencing of the insert obtained from PCR amplification of this clone confirmed that the gene for microsomal glutathione trans-
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DISCUSSION
The human microsomal glutathione transferase gene (GST12) spans 12.8 kb and consists of four exons (Fig. 1). With the exception of the 3* end of exon 1, all exon– intron junctions follow the general consensus sequence for exon–intron junctions. Two nucleotide differences, compared to a partial cDNA sequence previously reported, were noted in the exons. These two changes were silent, as they did not result in amino acid changes nor did they alter restriction enzyme sites. The presence of an in-phase stop codon and an upstream false start codon (from the authentic start codon) in the 5*-untranslated region was confirmed. The
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KELNER ET AL.
FIG. 1. Structure of the human microsomal glutathione transferase (GST12) gene. Black boxes indicate exons of the gene. Numbers below the exons refer to the corresponding nucleotides in the mRNA sequence previously reported (DeJong et al., 1988). Regions sequenced, and their corresponding GenBank accession numbers, are noted above the map.
presence of the in-phase stop codon eliminates the possibility of an encoded microsomal leader sequence, so cotranslation insertion must be operative using internal sequence signals. The presence of the in-phase stop codon prior to the authentic start codon and the lack of a second in-phase start codon more than 300 bases upstream of the mRNA site are intriguing. According to the scanning model for eukaryotic translation initiation, the presence of an upstream start codon that codes for a heptadecapeptide that terminates past the authentic start codon should depress translation initiation at the authentic AUG (Kozak, 1987a, 1989). Ribosomes, however, may reinitiate at a downstream AUG provided a termination codon exists 5* in close proximity to the upstream codon and is functionally active (Kozak, 1987b). The in-phase stop codon is strategically located to alleviate the inhibition expected by the heptadecapeptide open reading frame if it is functionally active and terminates for another upstream reading frame. Such an arrangement would allow for translation initiation at the downstream authentic start codon (Kozak, 1987b). This arrangement, whereby the upstream inphase TGA terminates a second mini-cistron, requires the presence of another in-phase start codon in the
early 5*-untranslated region of mRNA. This arrangement would allow for translation and initiation at the downstream authentic start codon. This arrangement, however, does require the presence of another in-phase start codon in the early 5* region of the mRNA. As the initial cDNA clone was known not to be full length (DeJong et al., 1988) but to be missing approximately 40 nucleotides of the initial 5* region due to the presence of an internal EcoRI restriction enzyme site, it was presumed that another in-phase AUG was present in this unrecovered region. However, we could not identify another start codon for greater than 200 bases prior to the internal EcoRI site. Thus, there is no second mini-cistron present, and initiation should be depressed at the authentic start codon per the scanning model for eukaryotic translation initiation. There are two mechanisms that may explain why the first start codon is suppressed. It is possible that the closeness of the stop codon to the first false start codon (TGA A ATG) suppresses initiation at the false start codon. The second possibility is that the first AUG codon occurs in a suboptimal context, as it is lacking the critical G position at 4/ (Kozak, 1986, 1987a, 1995; Cavener and Ray, 1991). The gene for microsomal glutathione transferase
TABLE 1 Nucleotide Sequences of the Exon/Intron Junctions of Microsomal Glutathione Transferase Exon-3* 1 2 3
CAAGTT AGAAAG ACGGAG
Consensus sequence
AG
5*-Intron-3* GTAAGT GTAAGA GTAAAC A GT-A G
779 bp Ç3000 bp Ç6100 bp
5*-Exon TTTAAG GGATAG CCACAG C -AG T
ATTCCA GGTTTT AGCCCA
2 3 4
NNN
Note. Over 200 bp of the 5*- and 3*-flanking regions surrounding each exon was sequenced. Six nucleotides on both sides of the boundaries are provided.
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MICROSOMAL GLUTATHIONE TRANSFERASE (GST12)
(GST12) was previously assigned to human chromosome 12 (DeJong et al., 1990). Several regions of the gene both upstream and downstream were sequenced (Fig. 1). Despite recent publication of a detailed map of chromosome 12 (Krauter et al., 1995), a search of worldwide data banks failed to find homology. Thus, at this time, we cannot identify the neighboring genes. ACKNOWLEDGMENT This work was supported by NIH Grants ES04989 and CA52310.
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