* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Complementary DNA Cloning, Messenger RNA
Survey
Document related concepts
Endogenous retrovirus wikipedia , lookup
Real-time polymerase chain reaction wikipedia , lookup
Messenger RNA wikipedia , lookup
Genetic code wikipedia , lookup
Two-hybrid screening wikipedia , lookup
Silencer (genetics) wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Point mutation wikipedia , lookup
Biosynthesis wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Genomic library wikipedia , lookup
Epitranscriptome wikipedia , lookup
Expression vector wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Gene expression wikipedia , lookup
Transcript
(CANCER RESEARCH 52, 314-318, January 15, 1992] Complementary DNA Cloning, Messenger RNA Expression, and Induction of «-ClassGlutathione S-Transferases in Mouse Tissues1 Timo M. Buetler and David L. Eaton2 Department of Environmental Health and Institute for Environmental Studies, University of Washington, Seattle, Washington 98195 ABSTRACT Glutathione 5-transferases (EC 2.5.1.18) are a multigene family of related proteins divided into four classes. Each class has multiple isoforms that exhibit tissue-specific expression, which may be an important determinant of susceptibility of that tissue to toxic injury or cancer. Recent studies have suggested that «-class glutathione 5-transferase isoforms may play an important role in the development of cancers. Several a-class glutathione 5-transferase isozymes have been character ized, purified, and cloned from a number of species, including rats, mice, and humans. Here we report on the cloning, sequencing, and mRNA expression of two a-class glutathione 5-transferases from mouse liver, termed mYa and inYi. While mYa was shown to be identical to the known a-class glutathione 5-transferase complementary DNA clone pGT41 (W. R. Pearson et al., J. Biol. Chem., 263:13324-13332,1988), the other clone, mYc, was demonstrated to be a novel complementary DNA clone encoding a glutathione 5-transferase homologous to rat Yc (subunit 2). The mRNA for this novel complementary DNA is expressed constitutively in mouse liver. It also is the major a-class glutathione 5transferase isoform expressed in lung. The levels of expression of the butylated hydroxyanisole-inducible form (mYa) are highest in kidney and intestine. Treatment of mice with butylated hydroxyanisole had little effect on the expression levels of mYc but strongly induced mYa expres sion in liver. Butylated hydroxyanisole treatment increased expression levels for both mYa and mYc to varying degrees in kidney, lung, and intestine. The importance of the novel mouse liver a-class glutathione 5transferase isoform (mYc) in the metabolism of aflatoxin B, and other carcinogens is discussed. INTRODUCTION GSTs3 are a multigene family of related proteins predomi nantly involved in detoxification reactions (1,2). They are found in all tissues and species, including bacteria (3), yeast (4), and plants (2). In higher organisms, three classes of GST protein, a, n, and 7T(1, 2), have been discriminated; a fourth class, 0, has been tentatively identified, although relatively little is known about this protein's structure and function (5, 6). Each class consists of several closely related gene products which are capable of forming dimeric proteins with other gene products of the same class, but not with protein subunits from other classes (1, 2). In the rat at least five members of the a class have been described at either the DNA or protein level. Three cDNA sequences and one gene (7) for rat a-class GSTs have been published. The cDNAs code for subunits Ya4 (8-10) (pGTB38; Ref. 11), Ya2 (pGTR261; Ref. 12), and Yc (pGTB42; Ref. 13). Recently the amino acid sequence for two distinct rat Received 8/5/91; accepted 10/31/91. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by NIH Grants ES05780, CA47561, and ES03933. 2To whom requests for reprints should be addressed, at Department of Environmental Health, SC-34, and Institute for Environmental Studies, Univer sity of Washington, Seattle, WA 98195. 3The abbreviations used are: GST, glutathione 5-transferase; AFB, aflatoxin B,; BHA, 2(3)-fert-butyIhydroxyanisole;cDNA, complementary DNA. ' Because of the disparity in numerical designations between mouse and rat GST isoenzymes (8, 9), which may confuse the comparison of mouse GST isoenzymes with rat GST isoenzymes, we adopted the "Y" designation of Hayes (10). a-class GST isozymes have been published (14, 15). These include the complete amino acid sequence for rat liver GST Yk (subunit 8) (14) and a partial amino acid sequence for rat liver GST Yl (subunit 10) (15). In the mouse, one a-class GST cDNA has been characterized (pGT41) (16). The sequence for a very closely related mouse a-class GST (mus Ya) has been characterized at the gene level (17). However, Southern blot analysis of the murine genome suggested the presence of at least four or five a-class GST genes (18). Indeed, analysis of GST protein has revealed the presence of at least two different a-class proteins in mouse liver (10, 19-21). Several investigators have demonstrated the presence of a constitutively expressed a-class GST isoform in mouse liver as well as a distinct isoform which is inducible by the antioxidant BHA. For example, Benson et al. (19), using glutathione transferase activity toward l-chloro-2,4-dinitrobenzene for detec tion, demonstrated the presence of (a) a constitutively active aclass GST isoform (GST 10.6) in male and female CD-I mice which was not significantly influenced by treatment with BHA and (b) a BHA-inducible a-class GST isoform (GST 10.3). McLellan and Hayes (20) showed that, in mice, BHA induced an a-class GST isoform, termed Ya]Yai, that was essentially absent from the livers of untreated mice. These investigators also purified a second isoform, termed YasYa3, which was constitutively expressed in mouse liver and was not affected by BHA treatment. The mouse YaiYai isoform shared a high structural identity with the rat YaYa (1-1) isoenzyme, while the constitutively expressed form was less homologous to the rat YaYa protein. Pearson et al. (16) isolated a mouse liver a-class GST cDNA clone (pGT41) which was 95% homologous to the rat Ya a-class GST sequence (pGTB38). Northern blot studies showed that constitutive mRNA levels measured with this clone were very low in liver but high in kidney. BHA treatment induced this mRNA about 50-fold in liver but produced only a slight induction in kidney. The RNA expression data from untreated mice (16) correspond well with the protein data published by Gupta et al. (22), who used antisera raised against human a-class GST protein to detect a-class GST protein(s) in mouse liver and kidney. The low levels of protein detected in liver by Gupta et al., relative to that detected in kidney, would suggest that the antibody used might specifically detect the BHA-induced a-class GST protein, since Pearson et al. have shown that the constitutive levels of expression of their a-class GST clone, pGT41, are low in livers and high in kidneys of untreated animals. Previous work from our laboratory (21) also demonstrated the presence of a constitutively expressed a-class GST isoform which was chromatographically distinct from a BHA-inducible isoform. Differences in the expression of specific a-class GSTs may have significant physiological implications. For example, Mon roe and Eaton (23) have demonstrated that GST activity toward the carcinogenic epoxide metabolite of aflatoxin Br (AFB epoxide) differs by 50-100-fold between rats and mice. Such differ ences appear to be related specifically to differences in the activity of «-classGSTs (21). Recently, a-class GST proteins have been implicated in the resistance of rat and mouse tissue 314 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1992 American Association for Cancer Research. GST EXPRESSION IN MOUSE TISSUES culture cells to chlorambucil (24, 25) and Adriamycin (26) and in the metabolism of the nitrogen mustard melphalan (27). Thus, while the existence of two a-class GST isoforms in mice is well established, their contribution to the protection against AFB-induced hepatocarcinogenicity and their role in the detox ification of nitrogen mustards used in cancer therapy are not known, and the relationship to their rat counterparts is unclear. In this paper we report on the isolation of two cDNA clones from mouse liver; one corresponds to the sequence published by Pearson et al. (16), which we term mYa, and the other is a novel sequence which shows the highest homology to the rat Yc (subunit 2) sequence (pGTB42; Ref. 13) and which we term mYc. Expression studies demonstrate that the mYc clone rep resents the constitutively expressed mouse liver a-class GST isoform, whereas the mYa clone represents the BHA-inducible isoform. MATERIALS AND METHODS Chemicals and Enzymes. The mouse cDNA library was purchased from Clontech Laboratories, Inc. (Palo Alto, CA). The mRNA for the library was derived from adult male BALB/c mouse liver, and the cDNA was constructed by 5'-stretch, oligo-thymidine, and random priming and cloned in Xgtll. Radioisotopes [35S]dATP, [«-"P]dCTP, and [7-32P]ATP were purchased from ICN Biomedicals, Inc. (Costa Mesa, CA). Restriction enzymes and other molecular biology-grade reagents were purchased from Bethesda Research Laboratories (Gaithersburg, MD), Stratagene (La Jolla, CA), International Biotechnolo gies, Inc. (New Haven, CT), United States Biochemical (Cleveland, OH), Boehringer Mannheim (Indianapolis, IN), and Sigma (St. Louis, MO). The Sequenase sequencing kit was purchased from United States Biochemical Corp. Pre-made Luria broth was purchased from Gibco/ BRL (Gaithersburg, MD). Male Swiss-Webster mice were obtained from Tyler Laboratories (Bellevue, WA). Plasmids. The plasmids pGTB38 [containing the rat Ya (subunit 1) cDNA] and pGTB42 [containing the rat Yc (subunit 2) cDNA] were a generous gift from Dr. Cecil Picket! (Merck Frosst Canada, Inc., Québec,Canada). Isolation of Mouse «-ClassGST cDNA Clones. The cDNA library in Xgtl 1 was plated at 50,000 pfu/150-mm plate on Luria broth agarose with Y1090 as indicator bacteria, following the protocol provided by the manufacturer (Clontech Laboratories, Inc., Palo Alto, CA). Two replica plaque lifts were made on Nytran filters (Schleicher & Schuell, Keene, NH). A probe was made by digesting the rat Ya plasmid pGTB38 with Pstl and isolating the 550-base pair insert fragment comprising most of the protein coding region. Both filters were washed at relatively low stringency (Ix standard saline citrate, composed of 150 HIMNaCl and 15 mivi Na citrate, pH 7.0, 0.1% sodium dodecyl sulfate, at 63°C).cDNA inserts were subcloned into pUCIS plasmid vector and transfected into Escherichia coli TG1 cells. Sequencing. cDNA clones were sequenced using the universal primer provided with the Sequenase kit (5'-GTAAAACGACGGCCAGT-3') and the reverse primer. Nested deletions in the insert sequence were created using the Exonuclease III/mung bean kit from Stratagene. The vector was cut with Sphl to protect the vector from digestion with £roIII in the 5' direction, and BamHl to allow ExolH action in the 3' direction into the insert sequence. The remaining procedures were conducted according to the protocol provided by the manufacturer. Briefly, at room temperature £xoIIIactivity was determined to be about 100 nucleotides/min. Exolll incubations with DNA at room tempera ture were stopped in 2.5-min intervals for five time points. Mung bean nuclease was then added to digest the single-stranded DNA from the 5' end, leaving blunt-ended DNA. These DNAs were ligated and transfected into E, coli TG1. Three to six colonies from each time point were grown in 3 ml of Luria broth containing 25 Mgampicillin/ml, and plasmid DNA was prepared using Plasmid Quick columns (Stratagene). Two /¿g of this preparation were used directly for double-stranded DNA sequencing. Sequences were analyzed with GENEPRO software (Ver sion 5.0; Riverside Scientific Enterprise, Bainbridge Island, WA) and compared to the GenBank sequence data bank (Edition 64). Oligonucleotide Synthesis. Oligonucleotides were synthesized on a Cyclone Plus DNA synthesizer from MilliGen/Biosearch (Division of Millipore, Novato, CA). The mouse Ya-specific oligo malO (5'CCA'TTA'GAG'GCC'AGT'ATC'TGC-3') and Yc-specific oligo mcl5 (5'-CTC'GTC'AGT'CAT'CAT'GTCTAC'CTG-3') were cho sen to have less than 17 of 21 nucleotides matching the other member of the a class. Animal Treatment and RNA Isolation. Mice were fed Wayne Rodent Blox ad libitum for 2 weeks prior to separation into two groups, one of which was fed BHA (0.75% w/w) incorporated into ground chow while the other was kept on control diet. After 5 days, the animals were killed by cervical dislocation, and liver, kidney, lung, and intestine were removed and placed in ice-cold phosphate-buffered saline. Intestinal contents were removed by washing with ice-cold phosphate-buffered saline. RNA was isolated from 0.3 g of tissue using the acid phenol extraction protocol of Chomczynski and Sacchi (28). 20 Mg of total RNA were separated on a 1% agarose/formaldehyde gel, blotted onto Nytran membrane, and hybridized to either the "P-end-labeled oligonucleotides or random-primed 12P-labeled cDNA inserts from both mouse a-class GST clones. An oligonucleotide of 20 nucleotides in length corresponding to the 18S ribosomal RNA (from 943-962 of the mouse 18S ribosomal RNA gene, 5'-CAC'CTC'TAG'AGG' CGC'AAT'AC-3') was used to reprobe the Northern blots to normalize the expression levels to variable amounts of RNA loaded. RESULTS A total of 300,000 plaques were screened with the rat Ya probe, and 13 independent clones were isolated and purified. Nine of these clones gave a strong signal with the rat Ya probe, while the other four hybridized only weakly with the Ya probe. Six of the strongly hybridizing clones were further analyzed. DNA was isolated, and the £coRI inserts were cloned into pUC18. Of these six cDNA inserts, only three were longer than 800 base pairs. From the cloning work of others in rats, humans, and mice and based on the size of the GST protein, an 800base pair insert was considered the minimal length required to contain a full-length cDNA insert. Restriction analysis of five of the six clones revealed two different restriction patterns. The pattern fordone 4.1 was similar to that of pGT41, the sequence reported by Pearson et al. (16). The pattern of the four other clones, two full-length (5.1 and 6.1) and two shorter (insert sizes of 600-650 base pairs) clones were similar but distinct from the pattern reported for any other mouse GST cDNA sequence. Clone 5.1 seemed to be identical to clone 6.1 (both about 1.0 kilobase in length) but was in inverted orientation in the plasmid vector. Partial sequencing from both ends using the universal primer and the reverse primer for pUCIS suggested the sequence identity of clone 4.1 with pGT41 and of clone 5.1 with clone 6.1. Clone 4.1 was completely sequenced in one direction, and absolute sequence identity between this clone and the published sequence for the mouse a-class GST clone pGT41 was found. Clone 4.1 was 65 base pairs shorter at the 5' end but still extended 29 base pairs 5' of the start codon and thus contained the complete protein coding region. At the 3' end, this clone was 22 base pairs longer and contained a polyadenylation signal. Since clones 5.1 and 6.1 most probably encoded the same RNA but were in inverted orientation in the cloning vector, each clone was sequenced only in one direction in order to obtain sequence confirmation of the opposite strand. Nested deletions were created from the 5' end of each insert and sequenced using the universal primer, which annealed in the pUCIS vector, 315 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1992 American Association for Cancer Research. GST EXPRESSION IN MOUSE TISSUES Table 1 Nucleolide and amino acid homology comparison ofa-class glutathione S-transferases between different species The sequences of «-classGSTs between different species are compared at the nucleotide and amino acid levels. The sequences obtained from clones 4.1 (mYa) and 5.1 6.1 (mYc) were compared to the GenBank databank (Edition no. 64) using the Genepro 5.0 software on an IBM computer. therefore bypassing the deletion. Complete sequencing of these two clones revealed their identity; no mismatch was found (Fig. 1). When this sequence was compared with the 63 different GST cDNA sequences in the GenBank (Edition No. 64) sequence data bank, the highest homology (84%) was found with the rat Yc sequence (13) (Fig. 1). Sequence homology to the mouse and rat Ya sequences was only 70% and 67%, respectively (Table 1). Thus, clone 5.1/6.1 apparently codes for a mouse GST isoform which is orthologous to rat Yc (subunit 2) and is referred to as mYc. The open reading frame of the mYc clone is 663 base pairs long and encodes a protein of 221 amino acids with a deduced M, of 25,360. Because rat Yl (subunit 10) and rat Yc (subunit 2) are distinct but closely related, both had a homology similar to that of the mouse Yc GST isozyme (85%) (Table 1). The rat Yk (subunit 8) protein is apparently more distantly related to other rat or mouse «-classGST isozymes, since it has only a 57-59% amino acid sequence homology with the other members of the a class. Northern blots of 20 fig of total RNA from liver, kidney, lung, and intestine of control and BHA-treated mice were hybridized with cDNA inserts from either clone 4.1 (mYa) or clone 5.1 (mYc). The mYa probe detected a specific signal at 0.8 kilobases, while the mYc-specific probe detected a signal at 1.0 kilobase (Fig. 2). The pattern of mYa expression was found Mouse Ya Mouse Yc 90 67 67 84 74 69 Nucleotide level Rat Yal Rat Ya2 Rat Yc Human Hal Human Ha2 87 69 73 72 Amino acid level Rat Ya Rat Yc Rat Yk° Rat Yl* 95 69 59 66 67 85 57 85 °Complete amino acid sequence of rat liver GST Yk (subunit 8) (14). * Partial amino acid sequence of rat liver GST Y1 (subunit 10) ( 15). to be different from that of mYc, and neither of the two probes cross-hybridized with the mRNA encoding the other isoform. Therefore, it can be concluded that each probe reacted only with its homologous mRNA, resulting in a specific signal. The specificity of the signal was confirmed by hybridizing Northern blots with the isoform-specific oligonucleotides malO for mYa mYc rYc -21 AACAAGA AAACCCAAGC AGA GGGAGCAGCTT TTT G T -43 AACTGCTGCC --T Met Ala Gly Lys Pro Val Leu His Tyr Phe Asp Gly Arg Gly Arg Met Glu Pro He Arg Trp Leu Leu Ala Ala ATG GCG c_- GGG AAG CCA GTC CTT CAT __c TAC TTT __c GAT GGC AGG GGA __G AGA ATG GAG CCT __c ATC CGG c__ TGG CTC TTG GCT GCA __A - Pro ------------- 25 15 ---------- Ala Gly Val Glu Phe Glu Glu Lys Phe Leu Lys Thr Arg Asp Asp Leu Ala Arg Leu Arg Ser Asp Gly Ser Leu 50 GCT GGT GTG GAG TTT GAA GAA AAA TTT CTG AAA ACT CGG GAT GAC CTG GCA AGG TTA CGA AGT GAT GGG AGT CTG 150 c__ -_c c__ A_G _A_ T._ ------Cln -----------ASn - Met Phe Gin Gin Vil Pro Met Val Glu Ile Asp Gly Met Lys Leu Val Gin Thr Lys Ala Ile Leu Asn Tyr He 75 ATG TTC CAG CAA GTG CCC ATG GTA GAG ATC GAC GGG ATG AAA CTG GTG CAG ACC AAA GCC ATT CTC AAC TAC ATT 225 --G --T _-T - —¿G - - - - _G_ Arg ----- - Ala Ser Lys Tyr Asn Leu Tyr Gly Lys Asp Met Lys Glu Arg Ala Ile Ile Asp Met Tyr Thr Glu Gly Val Ala 100 GCC A-TCC AAA TAC AAC CTC TAT GGG AAG GAC ATG AAG GAG AGA GCC ATC C— ATT --C GAC ATG TAC --T G-ACÕ GAA GGA GTG GCG 300 - Thr ------------- Leu - - - - Ala - - - - Fig 1 Sequence alignment of clone 5 I/ 6.1 with the rat Yc sequenceof clone PGTB42 Asp Leu Glu Ile Met Ile Leu Tyr Tyr Pro His Met Pro Pro Glu Glu Lys Glu Ala Ser Leu Ala Lys Ile Lys 125 GATCTG GAGATA ATG ATT CTC TAT TAC ccc CACATG ccc CCT GAGGAGAAAGAGGCAAGCCTT Gcc AAGATC AAG375 T T "£ GA' A*¡G" . £ .' "T £ '£ T T cTy "-' "-' "-' "-' "* ~ '" "-* ~ "-' (13). The translated ammo acid sequencesare Shown above and below the respective nucleotide sequences. -, identical nucleotide or amino acid at that position; *, a gap for best alignment. Nucleotide 1 is the A of the ATG Glu Gin Thr Arg Asn Arg Tyr Phe Pro Ala Phe Glu Lys Val Leu Lys Ser His Gly Sin Asp Tyr Leu Val Gly 150 GAACAAACCAGGAACCGT TAC TTC CCT GCCTTT GAAAAGGTGTTG AAGAGCCAT GGACAAGAT TAT CTC GTT GGC450 —¿-e A-- G-A —¿â€”¿â€”¿â€”¿â€”¿T —¿â€”¿â€”¿â€”¿â€”¿â€”¿â€”¿â€”¿â€”¿â€”¿ —¿â€”¿â€”¿â€”¿â€”¿ Asf L** *la ~ '' °' Asn Arg Leu Ser Arg Ala Asp Ile Ala Leu Val Glu Leu Leu Tyr His Val Glu Glu Leu Asp Pro Gly Val Val 175 AAC AGG CTG AGC AGG GCT GAT ATT GCC CTG GTT GAA CTC CTC TAC CAT GTG GAA GAG CTG GAC CCC GGC GTT GTG 525 —¿T G— TA A --- c— G-T A c. T— - Val Tyr - - Gin Val - - - - - - - - - Ser Ala Leu Asp Asn Phe Pro Leu Leu Lys Ala Leu Arg Ser Arg Val Ser Asn Leu Pro Thr Val Lys Lys Phe Leu Gin Pro 200 GAC AAC TTC CCT CTC CTG AAA GCG CTG AGA AGC AGA GTC AGC AAC CTC CCC ACA GTG AAG AAG TTT CTT CAA CCT 600 _c_ -_G -_c -c__G Aja --------r/]r -------------Gly Ser Gin Arg Lys Pro Phe Asp Asp Ala Lys Cys Val Glu Ser Ala Lys Lys Ile Phe Ser GGC AGC CAG AGG AAG CCT --A TTT —¿A -_G GAT _-T GAC -AG GCA AAA TGT GTT -_A GAG -_A TCA _-T GCA GTT AAG AAG ATT --c TTC AGT TAATTCAGGC ------ Leu Glu - Glu - - - ATAAGT ACATAGCCCC —¿GGA- CT-TA-- CACAAAGCCA ACCTTCTAAA G G GCTAAC AAGTTTTCTA --C -c AGGCGTCTGT -T--T GTCAATTCAG GTAGACATGA --T T A GAAACT CATGATCACT TG--A- TCCTCGGATA A TTTTCTTCTG --ACT--GAA - - ATTTTGCATC ACATTGAAGT GC A- ----C-C-"- - Val 221 AACT _T._ 615 - GTTTTGACTA A AGTGTTGACC -A CTACTTA«** GAAA 141 TTG -G-G 148 CTGACGAGGA T ACGGCCGGGA -TT--T TGCTCTCTAG TTGTAGTTAA A-T-G- T'TCAAT'AA AACAAAACAA -C A— TTCG GCTTCTTAGA CTCTGG AATT 823 824 316 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1992 American Association for Cancer Research. 923 915 GST EXPRESSION li ki .+.+.+. lu int * IN MOUSE TISSUES DISCUSSION BHA +•1.0 kb Ya «-0.8 kb + 1.0 kb Ye «-0.8 kb 18S rRNA Fig. 2. Expression of mYa and mV'c GST mRNA in mouse tissues. RNA was prepared from liver (//), kidney (ki), lung (lu), and intestine (ini) of mice fed either a control diet (—)or a diet containing 0.75% BHA (+) for 5 days prior to sacrifice. Twenty ng of total RNA were separated on agarose gels and blotted onto Nytran membranes. The blots were hybridized to one of the following probes: thecDNA insert of clone 6.1 (top), thecDNA insert of clone 4.1 (middle), or an oligonucleotide specific for 18S rRNA (bottom). and mclS for mYc. The resulting expression pattern was found to be identical to that using the cDNA probes, with no observ able cross-hybridization (data not shown). Thus, these probes detected a specific mRNA for mYa and mYc at 0.8 and 1.0 kilobase, respectively. These sizes correspond well with the sizes of the cDNA clones, 849 base pairs for mYa (pGT41) and 950 base pairs for mYc (5.1), with the mYc clone longer in its 3' noncoding region. Two cDNA clones were isolated from a commercially avail able mouse liver cDNA library using the insert of a rat Ya cDNA clone as a probe. One clone (4.1) was found to be identical to the published cDNA sequence of pGT41 (16), a mouse a-class GST, and was therefore termed mYa. The other clone encoded a novel sequence which showed the highest homology to the rat Yc (subunit 2) sequence (84% nucleotide homology) and was termed mYc. The homology between the rat and the mouse Yc isoforms proved to be significantly lower than for mouse and rat Ya isoforms (85% versus 95% homol ogy). At high stringency of hybridization we found that the mYa probe did not cross-hybridize with the mYc mRNA and vice versa. This may explain why Pearson et al. did not detect the constitutively expressed liver a-class GST isoform (mYc) with their pGT41 cDNA probe on a Northern blot (16). The specificity of the hybridization using the cDNA inserts was confirmed by hybridization with mYa- and mYc-specific oligonucleotides (malO and mclS, respectively). Although hybrid ization with the mYa-specific oligonucleotide resulted in a very weak signal, it was adequate to determine the expression pattern for mYa. Probing a Northern blot with the mYc-specific oli gonucleotide yielded results identical to those obtained with the mYc cDNA insert. Based on the protein expression pattern known from the literature, the two «-classGST clones described here represent the two major «-classGST isoenzyme forms found in liver and probably in other tissues of the mouse. In liver, mYc represents the constitutively expressed, and mYa the BHA-inducible, isoform. Previous studies from our laboratory have demonstrated that mice are resistant to the hepatocarcinogenic effects of aflatoxin B, and have 100-fold lower AFB-DNA adduci formation than rats (23). This relatively low level of DNA binding was associ ated with a 50-100-fold higher level of cytosolic GST activity toward AFB epoxide in mice relative to rats, even though specific activity toward l-chloro-2,4-dinitrobenzene was com parable between the two species (29). The efficient detoxifica tion of AFB epoxide by mice appears to be a common charac teristic of the mus species, inasmuch as nine different strains Quantitation of expression by densitometric scanning re vealed that mouse liver contained a low basal level of mYa message, which was induced about 15-fold by BHA (Fig. 3). The basal levels of expression of mYa in intestine and kidney were 3- and 4-fold greater than in liver, respectively, and undetectable in lung. BHA treatment resulted in a 7- to 15-fold induction of mYa message levels in intestine and a 3-fold induction in kidney. No expression or induction was observed in lung. These data are in good agreement with the data from Pearson et al. (16), although the extent of induction by BHA was not as large as that found by these investigators. In liver, mYc was found to be constitutively expressed at a level approximately 25-fold greater than mYa (Fig. 3). BHA had no significant effect on mYc expression levels in liver. Constitutive expression of mYc was substantial in lung (about 10% of the constitutive level in liver). BHA increased mYc expression in lung to 40% of the level found in liver. In contrast to mYa, mYc expression levels were very low to undetectable in normal kidney and intestine. BHA treatment induced mYc expression in kidney, lung, and intestine 10-, 3-, and 6-fold, respectively (Fig. 3). liver kidney lung intestine kidney lung intestine Fig. 3. Relative expression levels of mYa tissues. The amounts of RNA were determined GS-300; Hoefer Scientific Instruments, San expression levels, relative to 18S rRNA, from and mYc GST mRNA in mouse by densitometric scanning (Hoefer Francisco, CA). Columns, mean at least 5 independent scannings. 317 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1992 American Association for Cancer Research. GST EXPRESSION IN MOUSE TISSUES and man. Biochem. J., 274: 409-414, 1991. 6. Singhal, S., Ahmad, H., Sharma, R., Gupta. S., Haque, A. K., and Awasthi, Y. C. Purification and characterization of human muscle glutathione 5transferases: evidence that glutathione S-transferase 8 corresponds to a locus distinct from GSTI, GST2, and GST3. Arch. Biochem. Biophys., 285: 6473. 1991. 7. Telakowski-Hopkins, C. A., Rothkopf, G. S., and Pickett, C. B. Structural analysis of a rat liver glutathione S-transferase Ya gene. Proc. Nati. Acad. Sci. USA, 83: 9393-9397, 1986. 8. Jakoby, W. B., Ketterer, B., and Mannervik, B. Glutathione transferases: nomenclature; commentary. Biochem. Pharmacol., 33: 2539-2540, 1984. 9. Mannervik, B., Âlin, P., Guthenberg, C, Jensson, H., Tahir, M. K., Warholm, M., and Jörnvall,H. Identification of three classes of cytosolic gluta thione transferase common to mammalian species: correlation between struc tural data and enzymatic properties. Proc. Nati. Acad. Sci. USA, 82: 72027206, 1985. 10. Hayes, J. D. Selective elution of rodent glutathione 5-transferases and glyoxalase I from the S-hexylglutathione-Sepharose affinity matrix. Biochem. J., 255:913-922, 1988. 11. Pickett, C. B., Telakowski-Hopkins, C. A., Ding, G. J-F., Argenbright, L., and Lu, A. Y. H. Rat liver glutathione 5-transferases: complete nucleotide sequence of a glutathione ^-transferase mRNA and the regulation of the Ya, Yb and Yc mRNAs by 3-methylcholanthrene and phénobarbital.J. Biol. Chem., 259:5182-5188, 1984. 12. Lai, H-C. J., Li, N-Q., Weiss, M. J., Reddy, C. C., and Tu, C-P. D. The nucleotide sequence of a rat liver glutathione S-transferase subunit cDNA clone (Ya). J. Biol. Chem., 259: 5536-5542, 1984. 13. Telakowski-Hopkins, C. A., Rodkey, J. A., Bennet, C. D., Lu, A. Y. H., and Pickett, C. B. Rat liver glutathione S-transferase: construction of a cDNA clone complementary to a Yc mRNA and prediction of the complete amino acid sequence of a Yc subunit. J. Biol. Chem., 260: 5820-5825, 1985. 14. Alin, P., Jensson, H., Cederlund, E., Jörnvall,H., and Mannervik, B. Cyto solic glutathione transferases from rat liver. Biochem. J., 261: 531-539, 1989. 15. Meyer, D. J.. Gilmore, K. S., Coles, B., Dalton, K., Hubler, P. B., and Ketterer, B. Structural distinction of rat GSH transferase subunit 10. Biochem. J., 274: 619, 1991. 16. Pearson, W. R., Reinhart, J., Sisk, S. C., Anderson, K. S., and Adler, P. N. Tissue-specific induction of murine glutathione transferase mRNAs by butylated hydroxyanisole. J. Biol. Chem., 263:13324-13332, 1988. 17. Daniel, V., Sharon, R., Tichauer, Y., and Sarid, S. Mouse glutathione Stransferase Ya subunit: gene structure and sequence. DNA (NY), 6: 317324, 1987. 18. Czosnek, H., Sarid, S., Barker, P. E., Ruddle, F. H., and Daniel, V. Gluta thione S-transferase Ya subunit is coded by a multigene family located on a single chromosome. Nucleic Acids Res., 12:4825-4833, 1984. 19. Benson, A. M., Hunkeler, M. J., and York, J. L. Mouse hepatic glutathione transferase isoenzymes and their differential induction by anticarcinogens. Biochem. J., 261: 1023-1029, 1989. 20. McLellan, L. I., and Hayes, J. D. Differential induction of class o-glutathione £-transferases in mouse liver by the anticarcinogenic antioxidant butylated hydroxyanisole. Biochem. J., 263: 393-402, 1989. 21. Ramsdcll, H. S., and Eaton. D. L. Mouse liver glutathione S-transferase isoenzyme activity toward aflatoxin B,-8,9-epoxide and benzo[a]pyrene-7,8dihydrodiol-9,10-epoxide. Toxicol. Appi. Pharmacol., 705: 216-225, 1990. 22. Gupta, S., Medeh, R. D., Leal, T., and Awasthi, Y. C. Selective expression of the three classes of glutathione S-transferase isoenzymes in mouse tissues. Toxicol. Appi. Pharmacol., 104: 533-542, 1990. 23. Monroe, D. H., and Eaton, D. L. Comparative effects of butylated hydroxyanisol on hepatic in vivo DNA binding and in vitro biotransformation of aflatoxin B, in the rat and mouse. Toxicol. Appi. Pharmacol., 90:401-409, 1987. 24. Ciaccio, P. J., Tew, K. D., and LaCreta, F. P. The spontaneous and glutathi one 5-transferase-mediated reaction of chlorambucil with glutathione. Cancer Commun., 2: 279-286, 1990. 25. Yang, W. Z., Begleiter, A.. Johnston, J. B., Israels, L. G., and Mowat, M. R. A. The role of glutathione (GSH) and glutathione 5-transferase (GST) in chlormabucil (CLB) resistance. Proc. Am. Assoc. Cancer Res., 32:360, 1991. 26. Schisselbauer, J. C., Crescimanno, M., D'Alessandro, N., Clapper, M., Toulmond, S., Tapiero, H., and Tew, K. D. Glutathione, glutathione 5transferase, and related redox enzymes in Adriamycin-resistant cell lines with a multidrug resistant phenotype. Cancer Commun., /: 133-139, 1989. 27. Bolton. M. G., Colvin, O. M., and Hilton, J. Specificity of isozymes of murine hepatic glutathione S-transferase for the conjugation of glutathione with L-phenylalanine mustard. Cancer Res., 51: 2410-2415, 1991. 28. Chomczynski, P., and Sacchi. N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162: 156-159, 1987. 29. Monroe, D. H.. and Eaton, D. L. Effects of modulation of hepatic glutathione on biotransformation and covalent binding of aflatoxin I!, to DNA in the mouse. Toxicol. Appi. Pharmacol., 94: 118-127, 1988. 30. Borroz, K. !.. Ramsdell, H. R., and Eaton, D. L. Mouse strain differences in glutathione 5-transferase activity and aflatoxin B, biotransformation. Toxi col. Lett., 5«:97-105, 1991. had similarly high specific activities (30). We have also dem onstrated that the high specific activity of GST toward AFB epoxide in mice is largely attributable to an «-classGST (21). Thus, because normal (untreated) mice have high AFB epoxideconjugating activity and do not constitutively express GST mYaYa, it is tempting to speculate that the mYcYc isoform is responsible for the high AFB epoxide-detoxifying ability of mouse liver cytosol. In fact, preliminary results of bacterially expressed mouse and rat isozymes (Ya and Yc) suggest that indeed only the mouse YcYc isozyme has substantial AFB epoxide-conjugating activity.5 The mouse Yc subunit may also prove to play an important role in the acquired resistance of tumor cells to nitrogen mustards like melphalan (27), chlorambucil (24, 25), and/or Adriamycin (26). Bolton et al. (27) have demonstrated that only «-classGST protein(s) from un treated mice are capable of forming the melphalan-glutathione conjugate. Since mYc is the predominant «-classGST isoform in untreated mouse liver, it may well be involved in this meta bolic reaction. Yang et al. (25) have observed an increased expression of «-classGST mRNA in chlorambucil-resistant mouse tissue culture cells. Schisselbauer et al. (26) have ob served an increase in «-classGST protein in two Adriamycinresistant Friend erythroleukemia cell lines. Further experiments utilizing site-directed mutagenesis and chimeric cDNA constructs should provide a means of identify ing the importance of specific sequences within «-classGST proteins which may account for the differences in GST activity among various species and target tissues, thus enhancing the ability to predict potential sensitivity to epoxide carcinogens or antineoplastic drugs across species and target tissues. ACKNOWLEDGMENTS We are grateful to Drs. Curtis Omiecinski and Christopher Masseti for helpful discussions and review of the manuscript and to Dr. Cecil Pickett (Merck Frosst Center for Therapeutic Research, Québec,Can ada) for his generous gift of the rat cDNA clones pGTB38 and pGTB42. Note Added in Proof J.D. Hayes et al. (Biochem.J., 279:385-398,1991) recently reported the isolation of a new, nonconstitutively expressed rat «-classGST subunit from ethoxyquin-induced rat liver, termed Yc2. The partial (-70%) amino acid sequence for rat Yc2 was 91% homologous to the mouse Yc clone reported here and had high activity toward aflatoxin8,9-epoxide. Thus, it appears that this new rat Yc2 subunit may be the orthologous form to the mouse Yc subunit reported here. REFERENCES 1. Picket!, C. B., and Lu, A. Y. H. Glutathione S-transferases: gene structure, regulation, and biological function. Annu. Rev. Biochem.. 58:743-764, 1989. 2. Boyer, T. D. The glutathione S-transferases: an update. Hepatology (Balti more), 9: 486-496, 1989. 3. Di Ilio, C., Aceto, A.. Piccolomini, R., Allocati, N., Faraone, A., Cellini, L., Ravagnan, G., and Federici, G. Purification and characterization of three forms of glutathione transferase from Proteus mirabilis. Biochem. J.. 255: 971-975, 1988. 4. Tamaki. H., Kumagai, H., and Tochhikura. T. Glutathione 5-transferase in yeast: induction of mRNA, cDNA cloning and expression in Escherichìacoli. Biochem. Biophys. Res. Commun., 772: 669-675, 1990. 5. Meyer, D., Coles, B., Pemble, S. E., Gilmore, K. S., Fraser, G. M., and Réitérer, B. Thêta,a new class of glutathione transferases purified from rat ' Unpublished observations. 318 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1992 American Association for Cancer Research. Complementary DNA Cloning, Messenger RNA Expression, and Induction of α-Class Glutathione S-Transferases in Mouse Tissues Timo M. Buetler and David L. Eaton Cancer Res 1992;52:314-318. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/52/2/314 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1992 American Association for Cancer Research.