[CANCER RESEARCH 47, 5626-5630, November 1, 1987]

Structure and Expression of a Human Class TTGlutathione -S-TransferaseMessenger RNA1

Toshiyuki Kano,2 Masaharu Sakai, and Masami Muramatsu3

Department of Biochemistry, The University of Tokyo Faculty of Medicine, Hongo, Bunkyo-ku, Tokyo 113, Japan

ABSTRACT

We have used a rat glutathione S-transferase P (GST-P) complementary DNA as a probe to screen a human placenta complementary DNAlibrary constructed in the Xgtll vector. One of the positive clonescontained the complete coding region (630 base pair) and the entire 3'-noncoding region (78 base pair) of the putative human glutathione .V-transferase T (GST-x) subunit mRNA. From the nucleotide sequence wededuced the complete amino acid sequence of the GST-*- subunit. It

contained 209 amino acids with the relative molecular mass of M, 23,224.Comparison of the amino acid sequences between GST-ir and GST-Psubunits suggests that they are the corresponding enzymes in thesespecies. GST-ir and GST-P both consist of 209 amino acids and differ inonly 30 amino acids (85.6% homology). The difference in amino acidcomposition can explain the large difference in isoelectric point betweenGST-* subunit (pi 5.5) and GST-P subunit (pi 6.9). The expression ofGST-*- mRNA in some normal and cancerous tissues, including some

hepatoma cell lines, hepatoma, and colon carcinoma specimens wasdetermined using complementary DNA as a probe. The results indicatethat the mode of the expression of GST-i- in humans is different fromthat of GST-P in rats.

INTRODUCTION

GSTs4 (EC 2.5.1.18) are a family of enzymes that play an

important role in detoxication by catalyzing the conjugation ofmany hydrophobic and electrophilic compounds with reducedglutathione (1, 2). Multiple forms of GST have been demonstrated in a number of animal species (2-5). Recently, species-independent classification of the isoenzymes has been proposedaccording to amino terminal amino acid sequence homology,enzymatic substrate specificity, and common antigenicity (6).This classification divides the isoenzymes into three classes, a,n, and K. Both human placenta! GST (GST-ir) and rat placenta!GST (GST-P) belong to class ir enzyme, although they are notparticularly concentrated in placenta. At least the lung, kidney,and testis in the rat contain higher concentrations of GST-Pthan placenta. They appear to exist as a homodimer in the cell.

GST-P has been reported as a new marker enzyme for pre-neoplastic lesions arising during chemical carcinogenesis in ratliver (7-12). It increases nearly two orders of magnitude duringthis process. Assuming that interspecies similarities exist inneoplastic development, GST-ir might be expected to be a usefulmarker for preneoplastic lesions in human organs. Indeed,whether or not neoplastic lesions of liver or other organssimilarly express this enzyme has become a subject of muchinterest among investigators in oncology (4, 13-16). As a first

Received 5/4/87; revised 7/27/87; accepted 8/4/87.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported in part by Grants-in-Aid from the Ministry of

Education, Science and Culture (especially for Special Project Research, CancerBioscience) and from the Foundation for Promotion of Cancer Research backedby the Japan Shipbuilding Industry Foundation.

2Present address: Department of Dermatology, Jichi Medical School, Minamikawachi-machi, Kawachi-gun, Tochigi 329-04, Japan.

3To whom requests for reprints should be addressed.' The abbreviations used are: GST, glutathione 5-transferase; SSC, standard

saline citrate; SDS, sodium dodecyl sulfate; pi, isoelectric point; cDNA, complementary DNA.

step in analyzing this point, we have set out to clone the cDNAof the mRNA of GST-Tr subunit. Here we report a successfulcloning of the cDNA and its total amino acid sequence. Comparison of the sequence with that of the rat GST-P subunit isalso presented. Furthermore, we investigated the state of GST-,Texpression in some human tissues including neoplastic lesions, with the aid of the cloned cDNA.

MATERIALS AND METHODS

Materials. Restriction enzymes, polynucleotide kinase, T4 DNA ligase, Klenow fragment of DNA polymerase I, and alkaline phosphatasewere purchased from Takara (Japan) and BRL (USA). [32P]ATP and[32P]dCTP were from New England Nuclear (Boston, MA). The human

placenta! cDNA library, constructed in the Xgtl 1 vector, was a generousgift of Dr. W. J. Rutter (Department of Biochemistry, UCSF, CA).Deoxy-7-deaza-GTP was kindly given by Dr. S. Nishimura (NationalCancer Center Research Institute, Japan).

Library Screening. The human placenta! cDNA library was screenedwith 32P-labeled 140-base pair fragment corresponding to the 5' end ofrat GST-P cDNA clone pGP5 (10). Recombinant phages were platedat a density of approximately 3 x IO4 plaque forming units per 150-

nim dish. Plaque hybridization was done as described by Benton andDavis (17). Hybridization was carried out in 6x SSC (Ix SSC: 0.15 MNaCl, 0.015 M sodium citrate), 10 IHMEDTA, 5x Denhardt's solution(Ix Denhardt's solution: 0.02% bovine serum albumin, 0.02% polyvi-

nylpyrrolidone, 0.02% Ficoll), 0.1 % SDS, 100 Mg/ml denatured salmonsperm DNA and 32P-labeled DNA probe (2-4 x 10* cpm/ml) at 65°Covernight. Filters were washed twice in Ix SSC, 0.1% SDS at 45*C for30 min each and exposed for autoradiography at -80°C.

DNA Sequencing. cDNA insert was isolated from the DNA ofpurified phage clones, digested with £coRI and subcloned into theEcoRl site of the pUC19 plasmili. Appropriate restriction fragmentswere further subcloned into either M13mpl8 or M13mpl9. Single-stranded templates were prepared and sequenced by the dideoxy chaintermination method (18, 19). The Maxam and Gilbert method was alsoused for sequencing (20).

Tissue Samples and RNA Extraction. Samples were taken from twonormal livers, two hepatocellular carcinomas, one liver metastasisoriginated from colon carcinoma, two colon carcinomas, two normalcolons (only mucosa and submucosa), and one placenta. Li-7 cells (21),a transplantable hepatoma maintained in nude mice, were also used.Liver and colon specimens were obtained immediately after surgicalresection. One full term placenta was obtained soon after parturition.Ulcered and necrotic tissues were dissected from the tumor tissues andthe muscle layers were removed from colon samples. Total cellularRNAs extracted from various human tissues, Morris hepatoma 5123D(a well-differentiated hepatocellular carcinoma of the rat), and fromadult male and female mouse livers were prepared by the guanidiniumthiocyanate-CsCl method (22, 23). Cytoplasmic RNAs from HuH7 andHepG2 [both human hepatocellular carcinoma cell lines (24, 25)] wereprepared by the method of Favaloro et al. (26) as modified by Maniatiset al. (23). Briefly, monolayer cells were washed with cold phosphate-buffered saline, harvested by a policeman, and were collected by a lowspeed centrifugation. Cell pellets were suspended in a lysis buffer [10IHM Tris-HCl (pH 6.8), 0.14 M NaCl, 1.5 HIM MgC!2J. NP-40 wasadded to 0.5% and vortexed. After being layered on a 24% sucrosecushion in lysis buffer and centrifuged, supernatant was extracted withphenol/chloroform (50/50).

RNA Dot Blot Hybridization Analysis. Total RNAs treated with 10HIM methylmercuryhydroxide were spotted on nitrocellulose filters

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STRUCTURE AND EXPRESSION OF HUMAN GST-T

which had been presoaked in 20x SSC and dried. Hybridization wasperformed with the isolated insert of GST-ir cDNA clone or GST-PcDNA clone as described in "Library Screening."

Northern Blot Hybridization Analysis. About 10 ng each of totalRNA was denatured by 2.2 M formaldehyde and 50% formamide at55°Cfor 15 min. Denatured RNAs were electrophoresed on a 1.2%

agarose gel containing 2.2 M formaldehyde (23, 27). rRNAs were usedas size markers. After transfer onto a nitrocellulose filter, they werehybridized with the cDNA probe as described in "Library Screening."The filter was washed twice in 2x SSC, 0.1% SDS at 60°Cfor 30 min

each time.

RESULTS AND DISCUSSION

cDNA Cloning and the Nucleotide Sequence of GSTir mRNA.Approximately 2 x 10s recombinant phages were screened with32P-labeled EcoRl-BstEll fragment (140 base pair) of pGP5

(10). Ten positive plaques were isolated and analyzed by restriction enzymes. The largest insert was subcloned into the plasmidpUC19. This clone, designated pGPi2, was large enough forthe complete sequencing of GST-ir. The restriction map andthe strategy for sequence analysis of this clone are shown inFig. \A. In the course of sequencing by the dideoxy chaintermination method of Sanger et al. (19), two parts (nucleotidenumbers 45-52 and 543-550, Fig IA) were impossible to readin the ladders because of the compression of the bands. Alternative use of the Maxam and Gilbert method (20) did notimprove the results, the sequence ladder of both parts beingstill ambiguous. The use of deoxy-7-deaza-GTP in place ofcK¡TI'in the dideoxy chain termination method (28) clarified

the ladders and the sequences could be determined unequivocally. Fig. Ißshows the nucleotide sequence and deduced aminoacid sequence of the pGPi2 cDNA. A 714 nucleotide-long

insert, not including the poly(A) tract, contains 630 nucleotidesof open reading frame encoding 210 amino acids including theinitiator methionine. The 5'- and 3'-noncoding sequences were6 and 78 nucleotides, respectively. In the 3'-noncoding se

quence, a poly(A) addition signal, AATAAA, is located 21nucleotides upstream from the poly(A) stretch. The amino acidsequence deduced from the nucleotide sequence was in fullagreement with the W-terminal amino acid sequence of GST-irfrom protein analysis (29, 30), except for initiating methionine.Initiating methionine residue must have been removed post-translationally. Furthermore, the predicted amino acid composition agreed well with the published values for GST-ir fromprotein analysis (31). From the above results, we conclude thatthe pGPi2 cDNA clone encodes the GST-ir subunit. The GST-ir subunit consists of 209 amino acids and its relative molecularmass is MT23,224.

Comparison of GST-ir with GST-P. GST-ir and GST-P aresupposed to belong to the class ir of glutathione 5-transferaseisozymes. In Fig. 2, the amino acids that differ in GST-P areshown under the GST-ir sequence. It shows that both subunitsconsist of 209 amino acids and that they differ in only 30 aminoacids (85.6% homology). This high degree of sequence homol-ogy contrasts with the difference between GST-P (Yp) and Ya(32%), Yb (24%), and Yc (27%) (10, 32, 33), thus confirmingthat GST-ir is the orthologue of GST-P. Notwithstanding GST-ir and GST-P belong to the same class, a remarkable differencein pi exists between these molecules. The pi of GST-ir subunitis 5.5 (4) and that of the GST-P subunit is 6.9 (9). This fact isnow explained by comparing the amino acid compositions ofGST-ir and GST-P subunits (Fig. 2). From the GST-P sequenceto GST-ir sequence, there are four changes from basic to neutralamino acid (positions 84,125,198, and 201) and three changesfrom neutral to acidic amino acid (positions 40, 146, and 163).

a

Hpall Bglll Hpall

100 bp

ATG CCG CCC TAC ACC CTC GTC TAT TTC CCA GTT CCA CCC CCC TOCMET Pro Pro Tyr Thr Val Val Tyr Phe Pro Val Arg Cly Arg Cys

CCG CCC CTC CCC ATC CTC CTC CCA CAT CAC CCC CAC ACC TCC AACAla Ala Leu Arg Met Leu Leu Ala Asp Gin Cly Cln Ser Trp Lys

GAG GAG CTC CTC ACC CTC CAC ACC TCC CAC GAG CGC TCA CTC AAAClu Clu Val Val Thr Val Glu Thr Trp Cln Clu Cly Ser Leu Lys

CCC TCC TCC CTA TAC CGC CAC CTC CCC AAC TTC CAC CAC CCA CACAla Ser Cys Leu Tyr Cly Cln Leu Pro Lys Phe Cln Asp Gly Asp

CTC ACC CTC TAC CAC TCC AAT ACC ATC CTC CCT CAC CTC CGC CGC*

Leu Thr Leu Tyr Cln Ser Asn Thr lie Leu Arg His Leu Cly Arg

ACC CTT CGC CTC TAT CGC AAC CAC CAG CAC CAC CCA CCC CTG CTCThr Leu Gly Leu Tyr Gly Lys Asp Gin Cln Clu Ala Ala Leu Val

CAC ATC CTC AAT CAC CGC CTC GAC CAC CTC CGC TCC AAA TAC ATc'

Asp Met Val Asn Asp Gly Val Glu Aap Leu Arg Cys Lye Tyr lie

TCC CTC ATC TAC ACC AAC TAT GAG CCC CCC AAC CAT CAC TAT CTG*

Ser Leu Ile Tyr Thr Asn Tyr Glu Ala Gly Lys Asp Aap Tyr Val

AAC CCA CTC CCC CGC CAÕ CTC AAC CCT TTT GAC ACC CTC CTC TCC*

Lys Ala Leu Pro Cly Gin Leu Lys Pro Phe Glu Thr Leu Leu Ser

CAC AAC CAC CCA CGC AAC ACC TTC ATT CTC CCA CAC CAC ATC TCC*

Cln Asn Gin Gly Cly Lys Thr Phe Ile Val Gly Asp Gin Ile Ser

TTC GCT CAC TAC AAC CTC CTC CAC TTC CTC CTC ATC CAT GAG CTc"

Phe Ala Asp Tyr Asn Leu Leu Asp Leu Leu Leu [le His Clu Val

CTA GCC CCT CGC TGC CTC CAT GCC TTC CCC CTC CTC TCA CCA TATLeu Ala Pro Cly Cys Leu Asp Ala Phe Pro Leu Leu Sor Ala Tyr

141CTC GGC CCC CTC ACC CCC CGC CCC AAC CTC AAG GCC TTC CTC CCCVal Cly Arg Leu Ser Ala Arg Pro Lys Leu Lys Ala Phe Leu Ala

«MTCC CCT GAC TAC CTC AAC CTC CCC ATC AAT CGC AAC CGC AAA CACSer Pro Clu Tyr Val Asn Leu Pro Ile Asn Cly Asn Cly Lys Gin

TCA CGGTTCCCGÅ“ACTCTGACCCCCACGCACACTrTGCCTTCCTTTCTCCACCACCA

ATAAAATTTCTAACACAGCT(A)n

Fig. 1. Restriction map. sequencing strategy, and the nucleotide sequence of pGP¡2insert. In A, arrows show the direction and extent of the sequence analysis.Solid arrows, sequencing by the dideoxy chain termination method using deoxy-7-deazaguanosine triphosphate; Broken arrows, sequencing by Maxam and Gilbertmethod. D, 5'- and 3'-noncoding region; shaded area, coding region; •.poly(A) stretch; E, EcoRl linkers. In B: numbers, nucleotide positions starting from theinitiation codon. *, stop condón.The poly(A) addition signal, AATAAA, is underlined.

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STRUCTURE AND EXPRESSION OF HUMAN GST-T

Human : MET-Pro-Pro-Tyr-Thr-Val-Val-Tyr-Phe-Pro-Val-Arg-Gly-Arg-Rat: lie

Le'u- Leu-Ala- Asp- Gln-Gly-Gln-Ser-Trp-Lys-Glu-Clu- Val- Val-

Fig. 2. Comparison of the deduced aminoacid sequences between GST-T (human) andGST-P (rat). Top line of type, complete humanGST-jr sequence; bottom line of type, aminoacid residues of rat GST-P sequence that aredifferent from human sequence. Initiation me-iImmilli' residue must have been removed post-translationally.

Cys-Ala-Clu

Thr-Val-lie

Ala-Leu-Arg-Mec

Thr

Clu-Thr-Asp Val

Trp-GlnLeu

Glu-Gly-Ser-Leu-Lys-Ala-Ser-Cys-Leu-Tyr-Gly-Gln-Leu-Pro-Lys-Phe-

Gln Ser Ihr

Gln-Asp-Glu

Leu-Thr-Leu-Tyr-Gln-Ser-Asn-Thr-Ile-Leu-Arg-His-Leu-Cly-Ala

Gly-Lys-Asp-Gln-Gln-Clu-Ala-Ala-Leu-Va1-Asp-Net-Val-Asn-

Lys

Arg-Cys-Lys-Tyr-Ile-Ser-Leu-Ile-Tyr-Thr-Asn-Tyr

Cly Thr110Lys-Ala-Leu-Pro-Gly-Gln-Leu-Lys-Pro-Phe-Glu-Thr

His

Gly-Asp

Leu-Gly-Leu-Tyr

Val-Glu-Asp-Leu

-Asp-Asp-Tyr-Val

-Leu-Leu-Ser-tln-Asn-Cln-Gly-Gly

Glu-AlaAsn

Arg-Thr-Ser

Asp-Gly-

Cly-Lys

Lys-Thr-Phe-Ile-Val-Gly-Asp-Gln-ïle-Ser-Phe-Ala-Asp-Tyr-Asn-Leu-Leu-Asp-Leu-Leu

Ala Asn

Leu-Ile-His-Glu-Val-Leu-Ala-Pro-Gly-Cys-Leu-AspVal Gin

•Ala-PheAsn

-Pro-Leu-Leu-Ser-Ala-Tyr

Val-Gly-Arg-Leu-Ser-Ala-Arg-Pro-Lys-Leu-Lys-Ala-Phe-Leu-Ala-Ser-Ala Ile Ser

IM 10*Asn-Leu-Pro-Ile-Asn-Gly-Asn-Gly-Lys-Gln

Arg

Pro-GluAsp

•Tyr-ValHis Leu

On the contrary, the number of reverse changes (from acidic toneutral or neutral to basic) are only two (positions 15 and 56).Since one amino acid substitution from neutral to acidic or tobasic appears to shift the pi of the protein by approximately0.3 (34), the overall amino acid substitution to acidic aminoacids accounts well for the 1.4 difference of pi between GST-Tand GST-P.

Expression of GST-ir mRNA in Human Tumors and OtherTissues. It is of interest to know if the GST-ir gene in humansis regulated in the same manner as GST-P in the rat (7-10). Itmay also be related to the feasibility of this enzyme as a tumormarker for specific organs or tissues. Therefore, we examinedthe state of GST-ir expression in various human tissues withNorthern blot and dot blot hybridization analysis. First, theNorthern blot hybridization was done to determine the amountand the size of GST-ir mRNA in some representative tissueswith the GST-ir cDNA insert as a probe. The RNAs from ahuman hepatoma cell line Hull?, normal human liver andMorris 5123D hepatoma cells of the rat were analyzed. Asshown in Fig. 3, an intense band of about 750 nucleotides wasdetected in HuH7 (lane 1). This size is very close to that of ratGST-P mRNA (10) as shown by the cross-hybridization of theprobe to GST-P mRNA in Morris hepatoma (lane 3). Incontrast to I lui 17, no band was detected in normal human liverRNA (lane 2). For more detailed information on differentialexpression in various tissues, we have carried out dot blothybridization analysis as shown in Fig. 4. As expected from theNorthern blot data, HuH7 showed a high concentration ofGST-7T mRNA (lane 3). Another hepatocellular carcinoma cellline HepG2, however, had only a very low amount of GST-irmRNA (lane 2). In Li-7, another hepatoma maintained in nudemice, little mRNA could be detected (lane 4). For surgicalspecimens, neither hepatocellular carcinomas (lanes 5a and 6a)nor normal liver cells (lanes 5b and 6b) showed detectableamounts of GST-ir mRNA. By contrast, colon carcinomas(lanes 8a and Pa) as well as normal colons (lanes 8b and 9b)showed detectable amounts of GST-ir mRNA, though not ashigh as HuH7. The expression did not change after metastasisto the liver (lane 7). In spite of the fact that the GST-ir wasoriginally purified from placenta and the clones obtained froma placenta cDNA library, GST-ir mRNA was not detectable inthe total RNA of the placenta by dot blot analysis (Fig. 4, lune

1 2 3

-28s

-18s

Fig. 3. Northern blot analysis of GST-T mRNA. About 10 pg of total RNAsextracted from HuH7 (lane /), human liver (lane 2), and rat Morris hepatomaS123D (lane 3) were electrophoresed on a 1.2% agarose gel containing 2.2 Mformaldehyde, transferred onto a nitrocellulose filler and hybridized with ''!'

labeled pGPi2 insert DNA.

7). The mRNA content in this full term placenta must havebeen very low. The result is not surprising because only 0.005%of the clones from the placenta cDNA library was positive inthe screening (see cDNA Cloning and Nucleotide Sequencing).

Thus, the expression of GST-ir mRNA in normal humanliver is very low as is the case for GST-P mRNA in rat liver butit does not increase in the cancerous lesion as in the latter.Detectable amounts of mRNA are present in the colon but nosignificant differences were detected between normal and tumorregions of surgically resected specimens. It is difficult to conclude what cell type is responsible for this expression, becausethe nontumor portions of colon tissues contained submucosaas well as mucosa cells. In situ hybridization will certainly helpclarify this point. A hepatoma cell line Hull? expressed largeamounts of GST-7r mRNA but another hepatoma cell lineHepG2 did not. Although it is too early to conclude from thesmall number of the examples described above, the expression

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STRUCTURE AND EXPRESSION OF HUMAN GST-T

RNA (ug)16 4 1

1

2

3

RNA (yg)16 4 1

1

4

5a

b

6a

b

7 •

8a

b

9a

b

Fig. 4. Dot blot analysis of total RNAs from various human tissues and cellsusing pGPi2 DNA as a probe. Total RNAs from various sources were appliedonto nitrocellulose filters at doses of 16, 4, and 1 *ig from left to right andhybridized with "P-labeled pGPi2 insert DNA. Total RNAs were prepared from:placenta (lane 1), HepG2 (lane 2), HuH7 (lane 3), Li-7 (lane 4), hepatocellularcarcinomas (lanes Sa and fiai, respective normal portions of livers (lanes 5b and(>h).liver metastasis originated from a colon carcinoma (lane 7), colon carcinomas(lanes 8a and 9a), and respective normal portions of colons (lanes 8b and 9b).Lanes Sa and 5b, 6a and 6b, 8a and 8b, and 9a and 9b are from the sameindividuals, respectively.

of GST-7Tenzyme does not seen to correlate well with neoplastictransformation in human liver unlike the constant and dramaticderepression of GST-P during rat hepatocarcinogenesis (9-11).The expression patterns of class -r enzymes may be different in

humans than in rats, or else human hepatomas obtained bysurgical operation have already lost the expression of thisenzyme during the relatively long progression period in humanpatients. The test of very early hepatomas is required to clarifythis point.

Sex Difference in the Expression of GST-MII (Mouse Ortho-logue of GST-ir) in Mouse Liver. Fig. 5 shows the dot blothybridization analysis of mouse GST-MII, a mouse orthologue

3 • •

4

Fig. S. Dot blot analysis of total RNAs from rat and mouse liver using pGPSDNA as a probe. Total RNAs, 16, 4, and I UKfrom left to right, were appliedonto a nitrocellulose filter and hybridized with a ';l' labeled pGPS insert DNA.

Total RNAs were prepared from: Morris hepatoma 5123D (lane I), male ratliver (lane 2). male mouse liver (lane 3). and female mouse liver (lane 4).

of GST-ir, mRNA in male and female liver using rat GST-PcDNA as a probe. Rat liver does not express a detectableamount of GST-P mRNA irrespective of sex (lane 2 and datanot shown). By contrast, the male mouse liver showed a substantial amount of GST-MII mRNA (lane 3), whereas thefemale mouse liver showed less than one-10th expression. Inview of the sequence difference between rat and mouse mRNA,mouse mRNA might have hybridized more weakly than ratmRNA to the GST-P cDNA probe, leading to an underestimation of the mouse mRNA. Indeed, this sex-dependentexpression of GST-MII in mouse liver has been detected bydetermination of the enzyme level (35). Everything considered,the expression patterns of the class ir GST gene is differentfrom one species to another. The structure and regulation ofthe genes of the class ?r GST enzymes in various animals andtissues remain to be clarified. We have already cloned anddetermined the complete sequence of GST-P gene in the rat(36). The analysis of human and mouse genes appears veryimportant for further understanding the regulation of this family of the gene during cell differentiation and transformation.

ACKNOWLEDGMENTS

The authors thank Drs. W. J. Rutter, UCSF, CA, and S. Nishimuraand S. Hirohasi, National Cancer Center Research Institute, Japan, forsupply of the placenta! cDNA library, 7-deaza-dGTP and hepatoma Li-7, respectively.

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STRUCTURE AND EXPRESSION OF HUMAN GST-x

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1987;47:5626-5630. Cancer Res   Toshiyuki Kano, Masaharu Sakai and Masami Muramatsu  -Transferase Messenger RNA

S Glutathione πStructure and Expression of a Human Class

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