GENOMICS
6,268-271
(1990)
Isolation and Chromosomal Localization of the Human Glutathione Peroxidase Gene SUNIL CHADA, MICHELLE M. LE BEAU,* LINDA CASEY, AND PETER E. NEWBURGER Departments
of Pediatrics and Molecular Genetics/Microbiology, University of Massachusetts Worcester, Massachusetts 0 1655; and ‘Joint Section of Hematology/Oncology, University of Chicago Medical School, Chicago, Illinois 60637 Received
July 7, 1989;
revised
We have isolated
cDNA clones for the gene, termed the major human selenoprotein, glutathione peroxidase. Sequence analysis coniirmed previous findings that the unusual amino acid selenocysteine is encoded by the opal terminator codon UGA. Southern blot analysis of human genomic DNA with the GPXI cDNA showed that restriction endonucleases without sites in the probe sequence produced three hybridizing bands at standard stringency, diminishing to one strongly and one weakly hybridizing band at high stringency. In situ hybridization localized the human GPXl gene to a single site on chromosome 3, at region 3qll-13.1. Thus, three genomic sites bear sequence homology to the GPXl cDNA, and the one most homologous maps to 3qll-13.1. o leso Acrdedc Inc.
INTRODUCTION
Glutathione peroxidase (EC 1.11.1.9) is an enzyme, widely distributed in eukaryotic cells, that catalyzes the reduction of hydroperoxides by glutathione (GSH) (Mills, 1957). The enzyme represents the major cellular defense against these toxic oxidant species.The protein is a soluble tetramer; each 22-kDa subunit contains one selenium atom in its reduced state, incorporated within a catalytically active selenocysteine residue (Forstrom et aZ., 1978). Sequence analysis of the murine glutathione peroxidase gene (GPXl) revealed the selenocysteine residue to be encoded by a TGA “termination” codon (Chambers et al., 1986). An identical codon and overall similar sequences have recently been reported for the human GPXl gene in clones isolated from human liver cDNA (Mullenbach et al., 1987), kidney cDNA (SuSequence data EME%L/GenBank 03&s7543po
from Data
this article have been deposited with Libraries under Accession No. M21304.
$3.00
Copyright 0 1990 by Academic Press, Inc. AlI rights of reproduction in any form reserved.
School,
11, 1989
kenaga et al., 1987), and genomic (Ishida et al., 1987) libraries. The chromosomal localization of the human GPXl gene has not been definitively made. An analysis of the electrophoretic separation of glutathione peroxidase speciesin human-Chinese hamster somatic cell hybrids first indicated an assignment of the GPXl gene to human chromosome 3 (Wijnen et al., 1978). Similar studies utilizing somatic cell hybrid clones containing rearrangements and deletions involving human chromosome 3 resulted in the further localization of GPXl to 3p14-q21, with the probable smallest region of overlap 3p13-q12 (Johannsmann et aZ., 1981). However, recent studies of hybridization of a human GPXl cDNA probe to genomic DNA from human-rodent somatic cell hybrids have demonstrated not only the locus on chromosome 3, but also additional cross-hybridizing sites on chromosome 21 and the X chromosome (McBride et al., 1988). The latter sequenceslack introns and show some sequence divergence; thus they may represent processed pseudogenes.
GPXl, encoding
Prau.
September
Medical
MATERIALS
AND
METHODS
Isolation and Sequence Analysis of cDNA Clones A previously described (Royer-Pokora et al., 1986) HL-60 human myeloid leukemia cell line cDNA library in Xgtll was probed with end-labeled oligonucleotides, synthesized using an Applied Biosystems DNA synthesizer. Individual positive plaques were purified by multiple rounds of screening and DNA was isolated from plate lysates as described (Helms et al., 1985). Inserts were subcloned into phage Ml3 and dideoxy sequenced @anger et al., 1977). The cDNA was sequenced in both orientations and analyzed using the DNAstar computer program package. Genomic DNA Analysis DNA was isolated from 107-lo8 cells, digested with restriction enzymes, and electrophoresed in a 1% aga-
the
268
HUMAN CTTGTTCGGGGCGCTCCGCTGGCTTCTTGGACAATTGCGC~TGTGCT 10" 20" 30" 40" 50" GCTCGGCTAGCGGCGGCGGCGGCCCAGTCGGTGTATGCCTTCTCGGCGCG 70* 60,' 80" 90" 100" CCCG~CCGGCGGGGAGCCTGTGAGCCTGGGCTCCCTGCGGGGCAAGG 11OA 120" 130" 14OA 150" TACTACTTATCGAGAATGTGGCGTCCCTC~GGCACCACGGTCCGGGAC 160" 170" 180* 190" 200" TACACCCAGATGAACGAGCTGCAGCGGCGCCTCGGACCCCGGGGCCTGGT 220" 210A 230" 240" 250" GGTGCTCGGCTTCCCGTGCAACCAGTTTGGGCATCAGGAGAACGCCAAGA 260" 270" 280" 29OA 300" ACGAAGAGATT~AATTCCCTCAAGTACGTCCGGCCTGGTGGTGGGTTC 320" 31OA 33OA 340" 350" GAGCCCAACTTCATGCTCTTCGAGAAGTGCGAGGTGAACGGTGCGGGGGC 370" 360" 380,' 390" 400" GCACCCTCTCTTCGCCTTCCTGCGGGAGGCCCTGCCAGCTCCCAGCGACG 410" 420" 430" 440" 450" ACGCCACCGCGCTTATGACCGACCCCAAGCTCATCACCTCATCACCTGGTCTCCGGTG 460" 470" 480" 490" 500" TGTCGCAACGATGTTGCCTGGAACTTTGAGAAGTTCCTGGTGGGCCCTGA 510" 520" 530" 550" 540" CGGTGTGCCCCTACGCAGGTACAGCCGCCGCTTCCAGACCATTGACATCG 570" 560" 580^ 590^ 600" AGCCTGACATCGAAGCCCTGCTGTCTCAAGGGCCCAGCTGTGCC~GGC 610" 620" 630" 640* 650" GCCCCTCCTACCCCGGCTGCTTGGCAGTTGCAGTGCTGCTGTCTCGGGGG 670" 660" 680" 690* 700" GGTTTTCATCTATGAGGGTGTTTCCTCTAAACCTACGAGGGAGGAACACC 710" 720" 730" 740^ 750" TGATCTTACAGAAAATACCACCTCGAGATGGGTGCTGGTCCTGTTGATCC 760" 770" 780" 790" 800" CAGTCTCTGCCAGACCAAGGCGAGTTTCCCCACTAATMAGTGCCGGGTG 810" 820^ 830" 840" 850,' TCAGCAAAAAAAAAAAAA 860"
FIG. 1. Human glutathione peroxidase cDNA sequence. The ATG initiator, TGA (selenocysteine) codon, TAG terminator, polyadenylation sequence, and the codons containing possible polymorphisms (bases 105-107 and 312-314) are underlined and printed in boldface.
rose gel (Maniatis et al., 1982). The DNA was hydrolyzed by acid depurination in 0.25 M HCl and denatured in 0.5 M NaOH-1.5 M NaCl. Transfer, prehybridization, and hybridization were performed as previously described (Gatti et al., 1984), with hybridization performed at 42°C in 50% formamide. The GPXl cDNA probe consisted of a 543-bp EcoRl restriction fragment (extending from the coding region to the end of the 3’-untranslated region) labeled by oligonucleotide priming (Feinberg and Vogelstein, 1984). After hybridization, filters were washed in 0.1% SSC, 0.1% SDS (Maniatis et aZ., 1982) at temperatures from 55 to 80°C.
GPXl
269
GENE RESULTS
A family of synthetic l&mers, corresponding to codons for amino acids 101-106 of bovine glutathione peroxidase (Zakowski et al., 1978), was used to screen a cDNA library from differentiating HL-60 cells in Xgtll. Inserts from positive plaques were subcloned into phage Ml3 and sequenced,the assembledsequence in shown in Fig. 1. Analysis of the GPXl cDNA sequence confirmed that the selenocysteine residue at amino acid 47 is encoded by the opal terminator TGA. Southern blot analysis of normal human leukocyte genomic DNA probed with GPXl cDNA revealed different patterns at standard and high stringencies. Normal human DNA was digested with a variety of endonucleases that do not have restriction sites within the GPXl cDNA sequence, hybridized with the GPXl probe, and washed at 68°C (Fig. 2, left) and then at 78°C (Fig. 2, right). Under standard conditions (Fig. 2, left) most lanes reveal three bands, indicating the presence of at least three genomic sequencesthat hybridize with the GPXl probe but possess different flanking regions. At very high stringency (Fig. 2, right), the digests produce only one strongly hybridizing band, with several lanes also showing a weaker band. This reduction in band number with increasing stringency suggeststhat one site bears closer sequence homology to the cDNA, but the high temperature requirement for the change and incomplete loss of somebands imply only slight sequence divergence in the alternate sites. To determine the chromosomal localization of the human GPXl gene, we hybridized a GPXl cDNA probe to normal human metaphase chromosomes. The dis-
-5
In Situ Chromosomal Hybridization Human metaphase cells were prepared from phytohemagglutinin-stimulated peripheral blood lymphocytes. A radiolabeled GPXl cDNA probe (from the same restriction fragment used for Southern blots) was prepared by nick translation of the entire plasmid with all four 3H-labeled deoxynucleoside triphosphates to a specific activity of 5.0 X 10’ dpm/pg. In situ hybridization was performed as described previously (Le Beau et al., 1984). Metaphase cells were hybridized at 2.0 and 4.0 ng of probe/ml of hybridization mixture. Autoradiographs were exposed for 11 days.
-2 -1 to FIG. 2. Southern blot analysis of GPXl cDNA hybridization normal human genomic DNA. Human lymphocyte DNA was digested with the indicated restriction endonucleases, which do not possess sites within the cDNA probe utilized. Electrophoresis, transfer, prehvbridisation. and hvbridization were oerformed as described under Materials andMethods, with washes at68’C (left) and 78’C (right).
270
CHADA
13
14
15
16
17
I8
CHRONOSOHE
19
ET
20
AL.
21
22
X
Y
3
NUMBER
FIG. 3. In situ chromosomal hybridization of the GPXI cDNA probe. (A) Distribution of labeled sites in cells from phytohemagglutinin-stimulated peripheral blood lymphocytes that were hybridized with the GPXI was observed on chromosome 3, at bands qll-13. (B) Distribution of labeled sites on chromosome 3. The largest at 3q12-13.1. Each dot indicates one labeled site observed in the corresponding band. Sixty-eight percent chromosome 3 were located at qll-13; this cluster represented 21% (38/181) of all labeled sites.
tribution of labeled sites is illustrated in Fig. 3. Specific labeling occurred only on chromosome 3 (Fig. 3A); of 100 metaphase cells examined from this hybridization, 30 were labeled on region ql of one or both chromosome 3 homologs. Of 181 labeled sites observed, 56 (31%) were located on this chromosome. These sites were clustered at bands qll-13 (Fig. 3B); this cluster represented 21% (38/181) of all labeled sites (cumulative probability for the Poisson distribution Q 0.0005). The largest number of grains was observed at 3q12-13.1. Similar results were obtained in a second hybridization experiment. DISCUSSION Using oligonucleotides directed against regions of the bovine amino acid sequence (Zakowski et ab, 1978), we have isolated cDNA clones for the major human selenoprotein, glutathione peroxidase. The human GPXl mRNA utilizes an opal terminator (UGA) codon to specify incorporation of selenocysteine, as previously reported in human GPXl clones isolated from human kidney cDNA (Mullenbach et aL., 1987), iiver cDNA (Sukenaga et al., 1987), and genomic (Ishida et al., 1987) libraries, as well as in mouse glutathione peroxidase (Chambers et aZ., 1986), in which it was first described. The nucleotide sequence we obtained for GPXl exactly confirms those reported by Sukenaga’s group (Sukenaga et al., 1987; Ishida et al., 1987) but differs at two coding region bases from that reported by Mullenbach’s laboratory (Mullenbach et al., 1987): at amino acid 22, we found leucine to be encoded by a TTG codon, whereas Mullenbach reported the same amino acid encoded by CTG; and at amino acid 91, we found leutine, encoded by CTG, and Mullenbach reported glutamate, encoded by CAG. These variations may rep-
100 normal human metaphase cDNA probe. Specific labeling number of grains was observed (38/56) of the labeled sites on
resent allelic differences. The substitution at amino acid 91 could constitute the basis of a known electrophoretic polymorphism (Beutler et al., 1974). The present Southern blot studies confirm the presence of three genomic copies of the sequence encoding glutathione peroxidase. Most likely the most strongly hybridizing at highest stringency is entirely homologous to the cDNA probe and therefore represents the GPXl gene. The others probably represent the other loci identified by McBride et al. (1988). Our in situ hybridization studies localize the human GPXl gene at chromosome 3qll-13, confirming and refining the prior chromosomal assignment (Johannsmann et aZ., 1981). This chromosomal band assignment also excludes the possibility, based on the previous broader localization, that GPXl is involved in the genomic alterations observed in 3~12-14 in small cell carcinoma of the lung (Naylor et al., 1987) and renal cell carcinoma (Seizinger et al., 1988). The absence of specific labeling on other chromosomes, including chromosome 21 and the X chromosome, in our in situ hybridization experiments probably reflects the stringency used in our studies as well as other factors such as the length and nature of the GPXl probe. We cannot exclude the possibility that all three sites identified on Southern blot occur in a cluster on chromosome 3, but it is more likely that the previously identified (McBride et al., 1988) processed pseudogenes are present but were not detected by the in situ technique. ACKNOWLEDGMENTS We thank Dr. Stuart Orkin for the synthetic oligonucleotides and Rafael Espinosa for technical assistance. This work was supported by National Institutes of Health Grants CA38325 and DK41625 and by the University of Chicago Cancer Besearch Foundation. M.M.L. is a Scholar of the Leukemia Society of America,
HUMAN
REFERENCES
GPXl
271
GENE
ox&se
gene maps to human chromosomes 3, 21, and X.
BioFactors
1. BEUTLER, E., WEST, C., AND BEUTLER, B. (1974). Electrophoretie polymorphism of glutathione peroxidase. Ann. Hum. Genet. 38: 163-169. 2. CHAMBERS, I., FRAMPTON, J., GOLDFARB, P., AFFARA, N., MCBAIN, W., AND HARRISON, P. R. (1986). The structure of the mouse glutathione peroxidase gene: The selenocysteine in the active site is encoded by the “termination” codon, TGA. EMBO J. 5: 1221-1227. 3. FEINBERG, A. P., AND VOGELSTEIN, B. (1984). Addendum: A technique for radioiabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 137: 266. 4. FORSTROM, J. W., ZAKOWSKI, J. J., AND TAPPEL, A. L. (1978). Identification of the catalytic site of rat liver glutathione perox&se as selenocysteine. Biochemistry 17: 2639-2644. 5. GA?TI, R. A., CONCANNON,P., AND SALSER, W. (1984). Multiple use of Southern blots. Biotechniques 2: 148-155. 6. HELMS, C., GRAHAM, M., DUTCHIK, J. E., AND OLSON, M. V. (1985). A new method for purifying lambda DNA from phage lysates. DNA 4: 39-49. 7. ISHIDA, K., MORINO, T., TAKAGI, K., AND SUKENAGA, Y. (1987). Nucleotide sequence of a human gene for glutathione peroxidase. Nucleic Acids Res. 15: 10051. 8. JOHANNSMANN, R., HELLKUHL, B., AND GFUESCHIK, K. H. (1981). Regional mapping of human chromosome 3: Assignment of a glutathione peroxidase 1 gene to 3p13-3q12. Hum. Genet. 66: 361-363. 9. LE BEAU, M. M., WESTBROOK, C. A., DIAZ, M. O., AND ROWLEY, J. D. (1984). Evidence for two distinct c-src loci on human chromosomes 1 and 20. Nature (London) 312: 70-71. 10. MANIATIS, T., FRITSCH, E. F., AND SAMBROOK, J. (1982). “MOlecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 11. MCBRIDE, 0. W., LEE, B. J., HATFIELD, D. L., AND MULLENBACH, G. (1988). Gene for selenium-dependent glutathione par-
1: 285-292.
12. MILLS, G. C. (1957). Glutathione peroxidase, an erythrocyte enzyme which protects hemoglobin from oxidative breakdown. J. Biol. Chem. 229: 189-197. 13. MULLENBACH, G. T., TABRIZI, A., IRVINE, B. D., BELL, G. I., AND HALLEWELL, R. A. (1987). Sequence of a cDNA coding for human glutathione peroxidase confirms TGA encodes active site selenocysteine. Nucleic Acids Res. 15: 5484. 14. NAYLOR, S. L., JOHNSON,B. E., MINNA, J. D., AND SAKAGUCHI, A. Y. (1987). Loss of heterozygosity of chromosome 3p markers in small cell lung cancer. Nature (London) 329: 451-454. 15. ROYER-POKORA, B., KUNKEL, L. M., MONACO, A. P., GOFF, S. C., NEWBURGER, P. E., BAEHNER, R. L., COLE, F. S., CURNU’ITE, J. T., AND ORKIN, S. H. (1986). Cloning the gene for an inherited disorder-chronic granuiomatous disease-on the basis of its chromosomal location. Nature (London) 322: 3238. 16. SANGER, F., NICKLEN, S., AND COULSON, A. R. (1977). DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:
5463-5467.
17. SEIZINGER, B. R., ROULEAU, G. A., Oz~~nrs, L. J., et al. (1988). Von Hippel-Lindau disease maps to the region of chromosome 3 associated with renal cell carcinoma. Nature (London) 330: 266-269.
18. SUKENAGA, Y., ISHIDA, K., TAKEDA, T., AND TAKAGI, K. (1987). cDNA sequence coding for human glutathione peroxidase. Nucleic Acids Res. 15: 7178. 19. WIJNEN, L. M., MONTEBA VAN HEUVEL, M., PEARSON, P. L., AND MEERA KHAN, P. (1978). Assignment of a gene for glutathione peroxidase (GPXl) to human chromosome 3. Cytogenet.
Cell Genet.
22:
232-235.
20. ZAKOWSKI, J. J., FORSTROM, J. W., CONDELL, R. A., AND TAPPEL, A. L. (1978). Attachment of seienocysteine in the catalytic site of glutathione peroxidase. B&hem. Biophys. Res. Commun. 84: 248-253.
6,268-271
(1990)
Isolation and Chromosomal Localization of the Human Glutathione Peroxidase Gene SUNIL CHADA, MICHELLE M. LE BEAU,* LINDA CASEY, AND PETER E. NEWBURGER Departments
of Pediatrics and Molecular Genetics/Microbiology, University of Massachusetts Worcester, Massachusetts 0 1655; and ‘Joint Section of Hematology/Oncology, University of Chicago Medical School, Chicago, Illinois 60637 Received
July 7, 1989;
revised
We have isolated
cDNA clones for the gene, termed the major human selenoprotein, glutathione peroxidase. Sequence analysis coniirmed previous findings that the unusual amino acid selenocysteine is encoded by the opal terminator codon UGA. Southern blot analysis of human genomic DNA with the GPXI cDNA showed that restriction endonucleases without sites in the probe sequence produced three hybridizing bands at standard stringency, diminishing to one strongly and one weakly hybridizing band at high stringency. In situ hybridization localized the human GPXl gene to a single site on chromosome 3, at region 3qll-13.1. Thus, three genomic sites bear sequence homology to the GPXl cDNA, and the one most homologous maps to 3qll-13.1. o leso Acrdedc Inc.
INTRODUCTION
Glutathione peroxidase (EC 1.11.1.9) is an enzyme, widely distributed in eukaryotic cells, that catalyzes the reduction of hydroperoxides by glutathione (GSH) (Mills, 1957). The enzyme represents the major cellular defense against these toxic oxidant species.The protein is a soluble tetramer; each 22-kDa subunit contains one selenium atom in its reduced state, incorporated within a catalytically active selenocysteine residue (Forstrom et aZ., 1978). Sequence analysis of the murine glutathione peroxidase gene (GPXl) revealed the selenocysteine residue to be encoded by a TGA “termination” codon (Chambers et al., 1986). An identical codon and overall similar sequences have recently been reported for the human GPXl gene in clones isolated from human liver cDNA (Mullenbach et al., 1987), kidney cDNA (SuSequence data EME%L/GenBank 03&s7543po
from Data
this article have been deposited with Libraries under Accession No. M21304.
$3.00
Copyright 0 1990 by Academic Press, Inc. AlI rights of reproduction in any form reserved.
School,
11, 1989
kenaga et al., 1987), and genomic (Ishida et al., 1987) libraries. The chromosomal localization of the human GPXl gene has not been definitively made. An analysis of the electrophoretic separation of glutathione peroxidase speciesin human-Chinese hamster somatic cell hybrids first indicated an assignment of the GPXl gene to human chromosome 3 (Wijnen et al., 1978). Similar studies utilizing somatic cell hybrid clones containing rearrangements and deletions involving human chromosome 3 resulted in the further localization of GPXl to 3p14-q21, with the probable smallest region of overlap 3p13-q12 (Johannsmann et aZ., 1981). However, recent studies of hybridization of a human GPXl cDNA probe to genomic DNA from human-rodent somatic cell hybrids have demonstrated not only the locus on chromosome 3, but also additional cross-hybridizing sites on chromosome 21 and the X chromosome (McBride et al., 1988). The latter sequenceslack introns and show some sequence divergence; thus they may represent processed pseudogenes.
GPXl, encoding
Prau.
September
Medical
MATERIALS
AND
METHODS
Isolation and Sequence Analysis of cDNA Clones A previously described (Royer-Pokora et al., 1986) HL-60 human myeloid leukemia cell line cDNA library in Xgtll was probed with end-labeled oligonucleotides, synthesized using an Applied Biosystems DNA synthesizer. Individual positive plaques were purified by multiple rounds of screening and DNA was isolated from plate lysates as described (Helms et al., 1985). Inserts were subcloned into phage Ml3 and dideoxy sequenced @anger et al., 1977). The cDNA was sequenced in both orientations and analyzed using the DNAstar computer program package. Genomic DNA Analysis DNA was isolated from 107-lo8 cells, digested with restriction enzymes, and electrophoresed in a 1% aga-
the
268
HUMAN CTTGTTCGGGGCGCTCCGCTGGCTTCTTGGACAATTGCGC~TGTGCT 10" 20" 30" 40" 50" GCTCGGCTAGCGGCGGCGGCGGCCCAGTCGGTGTATGCCTTCTCGGCGCG 70* 60,' 80" 90" 100" CCCG~CCGGCGGGGAGCCTGTGAGCCTGGGCTCCCTGCGGGGCAAGG 11OA 120" 130" 14OA 150" TACTACTTATCGAGAATGTGGCGTCCCTC~GGCACCACGGTCCGGGAC 160" 170" 180* 190" 200" TACACCCAGATGAACGAGCTGCAGCGGCGCCTCGGACCCCGGGGCCTGGT 220" 210A 230" 240" 250" GGTGCTCGGCTTCCCGTGCAACCAGTTTGGGCATCAGGAGAACGCCAAGA 260" 270" 280" 29OA 300" ACGAAGAGATT~AATTCCCTCAAGTACGTCCGGCCTGGTGGTGGGTTC 320" 31OA 33OA 340" 350" GAGCCCAACTTCATGCTCTTCGAGAAGTGCGAGGTGAACGGTGCGGGGGC 370" 360" 380,' 390" 400" GCACCCTCTCTTCGCCTTCCTGCGGGAGGCCCTGCCAGCTCCCAGCGACG 410" 420" 430" 440" 450" ACGCCACCGCGCTTATGACCGACCCCAAGCTCATCACCTCATCACCTGGTCTCCGGTG 460" 470" 480" 490" 500" TGTCGCAACGATGTTGCCTGGAACTTTGAGAAGTTCCTGGTGGGCCCTGA 510" 520" 530" 550" 540" CGGTGTGCCCCTACGCAGGTACAGCCGCCGCTTCCAGACCATTGACATCG 570" 560" 580^ 590^ 600" AGCCTGACATCGAAGCCCTGCTGTCTCAAGGGCCCAGCTGTGCC~GGC 610" 620" 630" 640* 650" GCCCCTCCTACCCCGGCTGCTTGGCAGTTGCAGTGCTGCTGTCTCGGGGG 670" 660" 680" 690* 700" GGTTTTCATCTATGAGGGTGTTTCCTCTAAACCTACGAGGGAGGAACACC 710" 720" 730" 740^ 750" TGATCTTACAGAAAATACCACCTCGAGATGGGTGCTGGTCCTGTTGATCC 760" 770" 780" 790" 800" CAGTCTCTGCCAGACCAAGGCGAGTTTCCCCACTAATMAGTGCCGGGTG 810" 820^ 830" 840" 850,' TCAGCAAAAAAAAAAAAA 860"
FIG. 1. Human glutathione peroxidase cDNA sequence. The ATG initiator, TGA (selenocysteine) codon, TAG terminator, polyadenylation sequence, and the codons containing possible polymorphisms (bases 105-107 and 312-314) are underlined and printed in boldface.
rose gel (Maniatis et al., 1982). The DNA was hydrolyzed by acid depurination in 0.25 M HCl and denatured in 0.5 M NaOH-1.5 M NaCl. Transfer, prehybridization, and hybridization were performed as previously described (Gatti et al., 1984), with hybridization performed at 42°C in 50% formamide. The GPXl cDNA probe consisted of a 543-bp EcoRl restriction fragment (extending from the coding region to the end of the 3’-untranslated region) labeled by oligonucleotide priming (Feinberg and Vogelstein, 1984). After hybridization, filters were washed in 0.1% SSC, 0.1% SDS (Maniatis et aZ., 1982) at temperatures from 55 to 80°C.
GPXl
269
GENE RESULTS
A family of synthetic l&mers, corresponding to codons for amino acids 101-106 of bovine glutathione peroxidase (Zakowski et al., 1978), was used to screen a cDNA library from differentiating HL-60 cells in Xgtll. Inserts from positive plaques were subcloned into phage Ml3 and sequenced,the assembledsequence in shown in Fig. 1. Analysis of the GPXl cDNA sequence confirmed that the selenocysteine residue at amino acid 47 is encoded by the opal terminator TGA. Southern blot analysis of normal human leukocyte genomic DNA probed with GPXl cDNA revealed different patterns at standard and high stringencies. Normal human DNA was digested with a variety of endonucleases that do not have restriction sites within the GPXl cDNA sequence, hybridized with the GPXl probe, and washed at 68°C (Fig. 2, left) and then at 78°C (Fig. 2, right). Under standard conditions (Fig. 2, left) most lanes reveal three bands, indicating the presence of at least three genomic sequencesthat hybridize with the GPXl probe but possess different flanking regions. At very high stringency (Fig. 2, right), the digests produce only one strongly hybridizing band, with several lanes also showing a weaker band. This reduction in band number with increasing stringency suggeststhat one site bears closer sequence homology to the cDNA, but the high temperature requirement for the change and incomplete loss of somebands imply only slight sequence divergence in the alternate sites. To determine the chromosomal localization of the human GPXl gene, we hybridized a GPXl cDNA probe to normal human metaphase chromosomes. The dis-
-5
In Situ Chromosomal Hybridization Human metaphase cells were prepared from phytohemagglutinin-stimulated peripheral blood lymphocytes. A radiolabeled GPXl cDNA probe (from the same restriction fragment used for Southern blots) was prepared by nick translation of the entire plasmid with all four 3H-labeled deoxynucleoside triphosphates to a specific activity of 5.0 X 10’ dpm/pg. In situ hybridization was performed as described previously (Le Beau et al., 1984). Metaphase cells were hybridized at 2.0 and 4.0 ng of probe/ml of hybridization mixture. Autoradiographs were exposed for 11 days.
-2 -1 to FIG. 2. Southern blot analysis of GPXl cDNA hybridization normal human genomic DNA. Human lymphocyte DNA was digested with the indicated restriction endonucleases, which do not possess sites within the cDNA probe utilized. Electrophoresis, transfer, prehvbridisation. and hvbridization were oerformed as described under Materials andMethods, with washes at68’C (left) and 78’C (right).
270
CHADA
13
14
15
16
17
I8
CHRONOSOHE
19
ET
20
AL.
21
22
X
Y
3
NUMBER
FIG. 3. In situ chromosomal hybridization of the GPXI cDNA probe. (A) Distribution of labeled sites in cells from phytohemagglutinin-stimulated peripheral blood lymphocytes that were hybridized with the GPXI was observed on chromosome 3, at bands qll-13. (B) Distribution of labeled sites on chromosome 3. The largest at 3q12-13.1. Each dot indicates one labeled site observed in the corresponding band. Sixty-eight percent chromosome 3 were located at qll-13; this cluster represented 21% (38/181) of all labeled sites.
tribution of labeled sites is illustrated in Fig. 3. Specific labeling occurred only on chromosome 3 (Fig. 3A); of 100 metaphase cells examined from this hybridization, 30 were labeled on region ql of one or both chromosome 3 homologs. Of 181 labeled sites observed, 56 (31%) were located on this chromosome. These sites were clustered at bands qll-13 (Fig. 3B); this cluster represented 21% (38/181) of all labeled sites (cumulative probability for the Poisson distribution Q 0.0005). The largest number of grains was observed at 3q12-13.1. Similar results were obtained in a second hybridization experiment. DISCUSSION Using oligonucleotides directed against regions of the bovine amino acid sequence (Zakowski et ab, 1978), we have isolated cDNA clones for the major human selenoprotein, glutathione peroxidase. The human GPXl mRNA utilizes an opal terminator (UGA) codon to specify incorporation of selenocysteine, as previously reported in human GPXl clones isolated from human kidney cDNA (Mullenbach et aL., 1987), iiver cDNA (Sukenaga et al., 1987), and genomic (Ishida et al., 1987) libraries, as well as in mouse glutathione peroxidase (Chambers et aZ., 1986), in which it was first described. The nucleotide sequence we obtained for GPXl exactly confirms those reported by Sukenaga’s group (Sukenaga et al., 1987; Ishida et al., 1987) but differs at two coding region bases from that reported by Mullenbach’s laboratory (Mullenbach et al., 1987): at amino acid 22, we found leucine to be encoded by a TTG codon, whereas Mullenbach reported the same amino acid encoded by CTG; and at amino acid 91, we found leutine, encoded by CTG, and Mullenbach reported glutamate, encoded by CAG. These variations may rep-
100 normal human metaphase cDNA probe. Specific labeling number of grains was observed (38/56) of the labeled sites on
resent allelic differences. The substitution at amino acid 91 could constitute the basis of a known electrophoretic polymorphism (Beutler et al., 1974). The present Southern blot studies confirm the presence of three genomic copies of the sequence encoding glutathione peroxidase. Most likely the most strongly hybridizing at highest stringency is entirely homologous to the cDNA probe and therefore represents the GPXl gene. The others probably represent the other loci identified by McBride et al. (1988). Our in situ hybridization studies localize the human GPXl gene at chromosome 3qll-13, confirming and refining the prior chromosomal assignment (Johannsmann et aZ., 1981). This chromosomal band assignment also excludes the possibility, based on the previous broader localization, that GPXl is involved in the genomic alterations observed in 3~12-14 in small cell carcinoma of the lung (Naylor et al., 1987) and renal cell carcinoma (Seizinger et al., 1988). The absence of specific labeling on other chromosomes, including chromosome 21 and the X chromosome, in our in situ hybridization experiments probably reflects the stringency used in our studies as well as other factors such as the length and nature of the GPXl probe. We cannot exclude the possibility that all three sites identified on Southern blot occur in a cluster on chromosome 3, but it is more likely that the previously identified (McBride et al., 1988) processed pseudogenes are present but were not detected by the in situ technique. ACKNOWLEDGMENTS We thank Dr. Stuart Orkin for the synthetic oligonucleotides and Rafael Espinosa for technical assistance. This work was supported by National Institutes of Health Grants CA38325 and DK41625 and by the University of Chicago Cancer Besearch Foundation. M.M.L. is a Scholar of the Leukemia Society of America,
HUMAN
REFERENCES
GPXl
271
GENE
ox&se
gene maps to human chromosomes 3, 21, and X.
BioFactors
1. BEUTLER, E., WEST, C., AND BEUTLER, B. (1974). Electrophoretie polymorphism of glutathione peroxidase. Ann. Hum. Genet. 38: 163-169. 2. CHAMBERS, I., FRAMPTON, J., GOLDFARB, P., AFFARA, N., MCBAIN, W., AND HARRISON, P. R. (1986). The structure of the mouse glutathione peroxidase gene: The selenocysteine in the active site is encoded by the “termination” codon, TGA. EMBO J. 5: 1221-1227. 3. FEINBERG, A. P., AND VOGELSTEIN, B. (1984). Addendum: A technique for radioiabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 137: 266. 4. FORSTROM, J. W., ZAKOWSKI, J. J., AND TAPPEL, A. L. (1978). Identification of the catalytic site of rat liver glutathione perox&se as selenocysteine. Biochemistry 17: 2639-2644. 5. GA?TI, R. A., CONCANNON,P., AND SALSER, W. (1984). Multiple use of Southern blots. Biotechniques 2: 148-155. 6. HELMS, C., GRAHAM, M., DUTCHIK, J. E., AND OLSON, M. V. (1985). A new method for purifying lambda DNA from phage lysates. DNA 4: 39-49. 7. ISHIDA, K., MORINO, T., TAKAGI, K., AND SUKENAGA, Y. (1987). Nucleotide sequence of a human gene for glutathione peroxidase. Nucleic Acids Res. 15: 10051. 8. JOHANNSMANN, R., HELLKUHL, B., AND GFUESCHIK, K. H. (1981). Regional mapping of human chromosome 3: Assignment of a glutathione peroxidase 1 gene to 3p13-3q12. Hum. Genet. 66: 361-363. 9. LE BEAU, M. M., WESTBROOK, C. A., DIAZ, M. O., AND ROWLEY, J. D. (1984). Evidence for two distinct c-src loci on human chromosomes 1 and 20. Nature (London) 312: 70-71. 10. MANIATIS, T., FRITSCH, E. F., AND SAMBROOK, J. (1982). “MOlecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 11. MCBRIDE, 0. W., LEE, B. J., HATFIELD, D. L., AND MULLENBACH, G. (1988). Gene for selenium-dependent glutathione par-
1: 285-292.
12. MILLS, G. C. (1957). Glutathione peroxidase, an erythrocyte enzyme which protects hemoglobin from oxidative breakdown. J. Biol. Chem. 229: 189-197. 13. MULLENBACH, G. T., TABRIZI, A., IRVINE, B. D., BELL, G. I., AND HALLEWELL, R. A. (1987). Sequence of a cDNA coding for human glutathione peroxidase confirms TGA encodes active site selenocysteine. Nucleic Acids Res. 15: 5484. 14. NAYLOR, S. L., JOHNSON,B. E., MINNA, J. D., AND SAKAGUCHI, A. Y. (1987). Loss of heterozygosity of chromosome 3p markers in small cell lung cancer. Nature (London) 329: 451-454. 15. ROYER-POKORA, B., KUNKEL, L. M., MONACO, A. P., GOFF, S. C., NEWBURGER, P. E., BAEHNER, R. L., COLE, F. S., CURNU’ITE, J. T., AND ORKIN, S. H. (1986). Cloning the gene for an inherited disorder-chronic granuiomatous disease-on the basis of its chromosomal location. Nature (London) 322: 3238. 16. SANGER, F., NICKLEN, S., AND COULSON, A. R. (1977). DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:
5463-5467.
17. SEIZINGER, B. R., ROULEAU, G. A., Oz~~nrs, L. J., et al. (1988). Von Hippel-Lindau disease maps to the region of chromosome 3 associated with renal cell carcinoma. Nature (London) 330: 266-269.
18. SUKENAGA, Y., ISHIDA, K., TAKEDA, T., AND TAKAGI, K. (1987). cDNA sequence coding for human glutathione peroxidase. Nucleic Acids Res. 15: 7178. 19. WIJNEN, L. M., MONTEBA VAN HEUVEL, M., PEARSON, P. L., AND MEERA KHAN, P. (1978). Assignment of a gene for glutathione peroxidase (GPXl) to human chromosome 3. Cytogenet.
Cell Genet.
22:
232-235.
20. ZAKOWSKI, J. J., FORSTROM, J. W., CONDELL, R. A., AND TAPPEL, A. L. (1978). Attachment of seienocysteine in the catalytic site of glutathione peroxidase. B&hem. Biophys. Res. Commun. 84: 248-253.
