Human Glutamate Pyruvate Transaminase (GPT): Localization to 8q24.3, cDNA and Genomic Sequences, and Polymorphic Sites

GENOMICS

40, 247– 252 (1997) GE964604

ARTICLE NO.

Human Glutamate Pyruvate Transaminase (GPT): Localization to 8q24.3, cDNA and Genomic Sequences, and Polymorphic Sites MELANIE M. SOHOCKI,* LORI S. SULLIVAN,*,† WILBUR R. HARRISON,‡ ERICA J. SODERGREN,§ FREDERICK F. B. ELDER,‡ GEORGE WEINSTOCK,§ SUMIO TANASE,Ø AND STEPHEN P. DAIGER*,†,1 *Human Genetics Center, School of Public Health, †Department of Ophthalmology and Visual Science, ‡Department of Pathology and Laboratory Medicine, and §Department of Biochemistry, The University of Texas Health Science Center, Houston, Texas 77225; and ØDepartment of Biochemistry, Kumamoto University School of Medicine, Kumamoto-shi 860, Japan Received September 23, 1996; accepted December 21, 1996

Two frequent protein variants of glutamate pyruvate transaminase (GPT) (E.C.2.6.1.2) have been used as genetic markers in humans for more than two decades, although chromosomal mapping of the GPT locus in the 1980s produced conflicting results. To resolve this conflict and develop useful DNA markers for this gene, we isolated and characterized cDNA and genomic clones of GPT. We have definitively mapped human GPT to the terminus of 8q using several methods. First, two cosmids shown to contain the GPT sequence were derived from a chromosome 8-specific library. Second, by fluorescence in situ hybridization, we mapped the cosmid containing the human GPT gene to chromosome band 8q24.3. Third, we mapped the rat gpt cDNA to the syntenic region of rat chromosome 7. Finally, PCR primers specific to human GPT amplify sequences contained within a ‘‘half-YAC’’ from the long arm of chromosome 8, that is, a YAC containing the 8q telomere. The human GPT genomic sequence spans 2.7 kb and consists of 11 exons, ranging in size from 79 to 243 bp. The exonic sequence encodes a protein of 495 amino acids that is nearly identical to the previously reported protein sequence of human GPT-1. The two polymorphic GPT isozymes are the result of a nucleotide substitution in codon 14, coding for a histidine in GPT-1 and an asparagine in GPT-2, which causes a gain or loss of an NlaIII restriction site. In addition, a cosmid containing the GPT sequence also contains a previously unmapped, polymorphic microsatellite sequence, D8S421. The cloned GPT gene and associated polymorphisms will be useful for linkage and physical mapping of disease loci that map to the terminus of 8q, including atypical vitelliform macular dystrophy (VMD1) and epidermolysis bullosa simplex, type Ogna (EBS1). In addition, this will be a useful system for characterizing the telomeric region of 8q. Finally, determina1 To whom correspondence should be addressed at Human Genetics Center, School of Public Health, The University of Texas Health Science Center,P. O. Box 20334, Houston, TX 77225-0334. Telephone: (713) 500-9829. Fax: (713) 500-0900.

tion of the molecular basis of the GPT isozyme variants will permit PCR-based detection of this worldwide polymorphism. q 1997 Academic Press

INTRODUCTION

Glutamate pyruvate transaminase (E.C.2.6.1.2), also known as alanine aminotransferase 1, catalyzes an important reaction in amino acid metabolism and gluconeogenesis by converting L-alanine and a-ketoglutarate to L-glutamate and pyruvate (Welch, 1972). GPT is known to have both soluble and mitochondrial forms, and serum GPT activity has been used as an indicator of hepatocellular injury. Genetic polymorphism of the soluble GPT protein from human erythrocyte lysates was reported by Chen and Giblett in 1971, and for more than 25 years, GPT isozymes have been used as a serological marker for linkage studies (Roychoudhury and Nei, 1988). Studies reporting linkage of human diseases to the GPT locus include linkage to epidermolysis bullosa simplex, type Ogna (EBS1) (OMIM 131950), with a maximum lod score of 11 at 5% recombination (Olaisen and Gedde-Dahl, 1973) and linkage to atypical vitelliform macular dystrophy (VMD1) (OMIM 153840) with a maximum lod score of 4.3 at 5% recombination (Ferrell et al., 1983). Previous reports of the localization of human GPT produced conflicting results. Khan and Wijnen (1984) reported localization of the structural gene of soluble GPT-1 to human chromosome 16 using hybrids of human blood leukocytes with Chinese hamster fibroblasts. Other groups assigned the gene to chromosome 8 using hybrids of human liver fibroblasts and rat hepatoma cells (Jeremiah et al., 1984; Astrin et al., 1982). Linkage of GPT to RFLP markers that map to 8q has been reported, with a maximum two-point lod score of 5.4 between GPT and D8S39, but placement of GPT on ‘‘framework’’ linkage maps has been problematic (O’Connell et al., 1987; Spurr et al., 1994; and unpublished results). In addition, no human DNA sequence data have been published, although the amino acid se-

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FIG. 1. Human GPT cosmids and gene structure. (A) Restriction map of the GPT region of human chromosome 8. The region of chromosome 8 spanned by cosmids 68C8 and 105B9 comprises 54 kb. The respective 35- and 44-kb inserts of the cosmids are represented by bars below the map. The shaded region corresponds to a 2.7-kb XhoI fragment containing most of the GPT gene. (B) Human GPT structural diagram. Exons are represented by blocks and are designated with Roman numerals. 5*- and 3 *-untranslated regions are designated by hashed marks. Intron and exon lengths are shown in bp.

quence of the GPT-1 isozyme was reported previously (Ishiguro et al., 1991). Thus, human GPT has been mapped by serologic phenotype only, as the DNA sequence difference between the GPT-1 and GPT-2 alleles was previously unknown. In this paper we report the isolation and characterization of cDNA and genomic clones of human GPT. Based on these data, we present the gene structure, cDNA and genomic sequences, and definitive chromosomal localization of the gene. We also report the nucleotide differences accounting for the GPT isozymes that allow typing of this classical serological marker at a DNA level. MATERIALS AND METHODS Screening of a human liver cDNA library and isolation of human GPT cDNA. A human liver cDNA library, constructed by Dr. Sumio Tanase at the Kumamoto University School of Medicine, Japan, was screened by the in situ plaque hybridization method (Maniatis et al., 1982). The filters were hybridized at 657C with a nick-translated BamHI/XhoI fragment (504 bp) derived from rat gpt cDNA (GenBank D10354). One clone contained a cDNA insert of 1648 bp. This clone lacked the 5* part of the coding sequence that corresponds to the three amino-terminal residues of GPT (Ishiguro et al., 1991) but was otherwise complete. Isolation and restriction mapping of genomic clones encoding human glutamate pyruvate transaminase. A 32P-labeled 1800-bp rat gpt cDNA with 81% homology to the human GPT cDNA sequence was used to screen the human chromosome 8 cosmid library LA08NC01 (Wood et al., 1992). The rat cDNA (GenBank D10354) was provided by M. Funatsu, The Chemo-Sero-Theraputic Research Institute, Kumamoto, Japan. The hybridization was performed using

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Rapid Hyb buffer (Amersham) at 657C for 2 h. Following hybridization, filters were washed to a final high stringency of 0.11 SSC, 0.1% SDS, for 20 min at 657C. Confirmed positives were grown in LB medium with 50 mg/ml kanamycin, and cosmid DNAs were prepared using the Wizard ‘‘midipreps’’ DNA purification system (Promega). Two GPT-containing cosmids, 68C8 and 105B9, were identified by this process. Large-scale preparations of cosmid DNA were purified by CsCl gradient centrifugation (Maniatis et al., 1982). Single restriction digests (XhoI, EcoRI, BamHI, HindIII, NotI, EcoRV, and DraI) and double digests (XhoI / EcoRI, XhoI / BamHI, EcoRI / DraI, BamHI / DraI, EcoRI / BamHI, BamHI / NotI, HindIII / NotI, HindIII / EcoRI, HindIII / BamHI, EcoRV / BamHI, and EcoRV / EcoRI) were carried out in accordance with the manufacturer’s suggested conditions. Sizes of restriction fragments were determined by comparison with a 1-kb DNA ladder and a Lambda/HindIII marker (BRL). The accuracy of restriction fragment alignments was supported by analysis using the computer program ‘‘Double Digester’’ (v1.1b3 by Lawrence Wright, available by anonymous ftp at ftp.cs. yale.edu). The DNA fragments were transferred to positively charged nylon membranes (Amersham) according to the manufacturer’s recommendations. The rat gpt cDNA was labeled by the random primer method (Promega) and used to screen the restriction fragments for human GPT. The ‘‘half-YAC’’ containing the 8q telomere (Macina et al., 1995) was screened for GPT by PCR amplification using several primer pairs. Sequence characterization of human GPT. Sequencing of the GPT genomic clone in cosmid 68C8 was carried out using a primer walking technique, beginning with primers based on a human GPT cDNA sequence that lies within the 3*-untranslated region of the gene, and HGPT3F, 5*-CGCAGAGGTAACCGGAGCCA-3 *, which begins with the third codon of the gene. All oligonucleotides were synthesized by a commercial source (Gibco BRL). Sequencing was performed using the fmol DNA sequencing system (Promega). The sequencing PCR protocol consisted of a 2-min denaturation step at 947C followed by 30 cycles of 1 min at 947C and 1 min at 557C. The sequence comparison of

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FIG. 2. Localization of GPT/gpt by fluorescence in situ hybridization. (A) Human metaphase; small arrows indicate chromosome 8specific alpha satellite probe; large arrows indicate human GPT probe at 8q24.3. (B) G-banded rat metaphase; arrow indicates rat chromosome 7 and band 7q33 –q34. (C) FISH with rat gpt cDNA probe on the same metaphase as in B; arrow indicates signal at 7q33 – q34. the human genomic GPT sequence to the partial human cDNA sequence (GenBank D10355) was used to confirm gene sequence and determine intron/exon junctions. Web Signal Scan (http:// www.crc.nus.sg; Prestridge, 1991) was used to predict transcription factor binding sites. Sequence polymorphism identification and testing. Initial genomic sequencing of GPT homozygotes was performed in CEPH family DNAs 1421-01 (GPT 1-1) and 1421-02 (GPT 2-2). Once identified, the polymorphism was confirmed by sequencing 1344-02, 1346-01, and 1413-1 (GPT 1-1), and 1331-01, 1346-02, and 1413-02 (GPT 22). Sequencing across the polymorphic region was performed with an initial PCR amplification using a 5*-biotinylated reverse primer (HGPTB115R: 5*-bio-CCCACACGCCTGCAAGATGC-3 *) and an unlabeled forward primer (HGPTSQ3F: 5*-CCTTCCCGCCTGGTCTGGGT-3*). The amplified fragment was purified using Dynabeads M-280 Streptavidin (Dynal). The AmpliCycle Sequencing Kit (Perkin Elmer) was then used with a nested reverse primer for sequencing (HGPT47R2, 5*-AGCTCCAAGGCTCGCTGCAC-3 *). For GPT typing using restriction digestion, genomic DNA was amplified by PCR for 40 cycles using the primers HGPTSQ3F and HGPT47R2 (sequences above), which span the polymorphic region. The restriction enzymes Hsp92II or NlaIII were used to digest 12.5 ml of the PCR product in a 25-ml reaction. The resulting DNA fragments were separated on a 6% NuSieve 3:1 (FMC) agarose gel. Fluorescence in situ hybridization (FISH). Blood from a normal human female donor was obtained, and chromosome slides for FISH were prepared using standard cytogenetic techniques. The slides were stored in a dry environment at 377C prior to use. Cosmid 68C8 (Fig. 1A) DNA, which contains the entire GPT sequence, was labeled with digoxigenin-11 –dUTP (Boehringer Mannheim) by nick-translation. A probe mixture of 200 ng labeled cosmid DNA, 5 mg human Cot-1 DNA, 10 mg sheared salmon sperm DNA, and 1 ml chromosome

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8 alpha-satellite probe (Oncor) in 50% formamide/21 SSC with 10% dextran sulfate was denatured for 6 min at 727C and applied to a denatured slide (2 min at 727C in 70% formamide/21 SSC). Hybridization was carried out overnight at 377C in a moist chamber. Following hybridization the slides were washed for 5 min at 727C in 21 SSC, and signals were detected and amplified with successive layers of anti-digoxigenin-fluorescein, rabbit anti-sheep IgG, and anti-rabbit IgG-fluorescein (Boehringer Mannheim). Slides were counterstained with propidium iodide/antifade (0.2 mg/ml), and metaphases were photographed with Kodak Gold 100 film on a Zeiss Axioskop equipped with epifluorescence. Slides used for mapping the rat gpt gene were from normal Rattus norvegicus fibroblast cultures. The slides were G-banded and representative metaphases photographed. The slides were then destained in 70% ethanol and treated at room temperature for 2.5 min in 3% buffered formalin and for 5 min in 0.2 N HCl. Hybridization and detection using the 1800-bp rat gpt cDNA probe was performed in the same manner as described previously.

RESULTS

Chromosomal Localization of the GPT Gene G-banded rat chromosome spreads were probed using an 1800-bp rat glutamate pyruvate transaminase cDNA which was nick-translated with digoxigenin-11– dUTP. Gpt was localized to rat 7q33–q34, a region syntenic to human 8q24 (Levan, 1991). Screening of a human chromosome 8 cosmid library using the rat gpt cDNA yielded two positive clones, 68C8 and 105B9,

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FIG. 3. Human GPT sequence. (A) Sequence of GPT 5*-untranslated region and exon 1. Positions of PCR primers used to amplify for GPT RFLP genotyping are indicated by arrows. Codon 14 containing the polymorphism is boxed. (B) Coding sequence (introns not shown). Codon numbers are to the left; amino acid is below each codon.

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which overlap each other. Figure 1A shows the restriction map of the clones. The 2.7-kb XhoI fragment in the overlap region of the clones was shown to contain GPT by Southern hybridization with the rat gpt cDNA. Cosmid 68C8 was nick-translated with digoxigenin11–dUTP and used to probe human chromosome spreads. This GPT-containing probe hybridized consistently at 8q24.3, the most distal band on the long arm of human chromosome 8 (Fig. 2). Several human GPT-specific primer pairs amplify GPT sequences in a chromosome 8q half-YAC, that is, a YAC that must contain a human telomere to propagate the human insert (Macina et al., 1995). The human insert in the 8q half-YAC is approximately 190 kb. Thus the GPT gene sequence and, presumably, the GPT-containing cosmids map within approximately 200 kb of the 8q telomere in humans. Isolation and Structural Organization of the Human Glutamate Pyruvate Transaminase Gene Sequence analysis of the 68C8 clone indicated that the GPT gene is composed of 11 exons ranging in length from 79 to 243 bp. The human GPT genomic map spans approximately 3 kb and is graphically illustrated in Fig. 1B. The predicted 495-amino-acid sequence of clone 68C8 is consistent with the published GPT-1 amino acid sequence— with a single amino acid difference, a histidine at position 222 in our sequencing and an alanine at this position in Ishiguro et al. (1991). We have confirmed the histidine at this position in five individuals, of both GPT isozyme types, including the human GPT cDNA and cosmid 68C8. Using 68C8 as a template, the DNA sequences of all exons, of all introns, and of the 5*- and 3*-untranslated regions were determined (GenBank U70732). The 10 introns of this gene all begin with a 5*-gt and conclude with a 3*-ag terminus, consistent with the consensus sequences for splice junctions in eukaryotic genes (Mount, 1982). Figure 3A presents the human GPT genomic sequence 5* to the ATG, and it includes the sequence of the first exon. As the human cDNA lacked the first three codons, as well as the 5*-untranslated region, the WebSignal Scan program was used to predict potential transcription factor binding sites in the genomic sequence. The region immediately upstream of the ATG start codon (Ç250 bp) is enriched (70%) in GC content and includes several potential Sp1 recognition sites (Briggs et al., 1986). There are no apparent TATA sequences or CAAT boxes in the GPT promoter region. The GPT 3*-untranslated region contains no introns and contains a single AATAAA consensus polyadenylation signal (Proudfoot, 1991). Figure 3B presents the full-length, translated coding sequence for GPT based on the genomic sequence (GenBank U70732). These data extend and correct previously submitted cDNA data (GenBank D10355).

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Sequence Polymorphism Identification and Testing To identify the sequence difference between the GPT1 and GPT-2 alleles, genomic GPT was sequenced in unrelated individuals homozygous for GPT-1 or GPT2. To be certain that the sequenced individuals were typed correctly for the GPT isozymes, the chosen homozygotes were parent-pairs with differing GPT types, whose offspring were all heterozygous at this locus. After all coding sequences and intron/exon junctions were sequenced, a single nucleotide difference causing an amino acid substitution was observed. The codon CAT (His) is present at amino acid position 14 of the GPT-1 allele, whereas the codon AAT (Asn) is present in the GPT-2 allele. This polymorphism was confirmed by direct sequencing in four other unrelated, homozygous CEPH parent-pairs. The C/A nucleotide difference in the first position of codon 14 causes the loss of a NlaIII (Hsp92II) restriction site (CATG) in the GPT-2 allele. PCR was used to amplify a 163-bp fragment that spans the polymorphic site. Enzyme digestion of the PCR fragment and electrophoresis on a high-concentration agarose gel results in two constant bands (67 and 25 bp) in both GPT alleles, as well as a 71-bp fragment in the GPT-2 allele, or a 40- and a 31-bp fragment in the GPT-1 allele. This technique was used to confirm GPT types in 80 individuals of a five-generation family that had been typed previously by serologic methods (Ferrell et al., 1983). The presence or absence of the NlaIII site segregated consistently with the GPT-1 and GPT-2 alleles, respectively. In addition to the GPT polymorphism, preliminary sequencing of the 68C8 cosmid (containing a human insert of approximately 35 kb) revealed the presence of the D8S421 microsatellite. This polymorphic, tetranucleotide repeat has not been mapped previously and should be another useful marker for 8q24.3. DISCUSSION

Reports from the early 1980s of human GPT localization are in conflict, and since the serologic polymorphism has been used in linkage studies for many years, it was important to map this locus definitively using DNA probes. We localized rat gpt by FISH to rat chromosome 7q33 –q34, which is syntenic to human 8q24. We also isolated two human cosmids containing GPT, 68C8, and 105B9 from a chromosome 8 cosmid library. Cosmid 68C8 was then used as a probe for FISH and localized GPT to the most distal band of the long arm of human chromosome 8 at band 8q24.3. We next sequenced the human GPT genomic clone within cosmid 68C8. The resulting coding sequence is consistent with the published GPT-1 amino acid sequence (Ishiguro et al., 1991). This information was then used to sequence GPT 1-1 and 2-2 homozygotes, leading to detection of a single amino acid substitution accounting for the serologic differences. This polymor-

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phism causes the loss of an NlaIII (Hsp92II) restriction enzyme site. Therefore, a new RFLP GPT marker is now available, as the polymorphism is easily genotyped by restriction enzyme digestion of the GPT PCR product. Applying this technique in a large, five-generation family, we confirmed that the CAT codon at position 14 of the first exon segregates with the GPT-1 allele and that the AAT codon segregates with the GPT-2 allele. Based on sequencing data and inheritance of the codon 14 variant in individuals of known GPT type, clone 68C8 must represent the GPT-1 allele. Human GPT consists of 11 exons, which is consistent with other characterized aminotransferases such as ornithine-d-aminotransferase (OAT), an enzyme implicated in the blinding disease gyrate atrophy (Mitchell et al., 1988). The 267 bp 5* of the start codon are GCrich (ú70%), and there are several potential Sp-1 transcription factor binding sites in this 5*-untranslated region. The protein polymorphism at the GPT locus has been tested in over 90 human populations, ranging from Alaskan Eskimos to South African Bushmen (Roychoudhury and Nei, 1988). In all human populations studied, both the GPT-1 and the GPT-2 alleles are present, with heterozygosity in the range 40 to 50%. Additional rare alleles are also found, including a GPT-null allele with a frequency of at least 0.5% in some populations (Ko¨mpf and Ritter, 1979). Thus, the DNA-based polymorphisms at or near the GPT locus will be important tools for illuminating recent human evolution and, additionally, may be useful forensic markers. ACKNOWLEDGMENTS This work was supported by grants from the Foundation Fighting Blindness and the George Gund Foundation, and by NIH Grant EY07142. We thank Dr. Harold C. Riethman, Wistar Institute, Philadelphia, for providing the 8qter half-YAC; Dr. Dan Wells, University of Houston, for providing chromosome 8 cosmids; Dr. Bob Ferrell, University of Pittsburgh, for providing DNAs from the VMD1 family; and Dr. M. Funatsu, Chemo-Sero-Theraputic Research Institute, Kumamoto, Japan, for providing the rat gpt cDNA.

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