Murine Equivalent of the Human Histo-blood Group ABO Gene Is acis-AB Gene and Encodes a Glycosyltransferase with Both A and B Transferase Activity
2001; Elsevier BV; Volume: 276; Issue: 17 Linguagem: Inglês
10.1074/jbc.m010805200
ISSN1083-351X
AutoresMiyako Yamamoto, Xiaohong Lin, Yoshihiko Kominato, Yukiko Hata, Reiko Noda, Naruya Saitou, Fumiichiro Yamamoto,
Tópico(s)Pancreatic function and diabetes
ResumoWe have cloned murine genomic and complementary DNA that is equivalent to the human ABO gene. The murine gene consists of at least six coding exons and spans at least 11 kilobase pairs. Exon-intron boundaries are similar to those of the human gene. Unlike human A and B genes that encode two distinct glycosyltransferases with different donor nucleotide-sugar specificities, the murine gene is a cis-AB gene that encodes an enzyme with both A and B transferase activities, and thiscis-AB gene prevails in the mouse population. Cloning of the murine AB gene may be helpful in establishing a mouse model system to assess the functionality of the ABO genes in the future. We have cloned murine genomic and complementary DNA that is equivalent to the human ABO gene. The murine gene consists of at least six coding exons and spans at least 11 kilobase pairs. Exon-intron boundaries are similar to those of the human gene. Unlike human A and B genes that encode two distinct glycosyltransferases with different donor nucleotide-sugar specificities, the murine gene is a cis-AB gene that encodes an enzyme with both A and B transferase activities, and thiscis-AB gene prevails in the mouse population. Cloning of the murine AB gene may be helpful in establishing a mouse model system to assess the functionality of the ABO genes in the future. Histo-blood group A/B antigens are clinically important antigens in blood transfusion and organ transplantation. These antigens are oligosaccharide antigens whose immunodominant structures are defined as GalNAc α1→3 (Fuc α1→2) Gal- and Gal α1→3 (Fuc α1→2) Gal- for A and B antigen, respectively. Functional alleles at the ABO locus encode enzymes that catalyze the final step of synthesis. A alleles encode for A transferase, which transfers the GalNAc residues from the UDP-GalNAc nucleotide-sugar to the galactose residue of the acceptor H substrates defined by Fuc α1→2 Gal-. B alleles encode for B transferase that transfers the galactose residue from UDP-galactose to the same H substrates. O alleles are nonfunctional, null alleles. During the past decade, we have been studying the molecular genetic basis of the histo-blood group ABO system (1Yamamoto F. Vox Sang. 2000; 78: 91-103PubMed Google Scholar). From a human gastric carcinoma cell line cDNA library, we were able to clone human A transferase cDNA (2Yamamoto F. Marken J. Tsuji T. White T. Clausen H. Hakomori S. J. Biol. Chem. 1990; 265: 1146-1151Abstract Full Text PDF PubMed Google Scholar) based on the partial amino acid sequence of the soluble form of A transferase purified from human lung (3Clausen H. White T. Takio K. Titani K. Stroud M. Holmes E. Karkov J. Thim L. Hakomori S. J. Biol. Chem. 1990; 265: 1139-1145Abstract Full Text PDF PubMed Google Scholar). Using cross-hybridization with A transferase cDNA probes, we then cloned B transferase cDNA and nonfunctional O allelic cDNA from cDNA libraries made with RNA from colon adenocarcinoma cell lines that exhibited different ABO phenotypes (4Yamamoto F. Clausen H. White T. Marken J. Hakomori S. Nature. 1990; 345: 229-233Crossref PubMed Scopus (908) Google Scholar). Possible allele-specific mutations were identified. Four amino acid substitutions were discovered between A and B transferases. O alleles were more homologous to A alleles than to B alleles. A single base deletion was found near the N terminus of the coding sequence in most of the O alleles, which caused the codon frame to shift. This resulted in a truncated protein without glycosyltransferase activity. In addition to the three major alleles (A 1The abbreviations used are: PCRpolymerase chain reactionα-S-dGTP2′-deoxyguanosine-5′-O-(1-thio-triphosphate)α-S-dCTP2′-deoxycytidine-5′-O-(1-thio-triphosphate)RACErapid amplification of cDNA ends, B, and O), we also identified mutations that modified the enzymatic activity by determination of the partial nucleotide sequences of subgroup alleles (A2, A3, Ax, and B3) (5Yamamoto F. McNeill P.D. Hakomori S. Biochem. Biophys. Res. Commun. 1992; 187: 366-374Crossref PubMed Scopus (184) Google Scholar, 6Yamamoto F. McNeill P.D. Yamamoto M. Hakomori S. Harris T. Judd W.J. Davenport R.D. Vox Sang. 1993; 64: 116-119Crossref PubMed Scopus (113) Google Scholar, 7Yamamoto F. McNeill P.D. Yamamoto M. Hakomori S. Harris T. Vox Sang. 1993; 64: 171-174Crossref PubMed Scopus (100) Google Scholar). We also elucidated the molecular mechanisms of two phenomena named cis-AB and B(A) (7Yamamoto F. McNeill P.D. Yamamoto M. Hakomori S. Harris T. Vox Sang. 1993; 64: 171-174Crossref PubMed Scopus (100) Google Scholar, 8Yamamoto F. McNeill P.D. Kominato Y. Yamamoto M. Hakomori S. Ishimoto S. Nishida S. Shima M. Fujimura Y. Vox Sang. 1993; 64: 120-123Crossref PubMed Scopus (114) Google Scholar). Although the incidence was low, another type of O allele was discovered that lacked the single base deletion but contained an amino acid substitution at the residue crucial for nucleotide-sugar recognition/binding (9Yamamoto F. McNeill P.D. Yamamoto M. Hakomori S. Bromilow I.M. Duguid J.K. Vox Sang. 1993; 64: 175-178Crossref PubMed Scopus (108) Google Scholar). Although no functional analyses have been performed to disprove polymorphism, others have reported additional alterations (10Ogasawara K. Yabe R. Uchikawa M. Saitou N. Bannai M. Nakata K. Takenaka M. Fujisawa K. Ishikawa Y. Juji T. Tokunaga K. Blood. 1996; 88: 2732-2737Crossref PubMed Google Scholar, 11Ogasawara K. Bannai M. Saitou N. Yabe R. Nakata K. Takenaka M. Fujisawa K. 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The nucleotide and deduced amino acid sequences of a variety of ABO alleles are posted on the Blood Group Antigen Gene Mutation Database developed by Blumenfeld and colleagues (available on the World Wide Web). polymerase chain reaction 2′-deoxyguanosine-5′-O-(1-thio-triphosphate) 2′-deoxycytidine-5′-O-(1-thio-triphosphate) rapid amplification of cDNA ends A/B antigens are not restricted to humans but are widely present in nature (16Kabat E.A. Blood Group Substances: Their Chemistry and Immunochemistry. Academic Press, Inc., New York1956Google Scholar). We therefore investigated the presence/absence of homologous sequence(s) in the genomes of other species of organisms (17Kominato Y. McNeill P.D. Yamamoto M. Russell M. Hakomori S. Yamamoto F. Biochem. Biophys. Res. Commun. 1992; 189: 154-164Crossref PubMed Scopus (50) Google Scholar). Hybridization of zoo blots, using the radiolabeled human A transferase cDNA probe, showed weak signals in chicken genomic DNA but strong signals, comparable with the signal detected in human DNA, in genomic DNA from mice and other mammals. No signals were detected in genomic DNA from lower species of organisms in the evolutionary tree. We next determined the partial nucleotide sequences of the primate ABO genes (17Kominato Y. McNeill P.D. Yamamoto M. Russell M. Hakomori S. Yamamoto F. Biochem. Biophys. Res. Commun. 1992; 189: 154-164Crossref PubMed Scopus (50) Google Scholar). The glycosyltransferases responsible for A or B phenotypes in primates were shown to conserve amino acid substitutions corresponding to codons 266 and 268 in humans. A similar study was also reported by others (18Martinko J.M. Vincek V. Klein D. Klein J. Immunogenetics. 1993; 37: 274-278Crossref PubMed Scopus (56) Google Scholar). Through comparative sequence analyses of the ABO genes from humans and apes, we and others proposed a convergent hypothesis of evolution that ABO genes arose from independent mutations after the speciation of humans and apes (19Saitou N. Yamamoto F. Mol. Biol. Evol. 1997; 14: 399-411Crossref PubMed Scopus (117) Google Scholar, 20O'Huigin C. Sato A. Klein J. Hum. Genet. 1997; 101: 141-148Crossref PubMed Scopus (31) Google Scholar). No apparent disadvantages are recognized among any of the phenotypes involving the ABO polymorphism. Hemolytic disease of newborns may be a natural selection against specific combinations of blood groups between the mother and fetus. However, serious incompatibility cases are rare with ABO, since the natural antibodies against A and B antigens are mostly IgM and do not cross the placenta. Although some anti-A, B antibodies are IgG and capable of crossing the placenta, A/B antigens are not well developed in fetuses. Therefore, little damage is done. There should be some reason for the existence of ABO polymorphism in the population. It has been speculated that the possible role of the ABO system is to provide resistance against infection (21Pittiglio D.H. Wallace M.F. Gibbs F.L. Genetics and Biochemistry of A, B, H, and Lewis Antigens. American Association of Blood Banks, Arlington, VA1986: 1-56Google Scholar). Actually, Leb(Fucα1→2 Gal β1→3 (Fuc α1→4) GlcNAc-), an ABO-related structure, was demonstrated to be the receptor for a Gram-negative bacillus, Helicobacter pylori, a causative agent for gastritis, peptic ulcer, and possibly gastric cancer (22Boren T. Falk P. Roth K.A. Larson G. Normark S. Science. 1993; 262: 1892-1895Crossref PubMed Scopus (1002) Google Scholar). A and B transferases modify the Leb structure into ALeband BLeb structures, which H. pylori does not bind to in vitro. This may explain the earlier observation that group O individuals have a higher incidence of stomach ulcer than individuals in any other group (21Pittiglio D.H. Wallace M.F. Gibbs F.L. Genetics and Biochemistry of A, B, H, and Lewis Antigens. American Association of Blood Banks, Arlington, VA1986: 1-56Google Scholar). Antibodies against the α1→3 Gal epitope (Gal α1→3 Gal-) were demonstrated experimentally to block the interspecies infection of certain retroviruses (23Takeuchi Y. Porter C.D. Strahan K.M. Preece A.F. Gustafsson K. Cosset F.L. Weiss R.A. Collins M.K. Nature. 1996; 379: 85-88Crossref PubMed Scopus (238) Google Scholar). From this result, anti-A and anti-B antibodies have been suspected to play a role in inhibiting the epidemics of certain infections. Some type of selection based on the advantage/disadvantage of having antigens/antibodies may have been operating at the ABO locus to secure the survival of species from extinction during evolution. The report that an anti-A monoclonal antibody neutralized human immunodeficiency virus particles produced by lymphocytes from group A individuals but not from group B or O individuals (24Arendrup M. Hansen J.E. Clausen H. Nielsen C. Mathiesen L.R. Nielsen J.O. AIDS. 1991; 5: 441-444Crossref PubMed Scopus (38) Google Scholar) may support this hypothesis. To experimentally assess the functionality of the ABO genes, establishing an animal model is critical. As an initial step, we cloned and characterized the murine ABO gene equivalent. Mouse genomic DNA library (ML1044j), which was constructed by replacing the internal BamHI–BamHI stuffer fragment of the λ EMBL3 SP6/T7 vector with MboI-partially cleaved genomic DNA fragments of the BALB/c strain of mouse, was purchased from CLONTECH (Palo Alto, CA). A Marathon cDNA Amplification Kit and an AdvanTage PCR1 cloning kit were also from CLONTECH. A GeneClean kit was purchased from Bio101 (La Jolla, CA), and pT7T3α18 plasmid vector, α-S-dGTP, α-S-dCTP, and S-300 MicroSpin columns were from Amersham Pharmacia Biotech. LipofectAMINE was purchased from Life Technologies, Inc. [14C]UDP-GalNAc, [14C]UDP-galactose, and [α-32P]dCTP were from PerkinElmer Life Sciences, and 2′-fucosyllactose was from Oxford Glycosystems (Rosedale, NY) and from Calbiochem. Murine anti-A and anti-B monoclonal antibody mixtures were purchased from Ortho Diagnostic Systems (Raritan, NJ). BiotinylatedUlex europaeus agglutinin I, Vectastain Elite ABC kit, and 4-chloro-1-naphthol substrate kit were from Vector Laboratories (Burlingame, CA), and EZ-Link Sulfo-NHS-LC-Biotin was purchased from Pierce. Bluescript SKM13+ vector, Prime-It II kit, Duralose-UV membranes, and frozen competent XL1-blue strain ofEscherichia coli bacteria were purchased from Stratagene (La Jolla, CA). The ULTRAhyb hybridization buffer was obtained from Ambion (Austin, TX). Restriction endonucleases and nucleic acid-modifying enzymes were from Life Technologies, New England Biolabs (Beverly, MA), or Roche Molecular Biochemicals. dRhodamine dye terminator cycle sequencing ready reaction kits and BigDye cycle sequencing kits were purchased from PerkinElmer Life Sciences. Under low stringency conditions, ∼1 million plaques from the mouse genomic DNA library were screened using a human A transferase cDNA probe by the plaque hybridization method (25Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982Google Scholar). Radiolabeled probe was prepared by the random hexamer primer method, using a Prime-It II kit and [α-32P]dCTP (26Feinberg A.P. Vogelstein B. Anal. Biochem. 1983; 132: 6-13Crossref PubMed Scopus (16651) Google Scholar). After four rounds of screening, individual clones were isolated. Phage DNA was prepared, cleaved with restriction endonucleases, gel-electrophoresed, and Southern transferred. Hybridization was then performed to construct restriction enzyme cleavage maps. DNA from MABOφ16 phage clone was cleaved with HindIII andSalI and subcloned into the Bluescript SKM13+ vector. Nested deletion constructs were prepared by the ExoIII-mung bean nuclease method (27Chang D.W. Tartof K.D. Yeung A.T. Gene ( Amst. ). 1993; 127: 95-98Crossref PubMed Scopus (2) Google Scholar). Where no unique 3′-overhang restriction sites were available, the thioderivative fill-in reactions were performed with Klenow enzyme using α-S-dGTP and α-S-dCTP beforeExoIII treatment. After transformation of E. coliXL1-blue strain, plasmid DNA was prepared from individual clones and analyzed for insert size. The nucleotide sequences were determined by Sanger's dideoxy chain termination method using the dRhodamine dye terminator cycle sequencing ready reaction kit (28Sanger F. Nicklen S. Coulson A.R. Bio/Technology. 1992; 24: 104-108PubMed Google Scholar). Sequences were aligned using Lasergene SeqMan II sequencing project management software. RNA from the CMT-93 rectal carcinoma cell line (ATCC 223-CCL), established from a C57BL strain of mouse, was prepared and used for the 5′-RACE experiments (29Barnes W.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2216-2220Crossref PubMed Scopus (983) Google Scholar). We followed the Marathon cDNA amplification protocol provided by the manufacturer. Briefly, the first strand of cDNA was synthesized from RNA using Moloney murine leukemia virus reverse transcriptase and Marathon cDNA synthesis primer. After the second strand was synthesized with RNase H, E. coli DNA polymerase I, andE. coli DNA ligase, the Marathon cDNA adaptor was ligated. Nested PCR was performed, first with AP1 adaptor primer and MY-1 primer and then with AP2 and MY-2 primers. AP1 and AP2 primers were provided in the kit. The nucleotide sequences of MY-1 and MY-2 primers were complementary to the sequences in the coding region of the murine ABO genes. Their sequences were as follows: 5′-TTAGTTTCTGATTGCCTGATGGTCCTTGGGCAC and 5′-TCATGCCACACAGGCTCAATGCCGT for MY-1 and 2, respectively. PCR products were electrophoresed through a 3% agarose gel, and the DNA was gel-purified using the GeneClean kit. DNA fragments were then ligated with pT-Adv vector from the AdvanTage PCR cloning kit by the T-A cloning method. Nucleotide sequences of the inserts were determined. TheBamHI–XhoI fragment containing the last coding exon of the murine ABO gene was first subcloned from a murine ABO genomic clone, MABOφ11, into the pT7T3α18 plasmid vector. TheBamHI site was located in the intron preceding the last coding exon of the murine ABO gene. The XhoI site was in the λ EMBL3 SP6/T7 vector next to the BamHI site used to accommodate genomic DNA. This construct was then digested withSstI and SnaBI. The SstI site was within the pT7T3α18 plasmid vector, and the SnaBI site was located downstream of the stop codon of the mouse ABO gene coding sequence. The SstI–SnaBI fragment containing the coding sequence in the last coding exon of the mouse ABO gene was then isolated. The human B transferase expression construct with intron, pBBBB (30Yamamoto F. Hakomori S. J. Biol. Chem. 1990; 265: 19257-19262Abstract Full Text PDF PubMed Google Scholar), was cleaved with BamHI, blunt-ended by the Klenow filling-in reaction, and then digested with SstI. TheSstI site was in the intron preceding coding exon 7 of the human ABO gene; BamHI was in the eukaryotic expression vector (originally pSG-5). The SstI-blunt (BamHI) vector fragment containing the human B transferase cDNA sequence of exons 1–6 was then ligated to the mouseSstI–SnaBI fragment to produce the human-mouse chimeric gene (pHuman-mouse chimera). A murine cDNA eukaryotic expression construct was then constructed by replacing theEcoRI–AflII fragment from the chimeric construct with the EcoRI–AflII fragment from a 5′-RACE clone in the pT-Adv vector. The EcoRI site of the clone was in the plasmid and located 5′ upstream of the cDNA end. TheAflII was in the last coding exon. The EcoRI site in the pSG-5 vector was located downstream of the SV40 early promoter and upstream of the human cDNA sequence. The resultant construct (pMouse) contained the entire coding sequence of the mouse ABO gene cDNA. Plasmid DNA was prepared by the SDS-alkaline method (25Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982Google Scholar). The HeLa cell line derived from a human adenocarcinoma of uterus was used as a recipient of transient DNA transfection analyses. The HeLa cells express H antigens on their cell surfaces and have been successfully used in similar transfection experiments of A and B transferase expression constructs (5Yamamoto F. McNeill P.D. Hakomori S. Biochem. Biophys. Res. Commun. 1992; 187: 366-374Crossref PubMed Scopus (184) Google Scholar, 30Yamamoto F. Hakomori S. J. Biol. Chem. 1990; 265: 19257-19262Abstract Full Text PDF PubMed Google Scholar, 31Yamamoto F. McNeill P.D. J. Biol. Chem. 1996; 271: 10515-10520Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Following the manufacturer's protocol, we used LipofectAMINE for transfection. Seventy-two hours after transfection, the cells were washed and harvested. Cell pellets were then lysed in buffer (0.1 m NaCl, 25 mm sodium cacodylate, 10 mm MnCl2, and 0.1% Triton X-100). A/B transferase activity was determined by measuring the transfer of carbon-14 from [14C]UDP-GalNAc or [14C]UDP-galactose to the acceptor substrate 2′-fucosyllactose, as described previously (31Yamamoto F. McNeill P.D. J. Biol. Chem. 1996; 271: 10515-10520Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). After incubation, the reaction products were separated from unincorporated nucleotide-sugars by AG1-X8 anion exchange column chromatography. The incorporation of radioactivity was determined using a scintillation counter. Murine submaxillary glands were used for the expression analyses of A/B transferases. A and B transferase activities were measured by the incorporation of carbon-14 from [14C]UDP-GalNAc or [14C]UDP-galactose to 2′-fucosyllactose. Although the same reaction conditions were used, the reaction product was separated from the precursor substrate by paper chromatography rather than column chromatography. Genomic DNA was prepared by the proteinase K-SDS method and used to amplify a DNA fragment derived from the murine ABO gene. The names and nucleotide sequences of the primers used were as follows: SN-16, 5′-GAGACTGCAGAACAACACTT; SN-17, 5′-CAATGCCGTTGGCCTTGTC. The PCR-amplified DNA fragments were purified through chromatography using S-300 MicroSpin columns and subjected to direct DNA sequencing reactions with the BigDye cycle sequencing kit. After the sequencing reaction, DNA was purified and then analyzed using an ABI Prism 377 automatic DNA sequencer. Expression of the ABH antigens in murine submaxillary glands was examined immunologically using extracts spotted on a nitrocellulose membrane. Murine anti-A and anti-B monoclonal antibody mixtures were biotinylated using EZ-Link Sulfo-NHS-LC-Biotin, following the protocol provided by the manufacturer. After biotinylation, the unincorporated biotin was removed using Microcon 30 centrifugal filter devices. The submaxillary glands from C57BL and ICR strains of mice were homogenized in buffer containing 20 mm Tris-HCl (pH 7.5), 0.15 mNaCl, and 1% Triton X-100. After centrifugation, the supernatant was diluted with buffer containing 25 mm Tris-HCl (pH 7.5) and 0.1% SDS. The extract was then spotted onto a Duralose-UV membrane. As controls, the extracts similarly obtained from human colon adenocarcinoma SW48 cells (AB phenotype) and from group A and O porcine submaxillary glands were also spotted on the membrane. After drying for 15 min, the membrane was treated with 0.3% hydrogen peroxide and 0.3% fetal calf serum in phosphate-buffered saline for 5 min to block endogenous peroxidase activity. After washing, the membrane was incubated overnight in phosphate-buffered saline containing 4% bovine serum albumin at 4 °C. The membrane was then cut into four pieces, which were individually incubated with either biotinylated murine anti-A monoclonal antibody mixture, biotinylated murine anti-B monoclonal antibody mixture, biotinylated Ulex europaeus agglutinin I, or bovine serum albumin (negative control) for 1 h at room temperature. The filters were washed separately and then incubated collectively with the Elite ABC reagents for 15 min. After washing with phosphate-buffered saline, the membranes were treated with 4-chloro-1-naphthol substrate for color development. Certain mammalian cells exhibit α1→3 Gal epitopes. The cDNA encoding α1,3-galactosyltransferase that synthesizes this epitope was cloned from cow (32Joziasse D.H. Shaper J.H. Van den Eijnden D.H. Van Tunen A.J. Shaper N.L. J. Biol. Chem. 1989; 264: 14290-14297Abstract Full Text PDF PubMed Google Scholar), mouse (33Larsen R.D. Rajan V.P. Ruff M.M. Kukowska-Latallo J. Cummings R.D. Lowe J.B. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8227-8231Crossref PubMed Scopus (190) Google Scholar), and pig (34Strahan K.M. Gu F. Andersson L. Gustafsson K. Transplant. Proc. 1995; 27: 245-246PubMed Google Scholar, 35Strahan K.M. Gu F. Preece A.F. Gustavsson I. Andersson L. Gustafsson K. Immunogenetics. 1995; 41: 101-105Crossref PubMed Scopus (55) Google Scholar). Humans do not exhibit this epitope but possess the antibody against the epitope in sera (36Galili U. Macher B.A. Buehler J. Shohet S.B. J. Exp. Med. 1985; 162: 573-582Crossref PubMed Scopus (367) Google Scholar). Human sequence corresponding to this gene was shown to be a pseudogene due to frameshifts and nonsense mutations (32Joziasse D.H. Shaper J.H. Van den Eijnden D.H. Van Tunen A.J. Shaper N.L. J. Biol. Chem. 1989; 264: 14290-14297Abstract Full Text PDF PubMed Google Scholar, 37Larsen R.D. Rivera-Marrero C.A. Ernst L.K. Cummings R.D. Lowe J.B. J. Biol. Chem. 1990; 265: 7055-7061Abstract Full Text PDF PubMed Google Scholar). A/B transferases utilize the galactose substrate with fucose, whereas α1,3-galactosyltransferase utilizes the substrate without fucose. ABO genes and α1,3-galactosyltransferase genes share significant homology at both the nucleotide and deduced amino acid sequence levels (30Yamamoto F. Hakomori S. J. Biol. Chem. 1990; 265: 19257-19262Abstract Full Text PDF PubMed Google Scholar). Cloned canine cDNA encoding Forssman glycolipid synthetase (UDP-GalNAc:globoside α1,3-N-acetyl-d-galactosaminyltransferase) also exhibited sequence homology (38Haslam D.B. Baenziger J.U. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10697-10702Crossref PubMed Scopus (80) Google Scholar). Therefore, these genes are believed to have derived from the same ancestral gene and constitute the ABO gene family. Southern hybridization experiments of murine genomic DNA showed different banding patterns when murine α1,3-galactosyltransferase cDNA probe and human A transferase cDNA probe were used (17Kominato Y. McNeill P.D. Yamamoto M. Russell M. Hakomori S. Yamamoto F. Biochem. Biophys. Res. Commun. 1992; 189: 154-164Crossref PubMed Scopus (50) Google Scholar, 33Larsen R.D. Rajan V.P. Ruff M.M. Kukowska-Latallo J. Cummings R.D. Lowe J.B. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8227-8231Crossref PubMed Scopus (190) Google Scholar). Accordingly, the ABO gene equivalent was assumed to exist in the mouse genome (17Kominato Y. McNeill P.D. Yamamoto M. Russell M. Hakomori S. Yamamoto F. Biochem. Biophys. Res. Commun. 1992; 189: 154-164Crossref PubMed Scopus (50) Google Scholar). Results from our cloning experiments of the murine ABO gene described here concluded that mice actually do possess an ABO gene equivalent. We cloned the genomic DNA sequence encompassing most of the murine ABO structural gene. By screening 1 million phage plaques from a murine genomic DNA library, we obtained a total of nine independent clones that hybridized with the human A transferase cDNA probe. A preliminary mapping showed that two phage clones named MABOφ11 and MABOφ16 contained the entire coding sequence in the last coding exon. Since the MABOφ16 clone contained the sequence farther upstream, this clone was used for the nucleotide sequence determination. The MABOφ11 clone containing the farther downstream sequence was used to construct a human-mouse ABO gene chimeric expression construct as well as a murine gene expression construct. We sequenced the entire insert in the MABOφ16 clone (∼11.2 kilobase pairs) with more than 99.9% accuracy. Almost all of the coding sequence was contained in the sequenced region. The exon-intron boundaries were determined and are shown in Fig.1. Fig. 1, A and B, represents two probable splicing patterns, although other possibilities still exist because the sequence encoding the first few amino acid residues has not yet been identified. There are six coding exons in Fig. 1 A and seven in Fig. 1 B. Approximately 4.0 kilobase pairs and 70 base pairs upstream of the splicing acceptor site of coding exon 2 (cEXON 2) in Fig. 1 A, there was a CTCAGAG sequence and a TGAATCTCAG sequence, respectively. These sequences may be portions of the coding sequence, since they are found upstream in the cDNA preceding the sequence in cEXON 2. Fig.1 B depicts the case where GAATCTCAG of the latter TGAATCTCAG sequence represents the sequence in the preceding exon. In that case, the acceptor site of cEXON 3 needs to shift 2 nucleotides upstream, which would break up the GT-AG rule of splice junctions. We determined the entire nucleotide sequence contained in the MABOφ16 clone, which included ∼5.0 kilobase pairs of sequence upstream of the splicing acceptor site of cEXON2 in Fig. 1 A. No sequence corresponding to the 5′-untranslated region was found. Therefore, the promoter region of the murine ABO genes must reside farther upstream. The sequence corresponding to human coding exons 3 and 4 was found in one exon in the mouse gene. However, the number of amino acid residues (19 amino acids) was much smaller than that of human exons 3 and 4 combined (35 amino acids). Further studies are needed, since there may be an alternative splicing that would divide this small exon into two smaller exons with an intron in between. The entire insert sequence in the MABOφ16 clone and the entire cDNA sequence have been deposited in the DNA Data Bank of Japan (DDBJ) (accession numbersAB041038 and AB041039). The nucleotide and deduced amino acid sequences in the coding region of the murine cDNA were aligned with those of human A1-1 (A101) allele (accession number AF134412 in GenBankTM) by combining the Clustal method (40Higgins D.G. Sharp P.M. Gene ( Amst. ). 1988; 73: 237-244Crossref PubMed Scopus (2885) Google Scholar) and the J. Hein method (41Hein J. Methods Enzymol. 1990; 183: 626-645Crossref PubMed Scopus (350) Google Scholar) using the MegAlign software. Results are shown in Fig. 1. Especially high homology was observed in the coding sequence in the last two coding exons. The percentages of identical nucleotide and amino acid residues in the last two coding exons were 78% (642/822) and 81% (222/273) between the two species, respectively. The amino acid sequence of the murine gene was also aligned with the amino acid sequences of human A and B transferases, mouse α1,3-galactosyltransferase, and canine Forssman glycolipid synthetase. Results are shown in Fig.2 A. The percentages of the identical amino acid residues of the coding sequences in the last two coding exons are 47% (127 of 272) between the mouse ABO and α1,3-galactosyltransferase genes and 49% (132 of 272) between the mouse ABO gene and the dog Forssman synthetase gene. Fig. 2 Bhighlights the amino acid sequences of the region important for the recognition/binding of nucleotide-sugars. The phylogenetic tree is shown in Fig. 2 C. The cloned mouse gene was evolutionarily mapped closest to the human ABO gene. It was also mapped closer to the canine Forssman gene than the murine α1,3-galactosyltransferase gene. We examined whether the isolated mouse ABO gene sequence could encode a functional glycosyltransferase. We first constructed a human-mouse chimeric construct in an eukaryotic expression vector pSG-5. A DNA fragment containing coding sequence in the last coding exon of the mouse ge
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