Biochemical Genetics, Vol 28, Nos. 3/4, 1990

Evolution of the Glycophorin Gene Family in the Hominoid Primates Ann Rearden, 1'2 Huan Phan, I Shinichi Kudo, 3 and Minoru Fukuda 3 Received30 Oct. 1989--Final 2 Feb. 1990

Analysis of nucleotide sequences of the human glycophorin A (GPA) and glycophorin B (GPB) genes has indicated that the GPA gene most closely resembles the ancestral gene, whereas the GPB gene likely arose from the GPA gene by homologous recombination. To study the evolution of the glycophorin gene family in the hominoid primates, restricted DNA on Southern blots from man, pygmy chimpanzee, common chimpanzee, gorilla, orangutan, and gibbon was probed with cDNA fragments encoding the human GPA and GPB coding and 3'-untranslated regions. This showed the presence in all of the hominoid primates of at least one GPA-like gene. In addition, at least one GPB-like gene was detected in man, both chimpanzee species, and gorilla, strongly suggesting that the event that produced the GPB gene occurred in the common ancestor of man-chimpanzee-gorilla. An unexpected finding in this study was the conservation of E c o R I restriction sites relative to those of the other four enzymes used," the significance of this observation is unclear, but raises the question of nonrandomness of E c o R I restriction sites in noncoding regions. Further analysis of the evolution of this multigene family, including nucleotide sequence analysis, will be useful in clarification of the evolutionary relationships of the hominoid primates, in correlation with the structure and function of the glycophorin molecules, and in assessment of the role of evolution in the autogenicity of glycophorin determinants.

This work was supported in part by National Institutes of Health Grants AM33463 and CA33000. Department of Pathology, M-012, University of California San Diego, La Jolla, California 92093. 2 To whom correspondence should be addressed. 3 La Jolla Cancer Research Foundation, Cancer Research Center, La Jolla, California 92037.

2O9 0006-2928/90/0400-0209506.00/0 © 1990 Plenum PublishingCorporation

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Rearden, Phan, Kudo, and Fukuda

KEY WORDS: glycophorin A; glycophorin B; evolution; primates; chimpanzee; gorilla; orangutan; gibbon.

INTRODUCTION In man, the principal sialoglycoproteins of the red cell membrane are glycophorin A (GPA) and glycophorin B (GPB). Variability in antigen content of the glycophorin molecules in the hominoid primates has been shown with a panel of 10 monoclonal antibodies to human glycophorin determinants (Rearden, 1986); common and pygmy chimpanzee red cells were agglutinated by 8 antibodies, gorilla by 6, orangutan by 5, and gibbon by 2. This observation suggested that glycophorins might be a useful model to study evolutionary differences among the hominoid primates. Analysis of the structure of the human GPA and GPB genes has shown that the genes are homologous from the 5' flanking region to 1.0-kb downstream from the exon encoding the transmembrane region (Kudo and Fukuda, 1989). Analysis of direct repeats flanking the Alu sequences at the transition sites suggests that GPA most closely resembles the ancestral gene, whereas GPB arose by homologous recombination at the Alu repeats during or after gene duplication, and acquired 3'-end sequences from an unrelated gene. To define further the evolution of the glycophorin gene family, restricted DNA from the hominoid primates on Southern blots was probed with DNA fragments encoding the human GPA and GPB coding regions and their respective Y-untranslated regions. MATERIALS AND METHODS

Human and Primate Bloods. Peripheral bloods (17-34 ml) from three pygmy chimpanzees (Pan paniscus), four common chimpanzees (Pan troglodytes), seven gorillas (Gorilla gorilla), five orangutans (Pongopygmaeus), and five gibbons (Hylobates lar) were obtained from the Yerkes Regional Primate Research Center, Emory University, Atlanta, Georgia, through the courtesy of Dr. Harold McClure. Blood (20-30 ml) from an additional nine common chimpanzees (Pan troglodytes) was obtained from the Laboratory for Experimental Medicine and Surgery in Primates of the New York Medical Center, through the courtesy of Dr. Wladyslaw W. Socha. Bloods were collected in acid-citrate-dextrose, shipped overnight, and used the following day. Primate bloods were obtained with the approval of the University of California San Diego Animal Subjects Committee. Human peripheral blood was obtained under conditions approved by the University of California San Diego Human Subjects Committee. Preparation of DNA. Whole-blood samples were centrifuged at 1000g for 10 min. The "buffy coat" layer of white cells (slightly contaminated with

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red blood cells) was removed, washed with phosphate-buffered saline (PBS), and pelleted at 550g. The white cell pellet was suspended in 10 ml of red cell lysing buffer (150 mM NH4C1, 10 mM NaHCO 3, 10 mM disodium EDTA) and incubated at 37°C for 5 min. White cells were washed with PBS and lysed overnight at 37°C in 5 ml of a buffer containing 0.5% sodium dodecyl sulfate (SDS), 0.1 M NaC1, 0.5 M Tris, pH 8.0, 1 mM EDTA, and 0.1 mg/ml proteinase K. Samples were heated at 68°C for 5 min, augmented with 0.75 ml of 8 M KCL, and vortexed. Samples were deproteinized by extraction with 7.5 ml chloroform. Nucleic acids were precipitated from the aqueous layer with 2 vol of 100% ethanol. The precipitate was dried slightly and resuspended in 3 ml of TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA), containing 30 #1 of DNase-free RNase (10 mg/ml) and incubated at 37°C for 3 hr. Protein extraction was carried out with KC1 and chloroform as above. The aqueous layer was precipitated with 2 vol of 100% ethanol. The pellet was washed with 70% ethanol, dried, and resuspended in TE buffer. DNA concentration was determined by measuring the absorbance at 260 nm. Typically, between 1.0 and 2.0 mg of DNA was recovered for each animal. c D N A Probes. Figure 1 illustrates the probes used for nucleic acid hybridization. A1 and A2 were derived by E c o R I restriction of a cDNA encoding GPA; this recently isolated cDNA is, at 2580 bp, the largest GPA cDNA (Kudo and Fukuda, unpublished observations). A1 is a 780-bp fragment that encodes the entire coding region for GPA polypeptide plus 300 bp of 3'-untranslated sequences. A2 is an 1800-bp fragment that encompasses two-thirds of the 3'-untranslated sequences of GPA, including the polyadenylation site at the furthest Y-end. B1 and B2 were derived by HaeIII digestion of a 480-bp cDNA encoding GPB; B1 is a 290-bp fragment that encodes the entire coding region of GPB, and B2 is a 190-bp fragment that encodes only 3'-untranslated sequences of GPB. As shown previously (Siebert and Fukuda, 1987), the nucleotide sequences of GPA and GPB genes are homologous in two-thirds of the genes from the 5'-end. Thus it is expected that restricted fragments derived from those sequences hybridize to both A1 and B1 probes. Since GPA and GPB have entirely different sequences in their I

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3'-untranslated regions (Kudo and Fukuda, 1989), A2 and B2 are specific for the GPA and GPB genes respectively. Restriction Enzyme Digestion and Electrophoresis. Genomic D N A (5 #g) was digested at 37°C for 7 hr with 20 U EcoRI, HindlII, MspI, SstI, and BamHI. Enzymes were obtained from Bethesda Research Laboratories. Reaction conditions were as recommended by the manufacturer. D N A digested with EcoRI, HindIII, MspI, and SstI was separated on 0.7% agarose gels. DNA digested with BamHI was separated on a 0.5% agarose gel. Electrophoresis was carried out in 1 x TBE buffer (0.089 M Tris, 0.089 M boric acid, 2 mM EDTA) for 16 hr at 35-40 V. A lambda Hind]II digest and a 1-kb DNA ladder were used as size markers. Southern Transfer and DNA Hybridization. DNA was transferred from agarose gels onto nitrocellulose by the method described by Southern (1975). The nitrocellulose filters were prehybridized in hybridization solution (10% dextran sulfate, 4 x SSC, 7 mM Tris, p H 7.6, 0.8 x Denhardt's solution, 0.05 mg/ml herring sperm DNA, and 40% formamide) at 42°C, for more than 1 hr, in a volume corresponding to 0.5 m l / c m 2. Stock solutions were as described by Maniatis et al. (1982). DNA probes were labeled to an estimated specific activity of 5 x 107 to 5 x 108 cpm/~g by the random oligonucleotide primer extension method (Feinberg and Vogelstein, 1984) using a kit from Amersham. Probes were boiled for 5 min and hybridized to the filters in fresh hybridization solution for 12-16 hr at 42°C. Filters were washed three times with 1 x SSC, 0.5% SDS at 55°C (30 min per wash) after hybridization. Filters hybridized with probes A2 or B2 were washed additionally with 0.1 x SSC, 0.5% SDS at 62°C (3 x 30 min). Filters hybridized with probe B1 were washed additionally with 0.2x SSC, 0.5% SDS at 62°C (3 x 30 min). Hybridizing fragments were EcoRI

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Fig. 3. Restricted human (H), pygmy chimpanzee (P), common chimpanzee (C), gorilla (G), orangutan (O), and gibbon (Gi) DNA probed with the A2 fragment. Size markersin kb are on the left. visualized by autoradiography with Kodak X A R - 5 film and intensifying screen for 1-10 days. To allow reprobing, filters were striped by shaking for 20 rain in a solution of 0.1 x SSPE, 0.1% SDS. RESULTS Autoradiograms prepared from Southern blots containing human and hominoid primate D N A restricted with EcoRI, HindIII, SstI, MspI, and BamHI, and probed with the fragments A1 (Fig. 2), A2 (Fig. 3), B1 (Fig. 4), and B2 (Fig. 5), show a complex pattern of restriction fragments. Fragments hybridizing with probes A1, A2, and B2 were found, even with conditions of high stringency, in all hominoid primates. Fragments hybridizing with the B1 probe were found in man, pygmy chimpanzee, common chimpanzee, and EC__OR I

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Fig, 5. Restrictedhuman (H), pygmychimpanzee (P), commonchimpanzee (C), gorilla (G), orangutan (O), and gibbon(Gi) DNA probedwith the B2 fragment. Sizemarkers in kb are on the left. gorilla but not in orangutan or gibbon (except for 3.7-kb HindllI and 3.9-kb BamHI fragments in gibbon). Tables I-V show the sizes in kilobases of the restriction fragments obtained with each of the four probes with EcoRI (Table I), SstI (Table II), MspI (Table III), HindIII (Table IV), and BamHI (Table V). Contents of the EcoRI, HindIII, and SstI fragments in man were derived from several sources (Siebert and Fukuda, 1986; Huang et al., 1987; Rahuel et al., 1988; Kudo and Fukuda, 1989). Fragment size varied slightly from experiment to experiment; therefore, these sizes are taken as tentative until the genomic clones are isolated. Seven fragments (and the probes detecting them) conserved in all hominoid species were 16.8-kb EeoRI (B2), 3.5-kb EcoRI (A1), 2.4-kb EcoRI (A2), 2.0-kb EcoRI (A1), 1.3-kb EeoRI (A1), 9.6-kb SstI (A1, A2, B2), and 1.0-kb HindIII (A1). Five fragments detected in five of the six species were 2.8-kb EcoRI (A1) (except orangutan), 6.8-kb SstI (A1) (except gibbon), 3.7-kb HindIII (A1, B1) (except orangutan), 9.4-kb MspI (B2) (except orangutan), and 6.4-kb MspI (A1) (except man). The EcoRI fragments, which were remarkably conserved, were especially informative. As shown previously (Kudo and Fukuda, 1989) and indicated in Table I, both human GPA and GPB genes yield similar EcoRI fragments at the 5'-end (2.0-kb fragments containing exon two and 3.5-kb fragments containing exons three, four, and five). The AI probe hybridized to these fragments in all the hominoid primates, whereas the B1 probe hybridized only in man, both chimpanzee species, and gorilla. The 2.8-kb EcoRI fragment hybridized solely with the A1 probe; this fragment contains GPA exon six (this exon is absent in GPB). The 2.4-kb and 1.3-kb EcoRI fragments contain GPA-specific 3'-untranslated region sequences and hybridized solely with GPA-derived probes in all the hominoid primates. Ten fragments were present in man, pygmy chimpanzee, common chimpanzee, and gorilla but absent in orangutan and gibbon: three EcoRI fragments of 4.3 kb (B2), 3.5 kb (B1), and 2.0 kb (B1); four SstI fragments of 9.4 kb (B2) (the pygmy chimpanzee fragment showed faint hybridization in some

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