Evolution of the glycophorin gene family in the hominoid primates

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 ofEcoRI restriction sites relative to those of the other four enzymes used; the significance of this observation is unclear, but raises the question of nonrandomness ofEcoRI 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.     

Polymorphisms and gross structure of glycophorin genes in common chimpanzees

Using human α glycophorin cDNA probe and six restriction enzymes, we examined the homologues of human glycophorin genes in genomic DNA of 11 unrelated chimpanzees. We show that, in contrast to the human, the chimpanzee exhibits an unusual array of nonrandomly distributed restriction fragment length polymorphisms (RFLP). No clear correlation was found between the RFLP and the V-A-B-D blood-group phenotypes of the subjects, with one possible exception. However, pairs of allelic RFLP occurring at a relatively high frequency were identified. In addition, the homology of chimpanzee glycophorin genes to the human genes was examined using as probes synthetic oligonucleotides specifying distinct regions of human glycophorin genes. We show that the glycophorin gene family in the chimpanzee consists of at least three members that are homologous to the human α, δ, and E genes (glycophorins A, B, and E) and may share a similar gross structure and overall organization. This research was supported by National Institutes of Health Grant GM 16389.     

Polymorphism of the T-cell receptor gamma variable and constant region genes in a Chinese population

Summary The human T-cell receptor gamma gene region spans 160 kb genomic DNA. Restriction fragment length polymorphisms (RFLPs) have been previously documented for the constant region (TRGC) genes, the joining (TRGJ) segments and the variable (TRGV) genes. We have recently defined the alleles of the T-cell receptor gamma V, J and C genes and we have described seven haplotypes of the V gamma subgroup I genes characterized either by RFLPs or by deletion or insertion of V gamma genes. The number of VI genes may vary from 7 to 10 per haploid genome, the 9-gene haplotype being the most frequent. Allelic fragments can unambiguously characterize the TRGC2 gene with duplication or triplication of the exon 2. These alleles and haplotypes have been analyzed in four different populations (French, Lebanese, Tunisian and Black African). In this paper, we compare these allele and haplotype frequencies with those found in a Chinese population and we describe new TRGV allelic restriction fragments found only in the Chinese samples. These results and the previous data demonstrate the flexibility of the human T cell receptor gamma locus and the importance of unequal crossing-overs in the evolution of that locus. Moreover, they underline the importance of studying these polymorphisms in population genetics.     

Polymorphism of glycophorins in nonhuman primate erythrocytes

Using immunoblotting techniques and polyclonal antisera to human erythrocyte glycophorin, we show that erythrocytes of several species of nonhuman primates, including representatives of anthropoid apes (19 chimpanzees, 3 gorillas, 6 orangutans, and 3 gibbons) and Old World monkeys (3 baboons, 5 rhesus monkeys, and 6 cynomologus macaques), contain human glycophorin-like molecules. Each species displays a unique glycophorin profile; in anthropoid apes the profile is more complex than in Old World monkeys and more similar to that seen in humans. The chimpanzee was the only species in which human -like glycophorin was detected but it differed from its human counterpart in electrophoretic mobility and reaction with M-specific monoclonal antibody. In contrast to humans, highly polymorphic glycophorin profiles were observed in each species of anthropoid apes and three distinct patterns were defined in each. No such polymorphism has been found so far among the Old World monkeys in the limited number of animals studied. The major glycophorins in all species but the chimpanzees failed to react with M- or N-specific monoclonal antibodies, suggesting structural differences from the human within the amino terminal regions. The reaction with the minor glycophorins showed inter- and intraspecies variability. All glycophorins, except -like glycophorin in the chimpanzee, reacted with the antiserum to the carboxyl terminal fragment of human glycophorin, indicating a structural relation to the human in this region. An unexpected correlation was observed, in the chimpanzee, between the patterns of electrophoretically resolved glycophorins and the V-A-B-D blood-group phenotypes, allowing the assignment of each determinant to specific glycophorin bands. The basis for the differences observed between human and nonhuman primate glycophorins is not clear but the possibilities include a common nonpolymorphic ancestor and differences in selective pressures.This research was supported by National Institutes of Health Grant 5 RO1 GM16389.     

Structural studies of the M blood group determinant

Structural studies of homozygous glycophorin A^M were undertaken by monitoring the ¹³C methyl resonances of ¹³C reductively methylated glycophorin A^M (contains five $\text{N}{}^{\mathrm{?}}\text{,N-[}{}^{13}\text{C}\text{]}\text{dimethyl}$ Lys residues, and the N-terminal $\text{N}{}^{\mathrm{\alpha }}\text{,N-[}{}^{13}\text{C]}\text{dimethyl}$ Ser residues) in various forms of glycosylation. The results indicate that removal of the α-d-NeuAc residues does not affect the structure about the N-terminal Ser residue. However, removal of the fifteen O-linked oligosaccharide units results in a structural effect about the N-terminal Ser residue. Partial methylation experiments performed on native glycophorin A^M and deglycosylated glycophorin A^M indicate that methylation of the lysine residue(s) may influence the structure about the N-terminal Ser residue, especially in the case of deglycosylated A^M.     

DNA finger printing by oligonucleotide probes specific for simple repeats

Summary Interspersed simple repetitive DNA is a convenient genetic marker for analysis of restriction fragment length polymorphisms (RFLPs) because of the numbers and the frequencies of its alleles. Oligonucleotide probes specific for variations of the GA _C ^T A simple repeats have been designed and hybridized to a panel of human DNAs digested with various restriction enzymes. Numerous RFLPs were demonstrated in AluI and MboI digested DNA with pure GATA oligonucleotides as probes. The optimal length of the probe for RFLP analysis was 20 bases taking into account fragment lengths (1.5-7 kilobases = kb), signal to background ratio, and number of clearly evaluable RFLPs. By using different restriction enzymes individual-specific hybridization patterns (DNA fingerprints) can be established. Hypervariable simple repeat fragments are stably inherited in a Mendelian fashion. Advantages of this method are discussed.     

Polymorphisms at the GLUT1 (HepG2) and GLUT4 (muscle/adipocyte) glucose transporter genes and non-insulin-dependent diabetes mellitus (NIDDM)

Summary In order to determine the possible contribution of the GLUT1 (HepG2) glucose transporter gene to the inheritance of non-insulin-dependent diabetes mellitus (NIDDM), two restriction fragment length polymorphisms (RFLPs) and the related haplotypes at this locus were studied in 48 Italian diabetic patients and 58 normal subjects. Genotype frequencies for the XbaI polymorphism were significantly different between patients and controls (XbaI: ² = 9.80, df= 2, P < 0.0079). A significant difference was also found in the allele frequencies between NIDDM patients and controls (² =9.39, df = 1, P < 0.0022), whereas no differences were found for the StuI RFLP. No linkage disequilibrium was detected between the XbaI and StuI RFLPs in this sample. The analysis of the four haplotype frequencies (X1S1, X1S2, X2S1, X2S2) revealed a significant difference between diabetic patients and controls (² = 14.26, df =3, P < 0.002). By comparing single haplotype frequencies, a significant difference between the two groups was found for the X1S1 and X2S2 haplotypes. A two-allele RFLP at the GLUT4 (muscle/adipocyte) glucose transporter gene, detected with the restriction enzyme KpnI, was also examined; no differences were found between patients and controls for this RFLP. The finding of an association between polymorphic markers at the GLUT1 transporter and NIDDM suggests that this locus may contribute to the inherited susceptibility to the disease in this Italian population.     

No association between RFLPs at the porphobilinogen deaminase gene and schizophrenia

Summary An association study of restriction fragment length polymorphisms (RFLPs) in the porphobilinogen deaminase (PBGD) gene and schizophrenia was conducted. RFLPs detected by MspI, PstI, ApaLI and BstNI in intron 1 of the gene were studied in 49 patients and 79 controls. There were no significant differences between the groups in allele frequencies, genotype counts or haplotype distribution.     

Primate evolution of a human chromosome 1 hypervariable repetitive element

Summary The clone designated hMF #1 represents a clustered DNA family, located on chromosome 1, consisting of tandem arrays displaying a monomeric length of 40 bp and a repetition frequency of approximately 7×10³ copies per haploid genome. The sequence hMF #1 reveals multiple restriction fragment length polymorphisms (RFLPs) when human genomic DNA is digested with a variety of 4–6-bp recognition sequence restriction enzymes (i.e., Taq I, Eco RI, Pst I, etc.). When hamster and mouse genomic DNA was digested and analyzed, no cross-species homology could be observed. Further investigation revealed considerable hybridization in the higher primates (chimpanzee, gorilla, and orangutan) as well as some monkey species.The evolutionary relationship of this repetitive DNA sequence, found in humans, to that of other primates was explored using two hybridization methods: DNA dot blot to establish copy number and Southern DNA analysis to examine the complexity of the RFLPs. Homology to the hMF #1 sequence was found throughout the suborder Anthropoidea in 14 ape and New and Old World monkey species. However the sequence was absent in one species of the suborder Prosimii. Several discrepancies between established evolutionary relationships and those predicted by hMF #1 exist, which suggests that repetitive elements of this type are not reliable indicators of phylogenetic branching patterns. The phenomenon of marked diversity between sequence homologies and copy numbers of dispersed repetitive DNA of closely related species has been observed inDrosophila mice,Galago, and higher primates. We report here a similar phenomenon for a clustered repeat that may have originated at an early stage of primate evolution.     

Molecular analysis of cystic fibrosis in the Hungarian population

Summary Hungarian cystic fibrosis (CF) families (n = 33) including 114 family members have been analysed for the presence of the F508 mutation within the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and have been haplotyped with probes for restriction fragment length polymorphisms (RFLPs) known to be linked to the CFTR gene. The F508 deletion was present in 64% of CF chromosomes. As in many other populations, linkage disequilibrium was found between the CF locus and the haplotype B (XV-2c: allele 1, KM1-9: allele 2), which accounts for 95% of F508 CF chromosomes in our families.     

DNA polymorphism of the human complementC8β gene: formal genetics and intragenic localization

The eighth component of human complement consists of three subunits of different molecular mass, which are coded for by three separate genetic loci. Polymorphisms have been described at the protein level for the alpha and beta subunits by means of sodium dodecyl sulfate gel electrophoresis and isoelectric focusing. Using a full-length humanC8β cDNA probe, we have studied more than 100 individuals by Southern blot analysis to detect DNA polymorphisms. We have found two restriction fragment length polymorphisms (RFLPs) with the enzymesTaqI andBam HI. TheTaq I polymorphism is defined by two alleles, i.e., a single 4.9 kb fragment or two 2.8/2.1 kb fragments. The allele frequencies are 0.68 and 0.32, respectively. The second RFLP withBam HI is correlated with theTaq I variants: 3 kbBam HI; 4.9 kbTaq I and 3.3 kbBam HI; 2.8/2.1 kbTaqI. Both RFLPs could be mapped to the 3′ portion of theC8β gene. Based on the size of genomic restriction fragments, theC8β gene can be estimated to have a size of 32–36 kb. Because of the even frequency distribution, theC8β DNA polymorphisms may be useful in gene mapping and disease association studies.     

Biochemical characterization of theO-glycans on recombinant glycophorin A expressed in Chinese hamster ovary cells

Alterations inN- andO-linked glycosylation affect cell surface expression and antigenicity of recombinant glycophorin A expressed in transfected Chinese hamster ovary (CHO) cells. To understand these effects further, glycophorin A was purified by immunoaffinity chromatography from transfected wild type and glycosylation deficient CHO cells. TheO-glycans were characterized both biochemically, using gel filtration and high performance anion exchange chromatography, and immunologically, using carbohydrate specific monoclonal antibodies to probe Western blots. TheO-glycans of human erythrocyte glycophorin A consist mainly of short oligosaccharides with one, two, or three sialic acid residues linked to a common disaccharide core, Gal1-3GalNAc1-Ser/Thr, with the disialylated structure being the most abundant. With the exception of the trisialylated derivative, the same structures were found on recombinant glycophorin A expressed by wild type CHO cells. However, in contrast to human crythrocyte glycophorin A, the monosialylated oligosaccharide was the most abundant structure on the recombinant protein. Furthermore, recombinant glycophorin A was shown to express a small amount of the Tn antigen (GalNAc1-Ser/Thr). Recombinant glycophorin A had the sameO-glycan composition, whether purified from clones expressing high or moderate levels of the recombinant glycoprotein. This indicates that the level of expression of the transfected glycoprotein did not affect itsO-glycan composition. Deletion of theN-linked glycosylation site at Asn₂₆, by introducing the Mi.I mutation (Thr₂₈ Met) by site-directed mutagenesis, did not markedly affect theO-glycan composition of the resulting recombinant glycoprotein expressed in wild type CHO cells. This demonstrates that the presence or absence of theN-glycan did not influenceO-linked glycosylation of the recombinant glycoprotein. Finally, theO-glycans on recombinant glycophorin A expressed in the Lec 2 and Lec 8 glycosylation deficient CHO cells were characterized. TheO-glycans on Lec 2 cell glycophorin A were predominantly Gal1-3GalNAc1-Ser/Thr (T antigen), while those on Lec 8 glycophorin A were exclusively GalNAc1-Ser/Thr (Tn antigen). These results will lead to a better understanding of the cell biology and immunology of this important human erythrocyte glycoprotein.     

Human immunoglobulin variable region genes: V κ polymorphisms suggest variation in V-gene repertoires

The present study characterizes four potentially informative polymorphic bands of 5.2, 2.3, 1.9, and 1.2 kb, detected by Southern blot hybridization of Eco RI digests of human DNA using HK101/80 (an immunoglobulin V I probe). These restriction fragment length polymorphisms (RFLP) show Mendelian segregation and they are linked to each other and to Km(1), the allotypic marker on the kappa constant region. There is strong linkage disequilibrium between the 2.3 and 1.2 kb polymorphisms. A 0.7 kb Pst I polymorphic band and a 2.9 kb Sac I polymorphic band were also identified; the latter may reflect a region of tandem repeats in the V region. No bands representing the alternative forms of any of the polymorphic restriction sites were identified. This implies either that genes are missing from the V repertoire or that such bands are hidden because of comigration of fragments due to conservation of restriction sites. Evidence for comigration of fragments was obtained from independent V clones and suggests that dark bands on Southern blots of Pst I digests must often represent several superimposed genes which have conserved restriction sites. The demonstration of RFLP within the V region provides circumstantial evidence for polymorphic variation in the repertoire of V structural genes. The RFLP reported here should be useful as genetic markers in future studies on the immune response and disease susceptibility in man.     

Deletion mapping of the mouse ornithine decarboxylase-related locus Odc-rs8 within Igh-V

The Odc-rs8 locus belongs to a family of mouse DNA sequences related to the gene encoding ornithine decarboxylase (ODC). Odc-rs8 was mapped by recombinant inbred (RI) strain analysis to the region of Chromosome (Chr) 12 occupied by the variable region genes of the immunoglobulin heavy chain (Igh) complex. In the present study, alleles at Odc-rs8 were shown to cosegregate with those for Igh variable region (Igh-V or V _H) genes among 37 inbred mouse strains that had been characterized previously for their haplotypes at Igh. For a more precise definition of the location of Odc-rs8 relative to Igh-V, DNAs from 17 Abelson murine leukemia virus (A-MuLV)-transformed pre-B cell lines cultured from mice heterozygous at Igh and Odc-rs8 were analyzed for the presence of DNA restriction fragments (RFs) derived from each parental Odc-rs8 allele. These cell lines, each of which has rearranged one or both Igh genes, previously were employed in mapping members of nine V _H gene families by deletion analysis (Brodeur et al. 1988). Comparing the deletion profiles of the cell lines for Odc-rs8 with those for the V _H gene families has located Odc-rs8 ^b within the V_HJ558/V_H3609 gene cluster and Odc-rs8 ^c either within or upstream of the 5-most 9% of V_HJ558, identifying Odc-rs8 as a potentially useful marker for the 5 end of the Igh complex.     

Saccharum spontaneum L. ‘SES 208’ genetic linkage map combining RFLP- and PCR-based markers

A 527 marker linkage map ofSaccharum spontaneum L. SES 208 (2n = 64) was established by analyzing 208 single-dose (SD) arbitrarily primed PCR polymorphisms, 234 SD RFLPs, 41 double-dose (DD) and one triple-dose (TD) polymorphisms. A map hypothesis constructed using these markers (minimum LOD = 4.00, = 0.25 M) had 64 linkage groups with 13 SD, nine DD, and one TD markers unlinked. Eight chromosome homology groups were identified by using DD fragments as well as SD RFLPs that identified more than one linkage group. Linkages in repulsion phase were absent from the map, as found in two previous genetic studies of this species. Together, these data demonstrate that SES 208 displayed polysomic segregation, a genetic behavior typical of autopolyploid species. As with previous studies, it was concluded that SES 208 behaved like an auto-octoploid, which was also in agreement with the number of homology groups observed. A ² was used to test whether the 527 markers were randomly distributed throughout the genome: both arbitrarily primed PCR markers and RFLPs had a distribution that was statistically indistinguishable from random. The integrated arbitrarily primed PCR-RFLP map had a predicted genomic coverage of 93% (considering only 442 SD polymorphisms) and an average interval between markers of 6 cM. SD markers were used to estimate the genome size of SES 208 at ca. 33 00 cM.     

haplotypes identified by DNA polymorphisms at the apolipoprotein A-1 and C-III loci and hypertriglyceridaemia

Summary A Japanese group comprising 40 hypertriglyceridaemic and 35 normolipidaemic subjects were genotyped for two intragenic DNA restriction fragment length polymorphisms (RFLPs) at the A-1 and C-III gene loci. An Sst-1 polymorphism is located at the 3 end of the C-III gene and a Msp-1 polymorphism in the third intron of the A-1 gene. The polymorphic restriction sites are 3.8kb apart. The polymorphism with Sst-1 was present at allelic frequencies of 0.67 (S1 allele) and 0.33 (S2 allele), and the polymorphism with Msp-1 was present at allelic frequencies of 0.55 (M1 allele) and 0.45 (M2 allele). The alleles S1, S2, M1, and M2 are in linkage disequilibrium and three haplotypes were identified S1-M1, S1-M2, and S2-M2. Unlike the previously reported association of the S2 allele with hypertriglyceridaemia found in Caucasians there was no difference in the frequency of S2 allele between normolipidaemic and hyperlipidaemic Japanese. However one of the haplotypes S1-M2 was significantly increased in the hypertriglyceridaemic subjects (32% versus 11% P>0.025). Thus in Japanese there is an association with genotypes at this locus and hypertriglyceridaemia but with a different haplotype than in Caucasians.     

An unreported RFLP for probe 218 EP6 that is useful in linkage analysis of adult polycystic kidney disease

Probe 218 EP6 is known to recognise two restriction fragment length polymorphisms (RFLPs) after digestion of genomic DNA withPvuII. We report a rare allele that segregates in Mendelian fashion, in a family where adult polycystic kidney disease was being tracked using linked polymorphisms.     

Sequence and diversity of bovine T-cell receptor β-chain genes

The nucleotide sequences of 38 T-cell receptor (Tcr) -chain cDNA clones which were isolated from a cDNA library (2 × 10⁶ plaques) constructed from bovine peripheral blood lymphocytes were determined. Of 38 cDNA clones, 22 were rearranged and contained the functional variable (V) gene segments. These clones were tentatively divided into nine Tcrb-V gene families which correspond to the human Tcrb-V family. Among them, a Tcrb-V12 gene segment was isolated from 9 out of 22 clones, suggesting that this Tcrb-V family was expressed in the bovine peripheral blood lymphocytes. Two different constant (C) geen segments were found, and both C regions were composed of 178 amino residues. The amino acid sequences of bovine Tcrb-C regions are approximately 80%–82%, 78%, and 78% similar to those from human, mouse, and rabbit, respectively. To estimate Tcrb-V-associated restriction fragment length polymorphisms (RFLPs), Southern blot analysis was performed using liver DNAs from four bovine breeds, Holstein, Angus, Hereford, and Japanese Black. However, no significant difference was observed among genomic DNAs of Tcrb-V loci from these four breeds.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers D90121-40. Address correspondence and offprint requests to: N. Ishiguro.     

Ca2+-activated K+ conductance of human red cell membranes exhibits two different types of voltage dependence

Summary The voltage dependence for outward-going current of the Ca-activated K⁺ conductance (g _k (Ca)) of the human red cell membrane has been examined over a wide range of membrane potentials (V _m) at constant values of [K⁺]_ex, [K⁺]_c and pH_c, the intact cells being preloaded to different concentrations of ionized calcium. Outward-current conductances were calculated from initial net effluxes of K⁺ and the corresponding (V _m-E_k) values. The basic conductance, defined as the outward-current coductance at (V _m-E_k) 20 mV and [K⁺]_ex 3mM (B. Vestergaard-Bogind, P. Stampe and P. Christophersen,J. Membrane Biol. 95:121–130, 1987) was found to be a function of cellular ionized Ca. At all degrees of Ca activationg _K(Ca) was an apparently linear function of voltage (V _m range –40 to +70 mV), the absolute level as well as the slope decreasing with decreasing activation. In a simple two-state model the constant voltage dependence can, at the different degrees of Ca activation, be accounted for by a Boltzmann-type equilibrium function with an equivalent valence of 0.4, assuming chemical equilibrium atV _m=0 mV. Alternatively, the phenomenon might be explained by a voltage-dependent block of the outward current by an intracellular ion. Superimposed upon the basic conductance is the apparently independent inward-rectifying steep voltage function with an equivalent valence of 5 and chemical equilibrium at the givenE _K value.Abbreviations CCCP carbonyl cyanidem-chlorophenylhydrazone - DIDS 4,4-diisothiocyanostilbene-2,2-disul     

Molecular genetic evidence for a founder effect in familial hypercholesterolemia among French Canadians

Summary Familial hypercholesterolemia (FH), at a prevalence of about 1 in 200 in the French-Canadian population, is caused by a 10-kb deletion in the low-density lipoprotein (LDL) receptor gene in 60% of French-Canadian FH heterozygotes. We genotyped 159 FH patients who carry this common mutation and 221 healthy French-Canadian controls for five DNA restriction fragment length polymorphisms (RFLPs) of the LDL receptor gene. The allele numbers for four of the five RFLPs differed significantly (P < 0.001) between FH patients and control subjects. Our results suggest that the 10-kb deletion carrier allele is associated with a single haplotype (called the B haplotype). In a family study including a patient homozygous for the 10-kb deletion, we showed that the B haplotype cosegregates with the deletion in affected members and that the B haplotype is also associated with the normal allele in some members of the family. We identified 15 different haplotypes for the normal allele in 10-kb deletion carrier FH heterozygotes. These results offer strong support, at a molecular level, for the hypothesis of a founder effect for the 10-kb deletion in the French-Canadian population.This work was presented in part at the meeting: Advances in Gene Technology: the molecular biology of human genetic disease, Miami, Florida, 1991