Multiple restriction fragment length polymorphisms associated with the Vc determinant of the MN blood group-related chimpanzee V-A-B-D system

Twelve restriction fragment length polymorphisms (RFLPs) were detected in common chimpanzee using two restriction enzymes (HindIII andMspI) and four DNA probes to the coding regions of the human glycophorin A (GPA) and glycophorin B (GPB) genes and their 3-untranslated regions. Seven RFLPs correlated with red cell expression of the V^c determinant of the MN blood group-related V-A-B-D system and five RFLPs correlated with nonexpression of this antigen. Animals heterozygous for theV allele that encodes the V^c determinant had all 12 polymorphic restriction fragments and appeared to show reduced intensity of probe hybridization to these fragments, consistent with the presence of aV and a non-V allele. No RFLPs were detected withEcoRI,SstI, orBamHI, in spite of the relatively large segment of DNA (at least 20 kb) involved in the polymorphisms. The RFLPs were chimpanzee specific and were not found in man, gorilla, orangutan, or gibbon. Multiple RFLPs distinguishing primate species are rare and may be useful markers for molecular evolution.This work was supported in part by National Institutes of Health Grants AM 33463 and CA 33000.     

Evolution of glycophorin A in the hominoid primates studied with monoclonal antibodies, and description of a sialoglycoprotein analogous to human glycophorin B in chimpanzee

Comparison of human and primate erythrocyte membrane sialoglycoproteins showed that common chimpanzee, dwarf chimpanzee, gorilla, orangutan, and gibbon have major periodic acid Schiff-positive proteins resembling human glycophorin A (GPA) monomer and dimer in electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels. Immunoperoxidase staining of Western blots with monoclonal antibodies to human GPA showed that these primate bands express some GPA antigenic determinants. A new sialoglycoprotein analogous to human glycophorin B (GPB) was detected in common chimpanzee. Although human MN blood group phenotype results from an amino acid polymorphism of GPA, Western blots showed that in chimpanzee sialoglycoprotein (GPAch) always expresses the M blood group, whereas chimpanzee sialoglycoprotein (GPBch) expresses either the N blood group or a null phenotype. This result explains the detection of M and MN, but not of N, blood group phenotypes in chimpanzee. GPBch has higher apparent m.w. than human GPB, is present in the erythrocyte membrane in greater quantity than human GPB, and contains trypsin cleavage site(s) and the 10F7 determinant (both found on human GPA but not GPB). Expression of human GPA antigenic determinants was consistent with the phylogeny of the hominoid primates; common and dwarf chimpanzee expressed most of the determinants tested, gorilla and orangutan an intermediate number, and gibbon and siamang the least. Of the GPA antigenic determinants examined, the MN blood group determinants were most consistently expressed during evolution of the hominoid primates. The results suggested that variability in expression of GPA antigenic determinants between species was due to both differences in amino acid sequence and glycosylation.     

Rapidly evolving genes in human. I. The glycophorins and their possible role in evading malaria parasites

In an attempt to identify all fast-evolving genes between human and other primates, we found three glycophorins, GPA, GPB, and GPE, to have the highest rate of nonsynonymous substitutions among the 280 genes surveyed. The Ka/Ks ratios are generally greater than 3 for GPA, GPB, and GPE in human, chimpanzee, and gorilla, indicating positive selection. The uniformly high substitution rate across loci can be explained by the frequent sequence exchanges among genes. GPA is the receptor for the binding ligand EBA-175 of the malaria parasite, Plasmodium falciparum. The levels of nonsynonymous divergence and polymorphism of EBA-175 are also the highest in the genome of P. falciparum. We hypothesize that GPA has been evolving rapidly to evade malaria parasites. Both the high rate of nonsynonymous substitutions and the frequent interlocus conversions may be means of evasion. The support for the evasion hypothesis is still indirect, but, unlike other hypotheses, it can be tested specifically and systematically.     

Identification of the crossing-over point of a hybrid gene encoding human glycophorin variant Sta. Similarity to the crossing-over point in haptoglobin-related genes

One of the human glycophorin variants, Stones (Sta), has been shown to be the product of a hybrid gene of which the 5'-half derived from the glycophorin B (GPB) gene whereas the 3'-half derived from the glycophorin A (GPA) gene. The present study reveals the crossing-over point of this hybrid gene from the analysis of polymerase chain reaction products. The genomic sequences encompassing the region corresponding to exon 3 to exon 4 of GPA were amplified by polymerase chain reaction with oligonucleotide primers synthesized according to GPA and GPB genomic sequences (Kudo, S., and Fukuda, M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4619-4623). After subcloning the products, the nucleotide sequences derived from GPA, GPB, and putative Sta genes were determined. Comparison of the nucleotide sequences of GPA, GPB, and Sta genes indicate that the crossing-over took place 200 base pairs upstream from the first nucleotide of exon 4. Intriguingly, the nucleotide sequence surrounding the putative crossing-over point is homologous to the crossing-over point proposed for haptoglobin genes (Maeda, N., McEvoy, S.M., Harris, H.F., Huisman, T.H.J., and Smithies, O. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 7395-7399). These results suggest strongly that homologous recombination through unequal crossing-over can be facilitated by specific genomic elements, such as those in common in these two crossing-over events. The present study also revealed that this Sta individual has a variant GPA gene; substitution of adenine for guanine at the nucleotide for codon 39 results in substitution of lysine for arginine at amino acid 39, and loss of an SstI restriction site.     

Concerted evolution of the primate immunoglobulin alpha-gene through gene conversion.

We determined four nucleotide sequences of the hominoid immunoglobulin alpha (C alpha) genes (chimpanzee C alpha 2, gorilla C alpha 2, and gibbon C alpha 1 and C alpha 2 genes), which made possible the examination of gene conversions in all hominoid C alpha genes. The following three methods were used to detect gene conversions: 1) phenetic tree construction; 2) detection of a DNA segment with extremely low variability between duplicated C alpha genes; and 3) a site by site search of shared nucleotide changes between duplicated C alpha genes. Results obtained from method 1 indicated a concerted evolution of the duplicated C alpha genes in the human, chimpanzee, gorilla, and gibbon lineages, while results obtained from method 2 suggested gene conversions in the human, gorilla, and gibbon C alpha genes. With method 3 we identified clusters of shared nucleotide changes between duplicated C alpha genes in human, chimpanzee, gorilla, and gibbon lineages, and in their hypothetical ancestors. In the present study converted regions were identified over the entire C alpha gene region excluding a few sites in the coding region which have escaped from gene conversion. This indicates that gene conversion is a general phenomenon in evolution, that can be clearly observed in non-functional regions.     

Comparaison des séquences de fragments de restriction répétés spécifiques du chromosome Y humain chez le chimpanzé et le gorille

Analysis of the evolutionary conservation and change of the Y human specific Hae III 2.4 kb repeated sequence were studied between man and anthropoïd species after restriction with EcoRI endonuclease. In chimpanzee, blocks of these repeats are absent, the remaining copies being interspersed with other sequences. A close similarity of the restriction patterns of these sequences is found between gorilla and man.     

A novel gene member of the human glycophorin A and B gene family. Molecular cloning and expression

A new gene closely related to the glycophorin A (GPA) and glycophorin B (GPB) genes has been identified in the normal human genome as well as in that of persons with known alterations of GPA and/or GPB expression. This gene, called glycophorin E (GPE), is transcribed into a 0.6-kb message which encodes a 78-amino-acid protein with a putative leader peptide of 19 residues. The first 26 amino acids of the mature protein are identical to those of M-type glycophorin A (GPA), but the C-terminal domain (residues 27-59) differs significantly from those of glycophorins A and B (GPA and GPB). The GPE gene consists of four exons distributed over 30 kb of DNA, and its nucleotide sequence is homologous to those of the GPA and GPB genes in the 5' region, up to exon 3. Because of branch and splice site mutations, the GPE gene contains a large intron sequence partially used as exons in GPA and GPB genes. Compared to its counterpart in the GPB gene, exon 3 of the GPE gene contains several point mutations, an insertion of 24 bp, and a stop codon which shortens the reading frame. Downstream from exon 3, the GPE and the GPB sequences are virtually identical and include the same Alu repeats. Thus, it is likely that the GPE and GPB genes have evolved by a similar mechanism. From the analysis of the GPA, GPB and GPE genes in glycophorin variants [En(a-), S-s-U- and Mk], it is proposed that the three genes are organized in tandem on chromosome 4. Deletion events within this region may remove one or two structural gene(s) and may generate new hybrid structures in which the promoter region of one gene is positioned upstream from the body of another gene of the same family. This model of gene organization provides a basis with which to explain the diversity of the glycophorin gene family.     

Multiple recombinational events in primate immunoglobulin epsilon and alpha genes suggest closer relationship of humans to chimpanzees than to gorillas

Summary Immunoglobulin epsilon and alpha genes of chimpanzee and gorilla were isolated and their structures were compared with their human counterparts. Multiple deletions and duplications seem to have happened in both genes during hominoid evolution; the chimpanzee had deleted the entire C₂ gene after its divergence. In addition, the length of the C₁ hinge region of gorilla is distinct from those of chimpanzee and humans. Structural homology of the epsilon and alpha genes suggests that humans are evolutionarily closer to chimpanzees than to gorillas.     

Molecular analysis of a hybrid gene encoding human glycophorin variant Miltenberger V-like molecule

The genomic structure of a human glycophorin variant, Miltenberger class V-like molecule (MiV*), was examined. Southern blot analysis of total genomic DNA revealed that the 5' half of the MiV* gene derived from glycophorin A (GPA) gene whereas the 3' half derived from glycophorin B (GPB) gene. This structure is reciprocal to another glycophorin variant, Sta, which has a GPB-GPA hybrid structure. The genomic sequences around the crossing-over point were amplified by polymerase chain reaction, and the sequences were determined. Comparison of the nucleotide sequences of the GPA, GPB, and MiV* genes indicates that the crossing-over point is located in the region around the 3' end of intron 3 of the GPA gene. This place is different from the crossing-over point for Sta, which was found to be highly homologous to that for haptoglobin-related genes. However, the nucleotide sequences within the presumptive crossing-over point for the MiV* gene were found to be homologous in a reverse orientation to the crossing-over point proposed for haptoglobin-related genes. These results suggest strongly that homologous recombination through unequal crossing over can be facilitated by specific genomic elements such as those in common for formation of MiV*, Sta, and haptoglobin-related genes. The present study also localized the gene of the third glycophorin, GPE, at chromosome 4, q31.1 band, the same locus as for the GPA and GPB genes. The results indicate that GPE was not involved in generating MiV* or Sta hybrid gene despite the fact that it is localized adjacent to the GPA and GPB genes.     

Lineage-Specific Homogenization of the Polyubiquitin Gene Among Human and Great Apes

Ubiquitin is a highly conserved protein, and is encoded by a multigene family among eukaryote species. The polyubiquitin genes, UbB and UbC, comprise tandem multiple ubiquitin coding units without a spacer region or intron. We determined nucleotide sequences for the UbB and UbC of human, chimpanzee, gorilla, and orangutan. The ubiquitin repeat number of UbB was constant (3) in human and great apes, while that of UbC varied: 6 to 11 for human, 10 to 12 for chimpanzee, 8 for gorilla, and 10 for orangutan. The heterogeneity of the repeat number within closely related hominoid species suggests that a lineage-specific unequal crossover and/or gene duplication occurred. A marked homogenization of UbC occurred in gorilla with a low level of synonymous difference (p_s). The homogenization of UbC also occurred in chimpanzee and less strikingly in human. The first and last ubiquitin coding units of UbC were clustered independently between species, and less affected by homogenization during the hominoid evolution. Therefore, the homogenization of ubiquitin coding units is likely due to an unequal crossing-over inside the ubiquitin units. The lineage-specific homogenization of UbC among closely related species suggests that concerted evolution has a key role in the short-term evolution of UbC.     

Gorilla and orangutan c-myc nucleotide sequences: Inference on hominoid phylogeny

The nucleotide sequences of the gorilla and orangutan myc loci have been determined by the dideoxy nucleotide method. As previously observed in the human and chimpanzee sequences, an open reading frame (ORF) of 188 codons overlapping exon 1 could be deduced from the gorilla sequence. However, no such ORF appeared in the orangutan sequence.The two sequences were aligned with those of human and chimpanzee as hominoids and of gibbon and marmoset as outgroups of hominoids. The branching order in the evolution of primates was inferred from these data by different methods: maximum parsimony and neighborjoining.Our results support the view that the gorilla lineage branched off before the human and chimpanzee diverged and strengthen the hypothesis that chimpanzee and gorilla are more related to human than is orangutan. Correspondence to: F. Galibert     

Studies on human red-cell membrane glycophorin A and glycophorin B genes in glycophorin-deficient individuals.

1. Genomic DNA derived from individuals who lack glycophorin A (GPA), glycophorin B (GPB) or both of these proteins was subjected to Southern-blot analysis using GPA and GPB cDNA probes. 2. Bands on the Southern blots were assigned to the GPA gene, GPB gene or to a putative pseudogene. 3. Genomic DNA derived from an individual of the Mk phenotype was shown to have deletions in the GPA and GPB genes. The simplest model for the results obtained is that a single deletion spans the GPA and GPB genes in the individual studied.     

Molecular analysis of glycophorin A and B gene structure and expression in homozygous Miltenberger class V (Mi. V) human erythrocytes

In the Miltenberger class V (Mi. V) condition, red cells lack glycophorin A (GPA) and glycophorin B (GPB) but carry instead an unusual glycoprotein thought to be a hybrid molecule produced by the unequal crossing-over between the closely linked genes encoding for GPA and GPB. By Western blot analysis with rabbit anti-GPA antibodies specific for discrete domains of GPA, it was found that the Mi. V glycoprotein (donor F. M.) contains approximately 60 amino acid residues of GPA at its N-terminus. As a preliminary approach to the molecular analysis of this variant the restriction maps of the GPA and GPB genes were established by Southern blot analysis of genomic DNA and from genomic clones isolated from a human leukocyte library constructed in lambda EMBL4. The GPA and GPB genes cover about 30 kb of DNA and are organized into seven exons (A-1-A-7) and five exons (B-1-B-5), respectively. In addition to the normal genes, a third gene (named inv), closely resembling the GPA and GPB genes, was also identified. In the homozygous Mi. V individual the normal GPA and GPB genes were absent, but an unusual form of gene structure was detected by Southern blot analysis. The Mi. V glycoprotein gene was composed of exon B-1 of the GPB gene followed by exons A-2 and A-3 of the GPA gene and the exons B-3, B-4 and B-5 of the GPB gene. Exon B-1 can be distinguished from exon A-1 of GPA since it is located within a different restriction fragment, but both encode the same amino acid sequence (N-terminal region of the signal peptides). Using the polymerase chain reaction, the junction between exon A-3 and exon B-3 was confirmed by amplification of the DNA region where the putative crossing-over has occurred and it was deduced that the Mi. V glycoprotein is a hybrid molecule composed of amino acid residues 1-58 from GPA fused to amino acid residues 27-72 of GPB. In addition, the finding that part of the signal peptide and the 5'-untranslated region are derived from GPB suggests that the genetic background of the Mi. V variant is rather complex and may involve a cascade of recombination or gene conversion events.     

Evolution of the Hominoid Semenogelin Genes,the Major Proteins of Ejaculated Semen

The hominoid primates (apes and humans) exhibit remarkable diversity in their social and sexual behavioral systems. This is reflected in many ways in their anatomy and physiology. For example, the testes and seminal vesicles are relatively large in species with high sperm competition like the chimpanzee and small in species with low or no sperm competition like the gorilla. Additionally, the chimpanzee is the only hominoid primate known to produce a firm copulatory plug, which presumably functions in sperm competition by blocking insemination of subsequent males. Here we examine the molecular evolution of the semenogelin genes (SEMG1 and SEMG2), which code for the predominant structural proteins in human semen. High molecular weight complexes of these proteins are responsible for the viscous gelatinous consistency of human semen; their rodent homologs are responsible for the formation of a firm copulatory plug. Chimpanzees have an expanded SEMG1 gene caused by duplications of tandem repeats, each encoding 60 amino acids, resulting in a protein nearly twice as long as that of humans. In contrast, at both SEMG1 and SEMG2 we observed several gorilla haplotypes that contain at least one premature stop codon. We suggest that these structural changes in the semenogelin proteins that have arisen since the human–chimpanzee–gorilla split may be responsible for the physiological differences between these species ejaculated semen that correlate with their sociosexual behavior. Present address: (Jensen-Seaman) Human and Molecular Genetics Center, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA     

一种鉴定MNSs红细胞血型单克隆抗体类型的方法的建立

为鉴定MNSs血型单克隆细胞株6D7C9分泌的抗体类型,通过克隆、亚克隆、细胞转染等分子生物学技术建立了血型糖蛋白GPA、GPB的异源表达系统,并将其作为抗原,通过ELISA、Western 印迹法确定6D7C9分泌的McAb.结果显示,RT－PCR技术成功克隆获得了GPA、GPB血型糖蛋白编码基因,通过分别构建其重组逆转录病毒表达载体pEGZ/GPA及pEGZ/GPB,转染包装细胞293T,再感染L929细胞,经zeocin筛选2周后,RT PCR及流式细胞仪分析证实,L929/GPA和L929/GPB转基因细胞中分别有GPA、GPB目的基因的转录和蛋白表达.用稳定高表达GPA、GPB的转基因细胞通过ELISA和Western 印迹法证实单克隆细胞株6D7C9分泌的是抗GPA/GPB McAb.本研究成功地建立了血型糖蛋白GPA、GPB的异源表达系统,为MNSs血型McAb的检测及GPA、GPB蛋白的功能学研究奠定了基础.     

Evolution of the X-linked Zinc Finger Gene and the Y-linked Zinc Finger Gene in Primates

We have sequenced the partial exon of the zinc finger genes (ZFX and ZFY) in 5 hominoids, 2 Old World monkeys, 1 New World monkey, and 1 prosimian. Among these primate species, the percentage similarities of the nucleotide sequence of the ZFX gene were 96-100% and 91.2-99.7% for the ZFY gene. Of 397 sites in the ZFX and ZFY gene sequences, 20 for ZFX gene and 42 for ZFY gene were found to be variable. Substitution causes 1 amino acid change in ZFX, and 5 in ZFY, among 132 amino acids. The numbers of synonymous substitutions per site (Ks) between human and the chimpanzee, gorilla and orangutan for ZFY gene were 0.026, 0.033, and 0.085, respectively. In contrast, the Ks value between human and hominoid primates for the ZFX gene was 0.008 for each comparison. Comparison of the ZFX and ZFY genes revealed that the synonymous substitution levels were higher in hominoids than in other primates. The rates of synonymous substitution per site per year were higher in the ZFY exon than in the SRY exon, and higher in the ZFY exon than in the ZFY intron, in hominoid primates.     

Genetic organization and restriction enzyme cleavage map of the ksgA-pdxA region of the Escherichia coli chromosome

Summary Small multicopy plasmids carrying the Escherichia coli genes ksgA and pdxA were constructed by ligation in vitro of an EcoRI restriction fragment from ksg10 (Andrésson and Davies, 1980a) into the EcoRI sites of the ColE1 plasmids RSF2124 and pVH51. Cleavage maps of the plasmids were determined for 21 different restriction enzymes. The ksgA gene was located in a 750 basepairs (bp) region 1,450 bp clockwise of the EcoRI site in folA; pdxA is in a 2,040 bp region immediately clockwise of ksgA.     

Genome size and «C-heterochromatic-DNA» in man and the african apes

The genome sizes and the amounts of DNA after C-banding pretreatments (C-heterochromatic DNA) were measured by quantitative cytochemical methods in man and the African apes,Gorilla gorilla andPan troglodytes. As evaluated by flow cytometry on propidium-iodide-stained lymphocytes, gorilla and chimpanzee have genome sizes larger than man. On the basis of the different resistance of metaphase chromosome DNA to the C-banding procedure, two genome compartments were defined, i.e.,C-heterochromatic-DNA andeuchromatic-DNA. The latter proved to be fairly constant in man and the African apes (as well as in two hylobatid species), whereas the variable amounts ofC-heterochromatic-DNA account well for the interspecific differences of genome size among the hominoid species studied so far. During karyotype diversification, quantitative changes (with either gains or losses) ofC-heterochromatic-DNA seem to have taken place independently in the hylobatid and the man/African ape lineages.     

Analysis of GC-rich repetitive nucleotide sequences in great apes

The genomes of four primate species, belonging to the families Pongidae (chimpanzee, gorilla, and orangutan) and Hylobatidae (gibbons), have been analyzed for the presence and organization of two human GC-rich heterochromatic repetitive sequences: Satellite (Sat) and LongSau (LSau) repeats. By Southern blot hybridization and PCR, both families of repeats were detected in all the analyzed species, thus indicating their origin in an ape ancestor. In the chimpanzee and gorilla, as in man, Sat sequences showed a 68-bp Sau3A periodicity and were preferentially organized in large clusters, whereas in the orangutan, they were organized in DNA fragments of 550 bp, which did not seem to be characterized by a tandem organization. On the contrary, in each of the analyzed species, the bulk of LSau sequences showed a longer Sau3A periodicity than that observed in man (450–550 bp). Furthermore, only in the chimpanzee genome some of LSau repeats seemed to be interspersed within blocks of Sat sequences. This sequence organization, which also characterizes the human genome, is probably absent in the gorilla. In fact, the analysis of a gorilla genomic library suggested that LSau repeats are not preferentially in linkage with Sat sequences. Moreover, LSau sequences were found in a genomic sector characterized by the simultaneous presence of L1 and (CA) repeats, as well as of anonymous sequences and known genes. In spite of the different sequence organization, the nucleotide differences between complete human and gorilla LSau repeats were very few, whereas one gorilla LSau repeat, interrupted by a probably-truncated L1 transposon, showed a higher degree of divergence. Besides the gorilla, this unusual sequence organization was detected in man, and, to a lesser extent, in the chimpanzee. Correspondence to: R. Meneveri