Evolutionary history of the Rh blood group-related genes in vertebrates

Rh and its homologous Rh50 gene products are considered to form heterotetramers on erythrocyte membranes. Rh protein has Rh blood group antigen sites, while Rh50 protein does not, and is more conserved than Rh protein. We previously determined both Rh and Rh50 gene cDNA coding regions from mouse and rat, and carried out phylogenetic analyses. In this study, we determined Rh50 gene cDNA coding regions from African clawed frog and Japanese medaka fish, and examined the long-term evolution of the Rh blood group and related genes. We constructed the phylogenetic tree from amino acid sequences. Rh50 genes of African clawed frog and Japanese medaka fish formed a cluster with mammalian Rh50 genes. The gene duplication time between Rh and Rh50 genes was estimated to be about 510 million years ago based on this tree. This period roughly corresponds to the Cambrian, before the divergence between jawless fish and jawed vertebrates. We also BLAST-searched an amino acid sequence database, and the Rh blood group and related genes were found to have homology with ammonium transporter genes of many organisms. Ammonium transporter genes can be classified into two major groups (amt alpha and amt beta). Both groups contain genes from three domains (bacteria, archaea, and eukaryota). The Rh blood group and related genes are separated from both amt alpha and beta groups.     

The nucleotide sequence of a cDNA clone containing the entire coding region for mouse X-chromosome-linked phosphoglycerate kinase

A clone containing cDNA for X chromosome-linked phosphoglycerate kinase (PGK-1) was isolated from a mouse myeloma cDNA library. The nucleotide (nt) sequence of the cDNA has been determined, and the amino acid (aa) sequence of the enzyme thereby deduced. At the nt level, the coding region of mouse PGK cDNA has 93% homology with human X-linked cDNA and 60% homology with the yeast gene. Mouse PGK-1 protein contains 416 aa and is 98%, 96% and 64% homologous with human, horse, and yeast enzyme sequences, respectively.     

Gene arrangement at the Rhesus blood group locus of chimpanzees detected by fiber-FISH

The Rhesus (Rh) blood group system in humans is encoded by two genes with high sequence homology. These two genes, namely, RHCE and RHD, have been implied to be duplicated during evolution. However, the genomic organization of Rh genes in chimpanzees and other nonhuman primates has not been precisely studied. We analyzed the arrangement of the Rh genes of chimpanzees (Pan troglodytes) by two-color fluorescence in situ hybridization on chromatin DNA fibers (fiber-FISH) using two genomic DNA probes that respectively contain introns 3 and 7 of human RH genes. Among the five chimpanzees studied, three were found to be homozygous for the two-Rh-gene type, in an arrangement of Rh (5'-->3') - Rh (3'<--5'). Although a similar gene arrangement can be detected in the RH gene locus of typical Rh-positive humans, the distance between the two genes in chimpanzees was about 50 kb longer than that in humans. The remaining two chimpanzees were homozygous for a four-Rh-gene type, in an arrangement of Rh (5'-->3') - Rh (3'<--5') - Rh (3'<--5') - Rh (3'<--5') within a region spanning about 300 kb. This four-Rh-gene type has not been detected in humans. Further analysis of other great apes showed different gene arrangements: a bonobo was homozygous for the three-Rh-gene type; a gorilla was heterozygous for the one-Rh- and two-Rh-gene types; an orangutan was homozygous for the one-Rh-gene type. Our findings on the intra- and interspecific genomic variations in the Rh gene locus in Hominoids would shed further light on reconstructing the genomic pathways of Rh gene duplication during evolution.     

The human serum amyloid A (SAA)-encoding gene GSAA1: nucleotide sequence and possible autocrine-collagenase-inducer function

We have determined the genomic sequence of the human GSAA1 gene, a member of the family of acute-phase human serum amyloid A (SAA)-encoding genes. This sequence predicts a mature protein of 104 amino acids (aa), several of which differ from residues usually conserved in the sequence of SAA proteins isolated from serum. Despite coding differences, however, the four-exon structure of GSAA1 resembles that of other SAA genes in humans and mice. The N-terminal 25 aa of the mature GSAA1 protein are virtually identical to those of an 'SAA-like' autocrine collagenase inducer produced by rabbit synovial fibroblasts; the latter also differ from the corresponding aa found in SAA in serum. We propose that GSAA1 is the human gene coding for a protein closely related to the SAA, but which is adapted to this important autocrine cytokine function.     

Gibbon and marmoset c-myc nucleotide sequences.

The nucleotide sequences of the gibbon and marmoset myc loci have been determined by the dideoxy ribomethod. The number of mutations which occurred during evolution and the branches affected were deduced according to the principle of maximum parsimony, from a comparison with known mammal sequences. As previously observed for the human and chimpanzee myc genes, an Alu repeat belonging to subclass III was observed in the second intron of the gibbon myc gene. In contrast, no such element was found in the marmoset gene. Alignment of the Myc amino acid (aa) sequences provided clues for detecting which aa or which protein regions have been more heavily mutated. Conversely, some regions remained free of mutations and remained unchanged from mouse to human, most probably in connection with some important embedded property(ies). An intriguing feature of the human Myc protein is duplication of 50 aa out of 439. Strikingly, most of these aa remain unchanged in mouse, rat, cat, marmoset, gibbon, chimpanzee and human.     

Molecular cloning, characterization, and chromosomal localization of the mouse homologue of CD84, a member of the CD2 family of cell surface molecules

 CD84 is a member of the immunoglobulin gene superfamily (IgSF) with two Ig-like domains expressed primarily on B lymphocytes and macrophages. Here we describe the cloning of the mouse homologue of human CD84. Mouse CD84 cDNA clones were isolated from a macrophage library. The nucleotide sequence of mouse CD84 was shown to include an open reading frame encoding a putative 329 amino acid protein composed of a 21 amino acid leader peptide, two extracellular immunoglobulin (Ig)-like domains, a hydrophobic transmembrane region, and an 87 amino acid cytoplasmic domain. Mouse CD84 shares 57.3% amino acid sequence identity (88.7%, considering conservative amino acid substitutions) with the human homologue. Chromosome localization studies mapped the mouse CD84 gene to distal chromosome 1 adjacent to the gene for Ly-9, placing it close to the region where other members of the CD2 IgSF (CD48 and 2B4) have been mapped. Northern blot analysis revealed that the expression of mouse CD84 was predominantly restricted to hematopoietic tissues. Two species of mRNA of 3.6 kilobases (kb) and 1.5 kb were observed. The finding that the pattern of expression was restricted to the hematopoietic system and the conserved sequence of the mouse CD84 homologue suggests that the function of the CD84 glycoprotein may be similar in humans and mice. Received: 1 July 1998 / Revised: 31 August 1998     

Tempo and mode of evolution of the Rh blood group genes before and after gene duplication

The Rh blood group genes became duplicated in a common ancestor of human–chimpanzee–gorilla. We compared the evolutionary rates of the Rh blood group genes for each exon for branches connecting to humans, having duplicated Rh loci, and to orangutan, gibbon, and Old World monkeys, species having a single Rh locus. Our results show that evolutionary rates of nonsynonymous substitutions at exon 7 became accelerated in the human lineage. Furthermore, we surveyed the sequence variation in the region surrounding exon 7 of gibbons to clarify whether the diversity of the human exon 7 was introduced after the duplication or had been maintained before it. Two amino acid polymorphisms in white-handed gibbons were observed in the immediate vicinity of the D-specific motif in the human exon 7. Although the evolutionary rate of exon 7 was accelerated after the gene duplication, our results suggest that exon 7 had the potential for change even before the gene duplication. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular cloning,gene structure,expression profile and functional characterization of the mouse glutamate transporter (EAAT3) interacting protein GTRAP3-18

Glutamate is an important amino acid implicated in energy metabolism, protein biosynthesis and neurotransmission. The Na(+)-dependent high-affinity excitatory amino acid transporter EAAT3 (EAAC1) facilitates glutamate uptake into most cells. Recently, a novel rat EAAT3-interacting protein called GTRAP3-18 has been identified by a yeast two-hybrid screening. GTRAP3-18 functions as a negative modulator of EAAT3-mediated glutamate transport. In order to further understand the function and regulation of GTRAP3-18, we cloned the mouse orthologue to GTRAP3-18 and determined its gene structure and its expression pattern. GTRAP3-18 encodes a 188-residue hydrophobic protein whose sequence is highly conserved amongst vertebrates. Mouse and human GTRAP3-18 genes contain three exons separated by two introns. The GTRAP3-18 gene is found on mouse chromosome 6D3 and on human chromosome 3p14, a susceptibility locus for cancer and epilepsy. GTRAP3-18 protein and RNA were found both in neuronal rich regions of the brain and in non-neuronal tissues such as the kidney, heart and skeletal muscle. Mouse GTRAP3-18 inhibited EAAT3-mediated glutamate transport in a dose-dependent manner. These studies show that GTRAP3-18 is a ubiquitously expressed protein that functions as a negative regulator of EAAT3 function.     

Entire sequence of a mouse chromosomal segment containing the gene Rhced and a comparative analysis of the homologous human sequence

The mouse genomic sequence of the region containing the gene Rhced, the orthologue to the human gene RH30, was determined to elucidate the structure of Rhced and its flanking regions and to compare these with the corresponding human genomic region. Two genes, Smp1 and AK003528 (an orthologue of FLJ10747), flank Rhced. Neither sequences homologous to the characteristic nucleotide elements flanking the RHD gene in humans (rhesus boxes) nor an additional Rh gene were found within the mouse region sequenced. This result and that of a previous report demonstrate that this chromosomal region of the mouse comprises five genes (FLJ10747-RHCE-SMP1-NPD014-P29) that exhibit syntenic homology with the corresponding human region, which suggests that the RHD gene and rhesus boxes were inserted later. Evaluations of tissue distribution and subcellular localization of these genes indicate that the SMP1 orthologue has a ubiquitous tissue distribution and cytoplasmic localization, whereas AK003528 is expressed slightly higher in testis with a strong subcellular localization in the nucleus. Despite the steady improvements in the draft sequence of the human genome, this study demonstrates the continuing benefits of comparative genetic analyses in increasing our understanding of human genomic structure.     

Structure of the human and murine R-ras genes, novel genes closely related to ras proto-oncogenes

The human R-ras gene was isolated by low-stringency hybridization with a v-H-ras probe. The predicted 218 amino acid R-ras protein has an amino-terminal extension of 26 residues compared with H-ras p21, and shows 55% amino acid identity; conserved domains include the p21 GTP-binding site and the carboxy-terminal membrane localization sequence. R-ras has at least six exons, with the position of the first intron conserved relative to the Drosophila ras64B and Dictyostelium ras genes; there is no similarity in the exon-intron structure of the R-ras gene and of the mammalian H-, K-, and N-ras proto-oncogenes. Cloned mouse R-ras cDNAs exhibit 88% nucleotide and 94.5% predicted amino acid identity to human R-ras. Human R-ras was localized to chromosome 19, a site different from ras p21 genes. Mouse R-ras is syntenic with c-H-ras on chromosome 7.     

Human mutations in glucose 6-phosphate dehydrogenase reflect evolutionary history.

Glucose 6-phosphate dehydrogenase (G6PD) is a cytosolic enzyme encoded by a housekeeping X-linked gene whose main function is to produce NADPH, a key electron donor in the defense against oxidizing agents and in reductive biosynthetic reactions. Inherited G6PD deficiency is associated with either episodic hemolytic anemia (triggered by fava beans or other agents) or life-long hemolytic anemia. We show here that an evolutionary analysis is a key to understanding the biology of a housekeeping gene. From the alignment of the amino acid (aa) sequence of 52 glucose 6-phosphate dehydrogenase (G6PD) species from 42 different organisms, we found a striking correlation between the aa replacements that cause G6PD deficiency in humans and the sequence conservation of G6PD: two-thirds of such replacements are in highly and moderately conserved (50-99%) aa; relatively few are in fully conserved aa (where they might be lethal) or in poorly conserved aa, where presumably they simply would not cause G6PD deficiency. This is consistent with the notion that all human mutants have residual enzyme activity and that null mutations are lethal at some stage of development. Comparing the distribution of mutations in a human housekeeping gene with evolutionary conservation is a useful tool for pinpointing amino acid residues important for the stability or the function of the corresponding protein. In view of the current explosive increase in full genome sequencing projects, this tool will become rapidly available for numerous other genes.     

In silico cloning of evolutionarily conserved mouse F-LANa]

Differentially expressed genes between normal liver and hepatocellular carcinomas were investigated using differential display. In previous study, human F-LANa was identified as a differentially expressed gene, up-regulated in hepatocellular carcinoma. In this study, we developed an in silico cloning approach to rapidly and accurately characterize the mouse ortholog of the human F-LANa. Mouse F-LANa encodes a 239 aa protein exhibiting 97.9% similarity to the human ortholog gene. Homology analysis was carried out in various species and showed that F-LANa was evolutionarily conserved from yeast to human. Based on the alignment results, phylogenetic tree was established here.     

进化上保守的小鼠FLANa基因的计算机克隆

EST(expressed sequence tag,表达序列标签)是对cDNA克隆进行测序时得到的序列,一般位于该克隆的一端,序列为单次测序所得到的。现在EST已成为一个非常大的数据库,并被广泛运用于基因家族新成员的寻找,也可作为人染色体上的标志来寻找多态性位点,还可以用来比较某基因在不同组织和病理状态中的表达模式。在EST数据库中,     

Characterization of the mouse Cblc/Cbl3 gene

The mouse Cblc/Cbl3 gene was cloned and characterized. It comprises 12 exons and encodes a putative protein of 496 amino acid residues which shares an overall 67% identity with its human ortholog; it also shares 70% of amino acid identity with mouse CBL over their conserved SH2 and Ring finger domains. Mouse Cblc mRNA is expressed in embryo and adult tissues and has a rather ubiquitous distribution.     

Evolution of RH genes in hominoids: characterization of a gorilla RHCE-like gene

The human RH locus is responsible for the expression of the Rh blood group antigens. It consists of two closely linked genes, RHD and RHCE, that exhibit 92% similarity between coding regions. These observations suggest that they are derived from a relatively recent duplication event. Previously a study of nonhuman primate RH-like genes demonstrated that ancestral RH gene duplication occurred in the common ancestor of man, chimpanzees and gorillas. By amplification of intron 3 and intron 4 of gorilla RH-like genes, we have now shown that, like man, gorillas possess two types of RH intron 3 (RHCE intron 3 being 289 bp longer than the RHD intron 3) and two types of intron 4 (RHCE intron 4 being 654 bp longer than the RHD intron 4). Here we report the characterization of a cDNA encoded by a gorilla RH-like gene which possesses introns 3 and 4 of the RHCE type. A comparison of this gorilla RHCE-like coding sequence with previously characterized human and ape cDNA sequences suggests that RH genes experienced complex recombination events after duplication in the common ancestor of humans, chimpanzees and gorillas.     

Human membrane type-2 matrix metalloproteinase is defective in cell-associated activation of progelatinase A

Transfection of the mouse membrane type-2 matrix metalloproteinase (MT2-MMP) gene into COS-1 cells resulted in activation of progelatinase A; however, that of the human gene had no effect. Expression of human and mouse MT2-MMP chimeric proteins revealed the defect of human MT2-MMP which resides in the region between amino acid (aa) residues 155 and 271. Seven aa residues in this region were not conserved between human and mouse MT2-MMP. Substitution with the corresponding mouse residue, proline-183 to serine and glutamine-185 to aspartic acid, recovered cell-associated progelatinase A activation function. These residues are located in the insertion sequence-2 (IS-2), which was conserved in six clones of the human MT2-MMP gene from different sources, except that of proline-183 which was substituted with serine from HT1080 cells. These results indicate that human MT2-MMP is defective in cell-associated activation of progelatinase A, and this is attributed to IS-2. These findings emphasize the importance of IS-2 in MT2-MMP functionality.     

Immunopurification of the blood group RhD protein from human erythrocyte membranes

Rh proteins are membrane proteins encoded by genes at the blood group RH locus. They are of paramount importance in transfusion medicine, but their function is still unknown. Biochemical and biophysical studies of these proteins are scarce since only minute amounts of the very hydrophobic Rh proteins, can be purified from human erythrocytes. Recently, a human monoclonal antibody (LOR-15C9) was described as having the unique property to recognize the Rh30 protein carrying the major blood group D specificity (RhD protein), either in a membrane detergent extract or when blotted on a membrane. In this report, we describe one-step purification of the RhD protein from detergent extracts of red cell membranes, based on immunoaffinity chromatography carried out with immobilized LOR-15C9 IgG. The technique yielded RhD protein with high purity which was devoid of other associated proteins (RhAG, CD47, LW and GPB) that comprise the Rh complex in the erythrocyte membrane. By contrast immunoprecipitation performed with the same antibody led to co-isolation of both RhD and RhAG.