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Y Colin

Publications and source records attributed to Y Colin.

At least 73 records · Page 4Linked to original sources
PubMed1994

Organization of the gene (RHCE) encoding the human blood group RhCcEe antigens and characterization of the promoter region.

The human RH (rhesus) locus is composed of two genes, RHD and RHCE, encoding the D, Cc, and Ee blood group antigens. The RHCE gene was isolated from a human genomic library and characterized. It is organized into 10 exons distributed over 75 kb. Exons 4-8 are alternatively spliced in the different RNA isoforms previously identified. Primer extension analysis indicated that the transcription initiation site is located 83 bp upstream of the initiation codon. The 5' flanking region of the RHCE gene, from nucleotide -600 to +42, exhibited a significant transcriptional activity after transfection in the erythroleukemic cell line K562, but not in the nonhematopoietic cell line HeLa. This result was in agreement with Northern blot analysis, suggesting that the expression of the RH locus is restricted to the erythroid/megakaryocytic lineage. Accordingly, putative binding sites for SP1, GATA-1, and Ets proteins, nuclear factors known to be involved in the erythroid and megakaryocytic gene expression, were identified in this Rh promoter.

Base Sequence↗
PubMed1994

Molecular characterization of the Rh-like locus and gene transcripts from the rhesus monkey (Macaca mulatta).

The human Rh blood group locus consists of two structurally related genes (D and CcEe) in Rh-positive haplotypes but a single gene (CcEe) in Rh-negative haplotypes. The genome of rhesus monkeys (Macaca mulatta), while not expressing any of the human Rh D, C, c, E, or e specificities, carries a Rh-like locus strongly related to the human Rh locus. Southern blot analysis suggested the presence of only one Rh-like gene with an additional truncated fragment corresponding to the 5' region. RNA preparations from M. mulatta bone marrow cells contained Rh-like species of 1.7 kb. Two allelic Rh-like transcripts were amplified by PCR and sequenced. The predicted translation product of the first transcript was a 417-amino-acid protein closely similar to the human Rh counterpart. The predicted product of the second transcript consisted of a 361-amino-acid polypeptide truncated in the NH2 terminal region and differing from the former by a few substitutions. The macaque Rh-like protein sequences differed from those of human D and Cc/Ee polypeptides by 22-25%, whereas the degree of identity between the human proteins was 91.5%. Implications of these results in the analysis of the evolutionary pathway of the Rh locus are discussed.

Alleles↗
PubMed1994

Molecular genetics of chimpanzee Rh-related genes: their relationship with the R-C-E-F blood group system, the chimpanzee counterpart of the human rhesus system.

As the chimpanzee R-C-E-F blood group system appears to be the chimpanzee counterpart of the human Rhesus (RH) system, we have tried to determine whether chimpanzee Rh-like genes encode R-C-E-F-related proteins. Chimpanzee genomic DNA, digested by any of eight endonucleases and hybridized with three Rh exon-specific probes, exhibits a high degree of polymorphism. Analysis of DNA from unrelated individuals of different R-C-E-F types revealed that the presence of some restriction fragments is correlated with particular R-C-E-F types. The cosegregation of these fragments with R-C-E-F haplotypes was confirmed by family studies. Oligonucleotides complementary to regions flanking human exons were used as PCR primers on chimpanzee DNA; the resulting amplified fragments were identical in size to their human counterparts. Moreover, the nucleotide sequences of the fragments present a high degree of similarity to the corresponding human regions.

Animals↗
PubMed1994

PCR-based determination of Rhc and RhE status of fetuses at risk of Rhc and RhE haemolytic disease.

After anti-RhD, anti-Rhc is the most important red cell alloantibody which can cause haemolytic disease of the newborn (HDN) when the mother is Rhc-negative and the fetus Rhc-positive. We report here the development of polymerase chain reaction (PCR) assays which detect the presence of the Rhc alleles in amniotic cells by the use of allele-specific primers (ASP). It is expected that such determination will help in the management of pregnancies at risk of Rhc haemolytic disease. In the course of this study we have similarly performed PCR-ASP experiments to detect fetal RHE alleles since, in rare cases, anti-RhE can also cause HDN.

Amniotic Fluid↗
PubMed1993

Prenatal determination of fetal RhD type by DNA amplification.

BACKGROUND: An RhD-negative woman whose partner is heterozygous may have preexisting anti-RhD antibodies that may or may not affect a subsequent fetus, depending on whether it is heterozygous. A safe method of determining fetal RhD type early in pregnancy would eliminate the risks to an RhD-negative fetus of fetal-blood sampling or serial amniocenteses. METHODS: We determined the RhD type in 15 fetuses using the polymerase chain reaction in amniotic cells and serologic methods in fetal blood collected simultaneously. In another 15 fetuses, the RhD type determined from chorionic-villus samples was compared with that identified by typing of DNA from the fetus itself. RESULTS: RhD typing of DNA from amniotic cells correctly indicated the serologic type in every fetus. Of 10 fetuses with RhD-negative mothers, 4 were identified as RhD-negative and 6 as RhD-positive. Of five fetuses with RhD-positive mothers, four were identified as RhD-positive and one as RhD-negative. There was also complete agreement between the results of RhD typing of DNA from chorionic-villus samples and the results of typing of DNA from fetal tissue. Eleven fetuses were RhD-positive, and 4 were RhD-negative. Four RhD-positive fetuses had RhD-negative mothers. Three RhD-negative fetuses had RhD-positive mothers. There was no contamination by maternal RhD-positive DNA of the samples from RhD-negative fetuses. CONCLUSIONS: Determining fetal RhD type in amniotic cells without invading the fetomaternal circulation is a reliable method that will be valuable in the management of Rh alloimmunization.

Amniotic Fluid↗
PubMed1993

Structure and expression of the RH locus in the Rh-deficiency syndrome.

Red blood cell deficiency of Rh proteins is associated with morphologic and functional abnormalities of erythrocytes and with a chronic hemolytic anemia of varying severity. Rh-deficiency may be the result of homozygosity either for a silent allele at the RH locus (Rhnull amorph type) or for a recessive inhibitor gene(s) at an autosomal locus unlinked to RH locus (Rhnull regulator and Rhmod). In this report, we investigated the RH locus structure of Rh-deficient individuals by Southern analysis using cDNA and exon-specific probes deduced from the recent cloning of Rh genes (CcEe and D). As expected from family studies indicating that Rhmod and Rhnull regulator individuals are unable to express Rh antigens but are able to convey functional Rh genes from one generation to another, no alteration of the Rh genes was detected in these variants. Although Rhnull of the amorph type arose by inheritance of a pair of silent alleles at the RH locus, the general organization of the unique CcEe gene in the genome of the particular individual under examination was apparently normal and indistinguishable from a Rh-negative chromosome. More surprisingly, no mutation could be detected by sequencing the polymerase chain reaction (PCR)-amplified reticulocyte mRNAs, suggesting that the RH locus of this patient might be altered in its transcriptional activity. Through hybridization with exon-specific probes, we were also able to determine the zygosity for the D gene in DNA samples from individuals of known genotypes; using this approach, we found that Rhnull regulator variants could be either of the DD, Dd, or dd genotypes. These findings suggest that the postulated inhibitor gene(s) can negatively suppress the RH locus expression from chromosomes carrying either one or two of the Rh genes.

Alleles↗
PubMed1993

Molecular genetic basis of the human Rhesus blood group system.

The Rhesus (RH) blood group locus is composed of two related structural genes, D and CcEe, that encode red cell membrane proteins carrying the D, Cc and Ee antigens. As demonstrated previously, the RhD-positive/RhD-negative polymorphism is associated with the presence or the absence of the D gene. Sequence analysis of transcripts and genomic DNA from individuals that belong to different Rh phenotypes were performed to determine the molecular basis of the C/c and E/e polymorphisms. The E and e alleles differ by a single nucleotide resulting in a Pro226Ala substitution, whereas the C and c alleles differ by six nucleotides producing four amino acid substitutions Cys16Trp, Ile60Leu, Ser68Asn and Ser103Pro. With the recent cloning of the RhD gene, these findings provide the molecular genetic basis that determine D, C, c, E and e specificities.

Alleles↗
PubMed1993

Relationship between chimpanzee Rh-like genes and the R-C-E-F blood group system.

The antigenic closeness between the chimpanzee alloantigen Rc of the R-C-E-F system, and the human alloantigen Rho(D) suggests a phyloge-connection between their genes. To confirm at the molecular level the common origin of these genes, genomic DNA from 16 unrelated chimpanzees of various R-C-E-F phenotypes were digested by three restriction enzymes and analyzed by Southern blot using a human Rh cDNA probe and three exon-specific probes. Restrictions profiles displayed reach polymorphism. Correlations between some bands and certain R-C-E-F phenotypes demonstrate that the human Rh cDNA probe defines in chimpanzee genomic DNA some genes of the R-C-E-F system.

Animals↗
PubMed1993

Recent advances in molecular and genetic analysis of Rh blood group structures.

The Rh antigens are carried by 30-32 kDa integral proteins of the red cell membrane. The Rh locus is composed of two genes: RhD, which encodes the major D antigen and is present only in Rh-positive genomes, and RhCcEe, which encodes both the Cc and Ee polypeptides, most likely by alternative splicing events. The D and non-D Rh mRNAs have been cloned. Their sequence homology suggest that the two Rh genes have evolved by duplication of a common ancestor.

Amino Acid Sequence↗
PubMed1992

Molecular cloning and primary structure of the human blood group RhD polypeptide.

The RH (rhesus) blood group locus from RhD-positive donors is composed of two homologous structural genes, one of which encodes the Cc and Ee polypeptides, whereas the other, which is missing in the RhD-negative condition, encodes the D protein that carries the major antigen of the RH system. Recently, different splicing isoforms transcribed from the CcEe gene were isolated. We report now the characterization of two other Rh clones, RhII and RhXIII, generated by alternative choices for poly(A) addition sites that were identified as the RhD gene transcripts. That these cDNAs represented the RhD messenger and that the previously described Rh clones were derived from the CcEe gene was demonstrated by amplification of RhII/XIII sequences only from D-positive genomes and by cloning and sequencing of D- and CcEe-specific gene fragments. The predicted translation product of the RhD mRNA is a 417-amino acid protein (M(r) = 45,500) that exhibited a similar membrane organization with 13 bilayer-spanning domains compared with the polypeptide encoded by the CcEe gene. The D and Cc/Ee polypeptides differ by 36 amino acid substitutions (8.4% divergence), but the NH2- and COOH-terminal regions of the two proteins are well conserved. Similarly, five of the six cysteine residues of the Cc/Ee proteins were conserved in the D protein, including the unique exofacial cysteine, which is critical for antigenic reactivity. The sequence homology between the Cc/Ee and D proteins supports the concept that the genes encoding these polypeptides have evolved by duplication of a common ancestor gene.

Amino Acid Sequence↗
PubMed1992

Multiple Rh messenger RNA isoforms are produced by alternative splicing.

Three Rh-related cDNAs have been isolated from a human bone marrow cDNA library and by polymerase chain reaction (PCR) amplification of human bone marrow and erythroblast mRNAs. They potentially encode a family of Rh protein isoforms that exhibit several unexpected structural properties as compared with the Rh polypeptide encoded by the cDNA clone identified previously. These modifications include several peptide deletions, the predicted alteration of Rh protein topology within the cell membrane, variations in the number and surface exposition of cysteine residues, and the generation of new C-terminal polypeptide segments caused by frameshift mutations. The four Rh mRNAs now described correspond to different splicing isoforms transcribed from the same Rh gene, and all exist in the same cell lineage (erythroid). Moreover, PCR experiments indicated that at least three of these RNA species exist in reticulocytes from donors with different commonly expressed Rh phenotypes. Although the translated proteins have not yet been characterized, these results suggest that the two genes at the RH locus may direct the synthesis of several protein species possibly corresponding to different Rh antigenic variants.

Amino Acid Sequence↗
PubMed1991

Genetic basis of the RhD-positive and RhD-negative blood group polymorphism as determined by Southern analysis.

Several lines of evidence have previously indicated that the RhD, c, and E blood group antigens are most likely carried by three distinct but homologous red blood cell membrane proteins. To determine whether these polypeptides are encoded by one or several related genes, we have performed Southern blot analysis of genomic DNA prepared from donors of different Rh phenotypes. Using an entire Rh cDNA probe and several exon-specific probes covering the cloned gene from its 5' to 3' ends, we have shown that the Rh locus carried by the genome of RhD-positive individuals is composed of two different but strongly related genes of identical general organization whether they expressed the C or c and E or e antigens, and, surprisingly, even when they do not express these epitopes, as in the D-- phenotype. The only antigenic variation found to be associated with a consistent genomic polymorphism corresponded to the RhD-positive/RhD-negative phenotypes. Indeed, one of the two Rh genes was completely lacking when the genomes of several unrelated RhD-negative donors were analyzed. From the present study we conclude that one of the two genes of the Rh locus encodes the RhC/c and RhE/e polypeptides while the other encodes the RhD protein. The absence of any D gene and of its postulated allelic form d in the RhD-negative genome explains finally why no Rhd antigen has ever been shown.

Blotting, Southern↗
PubMed1991

Molecular basis for elliptocytosis associated with glycophorin C and D deficiency in the Leach phenotype.

Glycophorin C (GPC) and glycophorin D (GPD) are highly glycosylated integral membrane proteins of human erythrocytes encoded by the same gene and associated with expression of Gerbich blood group system antigens. GPC/D deficiency (the Leach phenotype) is a rare condition usually found after identification of Gerbich blood group system antibodies in persons undergoing prenatal or pretransfusion evaluation. In all cases, the Leach phenotype has been associated with elliptocytosis. Characterization of the molecular basis of this phenotype in three previously uninvestigated families has shown that the most common genetic basis of GPC/D deficiency is deletion of exons 3 and 4 of the GPC gene. However, in one family, the Leach phenotype appeared due to a deletion of one nucleotide in exon 3, causing a frameshift mutation in the messenger RNA and premature generation of a stop codon. The GPC and GPD protein sequences are therefore interrupted in the extracellular domain and probably intracellularly degraded.

Amino Acid Sequence↗
PubMed1991

Localization of the human Rh blood group gene structure to chromosome region 1p34.3-1p36.1 by in situ hybridization.

A cDNA clone, RhIXb (1384 bp), encoding the entire protein sequence of a human blood group Rh polypeptide has been used to map the Rh locus, by in situ hybridization, to the region p34.3-p36.1 of chromosome 1. Two other unrelated cDNA clones, pUCA2 (750 bp) and pUCIII (1600 bp), isolated during the cloning procedure of the Rh cDNA were investigated simultaneously, and assigned to chromosome 3p21.1-3p22 (clone pUCA2) and to chromosome 22q12.1-22q13.1 (clone pUCIII).

Chromosome Banding↗
PubMed1990

Characterization of a new erythroid/megakaryocyte-specific nuclear factor that binds the promoter of the housekeeping human glycophorin C gene.

Human glycophorin C (GPC) is an integral membrane protein found in many cell types but which exhibits increased expression in red cells. We have investigated the in vitro binding of nuclear protein factors to the GPC gene 5'-flanking region which was transcriptionally active when transfected into erythroid and non-erythroid cells. We found that in addition to known ubiquitous or erythroid trans-acting factors, a presently uncharacterized erythroid/megakaryocyte-specific protein binds the GPC promoter. This factor, that we called NFE-6, is present in embryonic, fetal, or adult-like human or mouse erythroleukemic cell lines, in a megakaryoblastic cell line, but not in lymphoid or non-hematopoietic cell lines. These data suggest that NFE-6, like the well characterized NFE-1 factor, is tissue- but not stage-specific and is conserved across species. It is proposed that NFE-6 might be involved in the quantitative change in the expression of the house-keeping GPC gene related to red cell specialization.

Base Sequence↗