The human Rhesus-associated RhAG protein and a kidney homologue promote ammonium transport in yeast.

The Rhesus blood-group antigens are defined by a complex association of membrane polypeptides that includes the non-glycosylated Rh proteins (RhD and RhCE) and the RHag glycoprotein, which is strictly required for cell surface expression of these antigens. RhAG and the Rh polypeptides are erythroid-specific transmembrane proteins belonging to the same family (36% identity). Despite their importance in transfusion medicine, the function of RhAG and Rh proteins remains unknown, except that their absence in Rh(null) individuals leads to morphological and functional abnormalities of erythrocytes, known as the Rh-deficiency syndrome. We recently found significant sequence similarity between the Rh family proteins, especially RhAG, and Mep/Amt ammonium transporters. We show here that RhAG and also RhGK, a new human homologue expressed in kidney cells only, function as ammonium transport proteins when expressed in yeast. Both specifically complement the growth defect of a yeast mutant deficient in ammonium uptake. Moreover, ammonium efflux assays and growth tests in the presence of toxic concentrations of the analogue methylammonium indicate that RhAG and RhGK also promote ammonium export. Our results provide the first experimental evidence for a direct role of RhAG and RhGK in ammonium transport. These findings are of high interest, because no specific ammonium transport system has been characterized so far in human.

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.

Candidate gene acting as a suppressor of the RH locus in most cases of Rh-deficiency.

The Rh antigen is a multi-subunit complex composed of Rh polypeptides and associated glycoproteins (Rh50, CD47, LW and glycophorin B); these interact in the red cell membrane and are lacking or severely reduced in Rhnull cells. As a result, individuals with Rhnull suffer chronic haemolytic anaemia known as the Rh-deficiency syndrome. Most frequently, Rhnull phenotypes are caused by homozygosity of an autosomal suppressor gene unlinked to the RH locus (Rhnull regulator or Rhmod types). We have analysed the genes and transcripts encoding Rh, CD47 and Rh50 proteins in five such unrelated Rhnull cases. In all patients, we identified alteration of Rh50--frameshift, nucleotide mutations, or failure of amplification--which correlated with Rhnull phenotype. We propose that mutant alleles of Rh50, which map to chromosome 6p11-21.1, are likely candidates for suppressors of the RH locus accounting for most cases of Rh-deficiency.

The challenge of understanding ammonium homeostasis and the role of the Rh glycoproteins.

Rh glycoproteins belong to the superfamily of ammonium transporters, but until recent functional studies their functional role was unknown. This review focuses on the functional results obtained in our laboratory after the heterologous expression of RhAG (the erythroid Rh glycoprotein) and RhCG (an epithelial Rh glycoprotein). RhAG and RhCG were expressed in two different expression systems (HeLa cells and Xenopus laevis oocytes) that differed in their endogenous membrane permeabilities for NH3 and NH4+. To check if RhAG and RhCG are ammonium transporters, we measured intracellular pH changes in cells exposed to an ammonium-containing solution, and analyzed the ammonium-induced NH3 and NH4+ transmembrane fluxes in control versus transfected cells. We observed that RhAG and RhCG expression induced an enhancement of the ammonium-induced initial alkalinization (related to NH3 influx into the cell) and secondary acidification (related to NH4+ influx into the cell). Moreover, sub-millimolar ammonium concentrations induced inward currents in voltage-clamped RhAG- and in RhCG-expressing oocytes. Taken together, these results show not only that RhAG and RhCG are ammonium transporters, but also that they are promoting the transmembrane transport of NH3 and of NH4+. Data from our laboratory and from other groups raise several questions that are discussed.

Regions flanking ori sequences affect the replication efficiency of the mitochondrial genome of ori+ petite mutants from yeast.

The mitochondrial genomes of progenies from 26 crosses between 17 cytoplasmic, spontaneous, suppressive, ori+ petite mutants of Saccharomyces cerevisiae have been studied by electrophoresis of restriction fragments. Only parental genomes (or occasionally, genomes derived from them by secondary excisions) were found in the progenies of the almost 500 diploids investigated; no evidence for illegitimate, site-specific mitochondrial recombination was detected. One of the parental genomes was always found to be predominate over the other one, although to different extents in different crosses. This predominance appears to be due to a higher replication efficiency, which is correlated with a greater density of ori sequences on the mitochondrial genome (and with a shorter repeat unit size of the latter). Exceptions to the 'repeat-unit-size rule' were found, however, even when the parental mitochondrial genomes carried the same ori sequence. This indicates that noncoding, intergenic sequences outside ori sequences also play a role in modulating replication efficiency. Since in different petites such sequences differ in primary structure, size, and position relative to ori sequences, this modulation is likely to take place through an indirect effect on DNA and nucleoid structure.

The initiation of DNA replication in the mitochondrial genome of yeast.

We report here the first direct demonstration that the active ori sequences of the mitochondrial genome of Saccharomyces cerevisiae are indeed origins of DNA replication, as previously postulated on the basis of compelling but indirect evidence. Basically, such sequences are formed by four regions: (i) GC clusters A and B, which are separated by a 29-bp AT stretch; (ii) a central 200-bp AT stretch, l; (iii) GC cluster C; (iv) a 16-bp AT stretch r, which comprises a site for transcription initiation. The ori sequences investigated, ori 1 and ori 5, have opposite orientations on the parental wild-type genome; ori 1 has but ori 5 does not have an additional 14-bp AT stretch r', between cluster C and sequence r; they were carried by the genomes of two spontaneous petites. In both ori sequences, nascent DNA chains using as template the strand containing sequence r (the 'r strand') start at the r end of cluster C, are elongated towards sequence l, and follow an RNA primer starting at sequence r. Nascent DNA chains copied on the 'non-r strand' start within cluster C, are elongated towards sequence r, and follow an RNA primer starting in sequence l just before cluster C. Ori 1 and 5 are, therefore, used as sites for RNA-primed bidirectional replication of mitochondrial DNA. Several aspects of this process are discussed.

Comparative analysis by two-dimensional iodopeptide mapping of the RhD protein and LW glycoprotein.

The RhD polypeptide and LW glycoprotein were separately immunopurified with monoclonal antibodies and compared by two-dimensional (2-D) iodopeptide mapping after digestion with alpha-chymotrypsin. These proteins have distinct 2-D maps, as seen after 125I-labeling tyrosine residues (chloramine-T procedure), and even more strikingly after labeling primary amine residues (Bolton-Hunter procedure). Of the more than 20 iodopeptides visualized, only five migrated identically when preparations of RhD and LW were directly compared, suggesting that RhD and LW are different proteins that may share some common protein domains. N-glycanase treatment of the iodopeptides did not modify the 2-D map of the RhD protein but greatly affected the LW map, further indicating that LW, but not RhD, carries N-linked carbohydrate chains. After deglycosylation the LW map was different from the RhD map, confirming that the RhD and LW polypeptides are different proteins. These findings demonstrate that LW is neither a glycosylated form of Rh protein nor is Rh a precursor of LW.

Surface orientation and antigen properties of Rh and LW polypeptides of the human erythrocyte membrane.

Time course digestion of intact human erythrocytes and right side-out vesicles with carboxypeptidase Y altered the Rh polypeptides and removed the 125I label that is normally incorporated by cell-surface radioiodination, but did not affect the RhD, Rhc, or RhE antigens. Under the same conditions, however, the LW antigens were rapidly destroyed. Digestion of inside-out and right side-out vesicles with aminopeptidase M was without any detectable effect on the Rh and LW antigens or polypeptides, although glycophorin A was degraded from right side-out but not from inside-out vesicles. These findings demonstrate that the C-terminal domain of the Rh and LW polypeptides is exposed at the external surface of human erythrocytes and indicate, in addition, that the LW antigens and tyrosine residue(s) of the LW and Rh proteins, respectively, are located close to the C termini of these polypeptides. Further studies using monoclonal and polyclonal antibodies showed that LW antigen expression is inhibited by treatment of red cells with EDTA and is selectively restored by Mg2+, but not by Mn2+ or Ca2+, whereas the Rh antigens were not affected under these conditions. In addition, O- and N-glycanase digestion of the LW glycoprotein removed its sugar chains, but did not alter significantly the epitopes recognized by the monoclonal anti-LW antibody.

Chimpanzee Rh-like blood group genes map to chromosome region 1p36.1-->p34.2 by in situ hybridization.

A human Rh cDNA probe was used to map the Rh-like genes in the chimpanzee. The data gathered made it possible to uniquely localize these genes to chimpanzee chromosome region 1p36.1-->p34.2. This chromosomal localization is homologous to the location of the Rh genes in the human genome.

Characterization of the recombination hot spot involved in the genomic rearrangement leading to the hybrid D-CE-D gene in the D(VI) phenotype.

In the Caucasian population, the RH locus of RhD-positive individuals is composed of two homologous genes, RHD and RHCE, arranged in tandem but of a single gene, RHCE, in RhD-negative individuals. Many variants recently characterized carry rearranged RH genes, most often by an unidirectional segmental DNA-exchange (gene-conversion) event. In D(VI) variants of type II, RHD is a D-CE-D hybrid gene in which the DNA fragment carrying exons 4-6 has been replaced by the corresponding sequences from the RHCE gene. To identify precisely and characterize the two transition sites, we have studied, by both PCR and sequence analysis, a genomic region between the 3' end of intron 3 and exon 7 in normal RHCE and RHD genes as well as in D(VI) DNA. We show that the D-CE breakpoint is located in intron 3, within a 250-bp fragment comprising an Alu S sequence, and that the CE-D breakpoint lies within a 39-bp fragment in intron 6. This Alu S sequence (and the 100-bp region immediately downstream) most likely defines a recombination hot spot, since there lies also the 5' breakpoint of different rearrangement events leading to D-CE and CE-D transitions in hybrid D(VI),DFR and Dc-,R(N) gene complexes, respectively.

Molecular analysis of the structure and expression of the RH locus in individuals with D--, Dc-, and DCw- gene complexes.

Rh blood group antigens of the D, C/c, and E/e series are carried by at least three red cell membrane polypeptides encoded by two highly related genes, RHD and RHCE. Homozygous individuals carrying the D--, Dc-, and DCw- gene complexes are characterized by a total or partial lack of expression of the RHCE-encoded antigens. Analysis of the molecular genetic basis of these rare conditions indicates that complete or partial expression defect of Cc/Ee antigens result from different alterations at the RH locus, but not from gross deletions. No rearrangement or mutation of the RHCE gene could be detected in donors homozygous for the D-- complex, suggesting that the lack of the Cc and Ee antigens might result from a reduced transcriptional activity of the RHCE gene. The Dc- and DCw- gene complexes, however, exhibited an important rearrangement of the RHCE gene. Instead of the normal RHCE gene, both variants carried a hybrid RHCE-D-CE gene in which exons 4 to 9 (Dc- complex) and 2 (or 3) to 9 (DCw- complex) of the RHCE gene, respectively, have been substituted by the equivalent region of the RHD gene. These gene conversion events provide an explanation for the well-described abnormal antigen profiles associated with the Dc- and DCw- complexes, like the increased expression of RhD, the reduced expression of RhC/c or RhCw, and the absence of RhE/e.

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.

Organization of the human RH50A gene (RHAG) and evolution of base composition of the RH gene family.

Human Rh (rhesus) antigens are expressed in the red cell membrane as a multi-subunit complex, the central core of which is presumably composed of a tetramer made of two Rh and two Rh50 protein subunits. The interaction between Rh and Rh50 polypeptides is thought to be crucial to the correct assembly and transport of the complex to the cell surface. Here, we show that the human RH50A gene (RHAG) is composed of 10 exons whose size and exon/intron junctions are well conserved compared to those of the RH genes. We have also analyzed the RH50A 5' flanking region where the transcription initiation site has been identified. These results conclusively establish that the RH50A and RH genes do belong to the same gene family. Moreover, we show that the RH50A and RH genes are embedded in different compositional genomic contexts (i.e., different isochores) that are likely to drive the evolution of these genes, the base compositions (G + C content) of which differ drastically. Finally, we propose a scenario in which an RH50-like gene is likely to have played a founding role in the evolution of the RH gene family.

The members of the RH gene family (RH50 and RH30) followed different evolutionary pathways.

The evolution of the RH gene family is characterized by two major duplication events, the first one originating the RH50 and RH30 genes and the second one giving rise to RHCE and RHD, the two paralogous RH30 genes which encode the Rh blood group antigens in human. The new sequence data obtained here for mouse RH50 and RH30 and for macaque RH50 allowed us to compare the evolutionary rates of the two genes and to show that RH50 evolved about 2.6 times more slowly than RH30 at nonsynonymous positions. This result implies that Rh50 proteins were evolutionarily more conserved compared to Rh30 polypeptides, thus being indicative of the functional significance of the former protein in species as distantly related as sponge and human. The duplication event leading to RH50 and RH30 genes was estimated to have occurred between 250 and 346 million years ago. Moreover, we could also estimate that the duplication event producing the RHCE and RHD genes occurred some 8.5 +/- 3.4 million years ago, in the common ancestor of human, chimpanzee, and gorilla. Interestingly, this event seems to coincide with the appearance in these species of a G-to-T mutation in the RH50 gene which created a stop codon in the corresponding transcript. This led to an Rh50 C-terminal cytoplasmic domain shorter than that found in orangutan and early primates.

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.

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.

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.

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.

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).

Rh-deficiency of the regulator type caused by splicing mutations in the human RH50 gene.

The Rh polypeptides and the glycoproteins Rh50, CD47, LW, and glycophorin B, which interact in the red blood cell membrane to form a multisubunit complex, are lacking or are severely reduced in the Rh-deficiency syndrome. We previously reported that in several Rhnull patients the RH50 gene was altered at the coding sequence level, resulting in either a single amino acid substitution or the synthesis of a truncated polypeptide. In the present report, we have detected two mutations in the intronic region of the RH50 gene that identify a new molecular mechanism involved in Rh-deficiency. The first mutation affected the invariant G residue of the 3' acceptor splice-site of intron 6, causing the skipping of the downstream exon and the premature termination of translation. The second mutation occurred at the first base of the 5' donor splice-site of intron 1. Both these mutations were found in homozygote state. RNase protection assays demonstrated that the Rh50 mRNA level was strongly reduced or undetectable in the 3' and 5' splice mutants, respectively. The different mutations affecting the RH50 gene are indicative of an heterogeneous mutational pattern, which further supports the hypothesis that the lack of the Rh50 protein may prevent the assembly or transport of the Rh membrane complex to the red blood cell surface.