The constitutive activity of the ALK mutated at positions F1174 or R1275 impairs receptor trafficking.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK), which is transiently expressed during development of the central and peripheral nervous system. ALK has been recently identified as a major neuroblastoma predisposition gene and activating mutations have also been identified in a subset of sporadic neuroblastoma tumors. Two hot spots of ALK mutations have been observed at positions F1174 and R1275. Here, we studied stably transfected cell lines expressing wild-type or F1174L- or R1275Q-mutated ALK in parallel with a neuroblastoma cell line (CLB-GE) in which the allele mutated at position F1174 is amplified. We observed that the mutated ALK variants were essentially intracellular and were largely retained in the reticulum/Golgi compartments. This localization was corroborated by a defect of N-linked glycosylation. Although the mutated receptors exhibited a constitutive activation, the minor pool of receptor addressed to the plasma membrane was much more tyrosine phosphorylated than the intracellular pool. The use of antagonist monoclonal antibodies suggested that the constitutive activity of the mutated receptors did not require the dimerization of the receptor, whereas adequate dimerization triggered by agonist monoclonal antibodies increased this activity. Finally, kinase inactivation of the mutated receptors restored maturation and cell-surface localization. Our results show that constitutive activation of ALK results in its impaired maturation and intracellular retention. Furthermore, they provide a rationale for the potential use of kinase inhibitors and antibodies in ALK-dependent tumors.

Phylogeographical variation of chloroplast DNA in holm oak (Quercus ilex L.).

Variation in the lengths of restriction fragments (RFLPs) of the whole chloroplast DNA molecule was studied in 174 populations of Quercus ilex L. sampled over the entire distribution of this evergreen and mainly Mediterranean oak species. By using five endonucleases, 323 distinct fragments were obtained. From the 29 and 17 cpDNA changes identified as site and length mutations, respectively, 25 distinct chlorotypes were distinguished, mapped and treated cladistically with a parsimony analysis, using as an outgroup Q. alnifolia Poech, a closely related evergreen oak species endemic to Cyprus where Q. ilex does not grow. The predominant role of Q. ilex as maternal parent in hybridization with other species was reflected by the occurrence of a single very specific lineage of related chlorotypes, the most ancestral and recent ones being located in the southeastern and in the northwestern parts of the species' geographical distribution, respectively. The lineage was constituted of two clusters of chlorotypes observed in the 'ilex' morphotyped populations of the Balkan and Italian Peninsulas (including the contiguous French Riviera), respectively. A third cluster was divided into two subclusters identified in the 'rotundifolia' morphotyped populations of North Africa, and of Iberia and the adjacent French regions, respectively. Postglacial colonization probably started from three distinct southerly refugia located in each of the three European peninsulas, and a contact area between the Italian and the Iberian migration routes was identified in the Rhône valley (France). Chlorotypes identical or related to those of the Iberian cluster were identified in the populations from Catalonia and the French Languedoc region, which showed intermediate morphotypes, and in the French Atlantic populations which possessed the 'ilex' morphotype, suggesting the occurrence of adaptive morphological changes in the northern part of the species' distribution.

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.

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.

Shift from Rh-positive to Rh-negative phenotype caused by a somatic mutation within the RHD gene in a patient with chronic myelocytic leukaemia.

We report a female patient whose Rh phenotype shifted from RhD-positive to RhD-negative over a 3-year period (1991-94), during which time she was treated with mastectomy (1992) and local irradiation for a low-grade recurrent breast cancer. She was diagnosed with chronic myeloid leukaemia in 1994, and has since then received chemotherapy. The patient was repeatedly typed as O, RhD-positive between 1965 and 1991 and was repeatedly found RhD-negative after 1994. Bcr-Abl transcripts typical of Ph1 chromosome were detected. Molecular analysis indicated that the patient was heterozygous at the RH locus, carrying one haplotype in which the RHD gene exhibited a single nucleotide deletion (G600) resulting in a frameshift and premature stop codon, and a normal RHCE gene (allele Ce). The second haplotype contained only the RHCE gene (allele ce) and was normal. Further analysis carried out on total leucocytes, purified neutrophils, EBV-lymphoblastoid cell line and cultured erythroblasts indicated that the G600 deletion was restricted to the myeloid lineage. No modification of other blood group antigens could be detected. These findings suggest a somatic mutation which most probably occurred in a stem cell common to the myeloid lineage.

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.

Molecular defects of the RHCE gene in Rh-deficient individuals of the amorph type.

The deficiency of Rh proteins on the red blood cells from individuals of the Rhnull amorph type may be the result of homozygosity for a silent allele at the RH locus. This phenotype is also associated with the lack or reduced expression of glycoproteins (Rh50, CD47, LW, and glycophorin B), which interact with Rh polypeptides to form the multisubunit Rh membrane complex. In this study, we describe two molecular alterations affecting the RHCE gene in two unrelated Rhnull amorph individuals bearing Rh50 and CD47 normal transcripts. The first type of mutation, located at the donor splice-site in intron 4, induced the activation of two cryptic splice-sites within this intron and one such site in exon 4 that all generated aberrant transcripts. The second type of mutation affected the coding region and introduced a frameshift and a premature stop codon resulting in a shorter predicted protein (398 v 417 residues), including a completely different C-terminus of 76 amino acids. This suggests that protein folding and/or protein-protein interaction mediated by the C-terminal domain of the Rh proteins may play a role in the routing and/or stability of the Rh membrane complex.

A novel single missense mutation identified along the RH50 gene in a composite heterozygous Rhnull blood donor of the regulator type.

Rare individuals who lack all of the Rh blood group antigens are called Rhnull and may be classified as "regulator" or "amorph" types. The suppression of Rh antigen expression for regulator types may be attributed to mutations of the RH50 gene, which is independent of the RH locus. The RH50 gene encodes a glycoprotein that interacts with the Rh proteins to form a functional complex within the red blood cell membrane. This report describes an RH50 gene mutation for a previously unclassified Rhnull donor. Sequencing cDNA clones from Rh50 mRNA revealed a single base change (G836A) yielding a missense and nonconservative mutation (Gly279Glu) within a predicted hydrophobic domain for this membrane protein. Genomic DNA studies using polymerase chain reaction (PCR) restriction analysis and sequencing showed that the Rhnull propositus was a composite heterozygote for this mutation, carrying two alleles with the A and G at nucleotide 836, respectively. In contrast, cDNA studies showed that only the A836 sequence was present, suggesting that the second allele with G836 was apparently silent (no transcript detected). Family studies showed that the mutant RH50 allele (836A) was inherited maternally, whereas the silent RH50 allele (836G) was from paternal transmission. These findings provide further evidence that rare but diverse genetic alterations may occur along the RH50 gene where the Rhnull syndrome of the regulator type occurs. The single amino acid change (Gly to Glu) provides insight into the critical value of these residues for assembly of the Rh antigen complex within the membrane.

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.

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.

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.

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.

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.

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.