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The human TUPLE1 gene encodes a putative transcriptional regulator and maps to chromosome 22, and therefore may play a role in Di-George syndrome (DGS), relo-cardio-facial syndrome (VCFS), or a related pathology. The murine TUPLE1 gene has also been cloned and shows strong sequence similarity to TUPLE1. Comparative mapping is useful in the study of chromosome evolution and is sometimes able to indicate possible mouse mutations that are potential models of human genetic disorders. As TIPLE1 is a candidate gene for the haploinsufficient phenotype in DGS, we mapped TUPLE1 to mouse chromosome 16A-B1. 6 refs., 1 fig.

Members of the Rho GTPase family regulate the organization of the actin cytoskeleton in response to extracellular growth factors. We have identified three proteins that form a distinct branch of the Rho family: Rnd1, expressed mostly in brain and liver; Rnd2, highly expressed in testis; and Rnd3/RhoE, showing a ubiquitous low expression. At the subcellular level, Rnd1 is concentrated at adherens junctions both in confluent fibroblasts and in epithelial cells. Rnd1 has a low affinity for GDP and spontaneously exchanges nucleotide rapidly in a physiological buffer. Furthermore, Rnd1 lacks intrinsic GTPase activity suggesting that in vivo, it might be constitutively in a GTP-bound form. Expression of Rnd1 or Rnd3/RhoE in fibroblasts inhibits the formation of actin stress fibers, membrane ruffles, and integrin-based focal adhesions and induces loss of cell–substrate adhesion leading to cell rounding (hence Rnd for “round”). We suggest that these proteins control rearrangements of the actin cytoskeleton and changes in cell adhesion.

DC-LAMP, a member of the lysosomal-associated membrane protein (LAMP) family, is specifically expressed by human dendritic cells (DC) upon activation and therefore serves as marker of human DC maturation. DC-LAMP is detected first in activated human DC within MHC class II molecules-containing compartments just before the translocation of MHC class II-peptide complexes to the cell surface, suggesting a possible involvement in this process. The present study describes the cloning and characterization of mouse DC-LAMP, whose predicted protein sequence is over 50% identical to the human counterpart. The mouse DC-LAMP gene spans over 25 kb and shares syntenic chromosomal localization (16B2-B4 and 3q26) and conserved organization with the human DC-LAMP gene. Analysis of mouse DC-LAMP mRNA and protein revealed the expression in lung peripheral cells, but also its unexpected absence from mouse lymphoid organs and from mouse DC activated either in vitro or in vivo. In conclusion, mouse DC-LAMP is not a marker of mature mouse DC and this observation raises new questions regarding the role of human DC-LAMP in human DC.

Human (h)Langerin/CD207 is a C-type lectin of Langerhans cells (LC) that induces the formation of Birbeck granules (BG). In this study, we have cloned a cDNA-encoding mouse (m)Langerin. The predicted protein is 66% homologous to hLangerin with conservation of its particular features. The organization of human and mouse Langerin genes are similar, consisting of six exons, three of which encode the carbohydrate recognition domain. The mLangerin gene maps to chromosome 6D, syntenic to the human gene on chromosome 2p13. mLangerin protein, detected by a mAb as a 48-kDa species, is abundant in epidermal LC in situ and is down-regulated upon culture. A subset of cells also expresses mLangerin in bone marrow cultures supplemented with TGF-β. Notably, dendritic cells in thymic medulla are mLangerin-positive. By contrast, only scattered cells express mLangerin in lymph nodes and spleen. mLangerin mRNA is also detected in some nonlymphoid tissues (e.g., lung, liver, and heart). Similarly to hLangerin, a network of BG form upon transfection of mLangerin cDNA into fibroblasts. Interestingly, substitution of a conserved residue (Phe244 to Leu) within the carbohydrate recognition domain transforms the BG in transfectant cells into structures resembling cored tubules, previously described in mouse LC. Our findings should facilitate further characterization of mouse LC, and provide insight into a plasticity of dendritic cell organelles which may have important functional consequences.

By systematic analysis of a human testis library, we have isolated the hH-Rev107-3 cDNA, identical to hH-Rev107-1 cDNA, which was previously described as a class II tumor suppressor gene (Husmann et al., 1998). In this study, two transcripts (1 and 0,8 kb) were detected by Northern blot in all human tissues, excepted in thymus. The strongest expression was found in testis, skeletal muscle and heart. These two mRNA are probably transcribed from only one gene that we mapped to the q12–q13 region of the chromosome 11. In human testis, hH-Rev107 gene expression was localized, by in situ hybridization, within the round spermatids. To investigate a possible role for hH-Rev107 protein in testicular malignant growth, we examined the expression of this gene in germ cell tumors. A strong hH-Rev107 gene expression was observed in normal testis as well as in samples with preinvasive carcinoma in situ but was completely absent in overt tumors, both seminomas and non-seminomas. By in situ hybridization, CIS was found hH-Rev107 positive and tumor negative. A semi-quantitative assessment of hH-Rev107 mRNA level in testicular germ cell tumors, by RT-PCR, exhibited a 9-fold decrease in the gene expression. No gross structural aberrations of hH-Rev107 gene were detected in these human primary tumors. The results suggest that down-regulation of hH-Rev107 may be associated with invasive progression of testicular germ cell tumors.

Editor—Mental retardation is a component of a large number of syndromes, most of which qualify individually as rare genetic diseases. Altogether, mental retardation affects 2-3% of the population and is unexplained in 40% of cases. According to Knight et al ,1 subtle telomeric chromosomal rearrangements are responsible for approximately 1% of unexplained mental retardation (with the proportion being highest, 7.4%, in the subclass of unexplained moderate to severe mental retardation). The identification of the genes responsible will require the precise delimitation of minimum deletion regions, which relies upon the collection of a large number of cases. Because of the low expected frequency of each telomeric deletion in mental retardation, the procedure to be applied should allow the screening of many patients at a low cost. It is not yet clear whether all chromosome ends are associated with mental retardation syndromes with similar frequency. Distal chromosome 1p36 deletions were initially detected cytogenetically because of an associated segmental imbalance.2-5 This syndrome results from both interstitial and terminal deletions of varying sizes and different breakpoints6 and the severity of the phenotype is related to the extent of the deletion.7 Clinical examination can efficiently detect a large proportion of cases, so that the number of reports of 1p36 deletions has increased significantly in the past few years which may give the false impression that this is a relatively frequent syndrome. We show here how highly polymorphic minisatellites located within a short region can provide an efficient prescreening of samples without the need for parental samples at the initial stage. The procedure was tested here using 1p36 minisatellites and provides an estimate of the frequency of 1p36 deletions in mentally retarded patients. A selection of five highly polymorphic minisatellite probes was used to search for 1p36.3 deletions in a …

Mutations in the XNP/ATR-X gene, located in Xq13.3, are associated with several X linked mental retardation syndromes, the best known being α thalassaemia with mental retardation (ATR-X). The XNP/ATR-X protein belongs to the family of SWI/SNF DNA helicases and contains three C2-C2 type zinc fingers of unknown function. Previous studies have shown that 65% of mutations of XNP have been found within the zinc finger domain (encoded by exons 7, 8, and the beginning of exon 9) while 35% of the mutations have been found in the helicase domain extending over 3 kb at the C-terminus of the protein. Although different types of mutations have been identified, no specific genotype-phenotype correlation has been found, suggesting that gene alteration leads to a loss of function irrespective of mutation type. Our aims were to understand the function of the XNP/ATR-X protein better, with specific attention to the functional consequences of mutations to the zinc finger domain. We used monoclonal antibodies directed against the XNP/ATR-X protein and performed immunocytochemical and western blot analyses, which showed altered or absent XNP/ATR-X expression in cells of affected patients. In addition, we used in vitro experiments to show that the zinc finger domain can mediate double stranded DNA binding and found that the DNA binding capacity of mutant forms in ATR-X patients is severely reduced. These data provide insights into the understanding of the functional significance of XNP/ATR-X mutations. Keywords: XNP/ATR-X; mutation; zinc finger; DNA binding activity

Lhx9 is a LIM-homeodomain (LIM-hd) transcription factor expressed in the embryonic mouse brain. We report the isolation of Lhx9alpha, a cDNA encoding a truncated isoform of Lhx9 that lacks the recognition helix of the homeodomain and differs from Lhx9 cDNA in its 3'-coding and 3'-UTR sequences. Isolation of the Lhx9 gene showed that Lhx9 and Lhx9alpha are coded by six exons spanning 10 kb of genomic sequence and that Lhx9alpha is an isoform generated by alternative splicing of the fifth exon. Lhx9 was mapped to the subtelomeric region of chromosome 1. Further molecular analysis showed that Lhx9 is a new candidate gene for the unidentified dreher (dr) mutation in mouse. The comparison of genomic structure and molecular phylogenetic analysis led to the identification of six groups of LIM-hd proteins, a basis for further classification and knowledge of their evolutionary relationships. To investigate a possible role for Lhx9alpha, the expression patterns of Lhx9 and Lhx9alpha were compared during embryogenesis. Lhx9alpha was expressed at lower levels than Lhx9, with a similar but distinct pattern in the brain, especially in the neocortex. We suggest that Lhx9alpha could function as an endogenous dominant-negative form of Lhx9 during development, both to regulate in space and time the transcriptional effects of Lhx9 and to add a degree of refinement to the LIM-hd code.

V(D)J recombination in differentiating lymphocytes is a highly regulated process in terms of both cell lineage and the stage of cell development. Transgenic and knockout mouse studies have demonstrated that transcriptional enhancers from antigen receptor genes play an important role in this regulation by activating cis-recombination events. A striking example is the T-cell receptor beta-chain (TCRbeta) gene enhancer (Ebeta), which in the mouse consists of at least seven nuclear factor binding motifs (betaE1 to betaE7). Here, using a well-characterized transgenic recombination substrate approach, we define the sequences within Ebeta required for recombination enhancer activity. The Ebeta core is comprised of a limited set of motifs (betaE3 and betaE4) and an additional previously uncharacterized 20-bp sequence 3' of the betaE4 motif. This core element confers cell lineage- and stage-specific recombination within the transgenic substrates, although it cannot bypass the suppressive effects resulting from transgene integration in heterochromatic centromeres. Strikingly, the core enhancer is heavily occupied by nuclear factors in immature thymocytes, as shown by in vivo footprinting analyses. A larger enhancer fragment including the betaE1 through betaE4 motifs but not the 3' sequences, although active in inducing germ line transcription within the transgenic array, did not retain the Ebeta recombinational activity. Our results emphasize the multifunctionality of the TCRbeta enhancer and shed some light on the molecular mechanisms by which transcriptional enhancers and associated nuclear factors may impact on cis recombination, gene expression, and lymphoid cell differentiation.

We describe the characterization of sialic acid-binding Ig-like lectin-7 (siglec-7), a novel member of the siglec subgroup of the immunoglobulin superfamily. A full-length cDNA encoding siglec-7 was isolated from a human primary dendritic cell cDNA library. Siglec-7 is predicted to contain three extracellular immunoglobulin-like domains that comprise an N-terminal V-set domain and two C2-set domains, a transmembrane region and a cytoplasmic tail containing two tyrosine residues embodied in immunoreceptor tyrosine-based inhibition motif-like motifs. Overall, siglec-7 exhibited a high degree of sequence similarity to genes encoding CD33 (siglec-3), siglec-5, OBBP1/siglec-6, and OBBP-like protein and mapped to the same region on chromosome 19q13.3. When siglec-7 was expressed on COS or Chinese hamster ovary cells, it was able to mediate high levels of sialic acid-dependent binding to human erythrocytes and soluble sialoglycoconjugates, suggesting that it may be involved in cell-cell interactions. Among human peripheral blood leukocytes, siglec-7 was found to be present at low levels on granulocytes, intermediate levels on monocytes, and relatively high levels on a major subset of natural killer cells and a minor subset of CD8(+) T cells. Immunoprecipitation experiments indicated that siglec-7 is expressed as a monomer of approximately 65 kDa.

CBL genes encode cytoplasmic proteins involved in signal transduction downstream of a number of receptors including tyrosine kinases, cytokine receptors, and T-cell or B-cell receptors. They seem to be transducers associated with negative regulation of signals, and, as such, may be potential tumor suppressors. Using a probe derived from an expressed sequence tag, we isolated a cosmid containing part of a new CBL gene, CBLc, related to the two characterized paralogous genes CBLa and CBLb. Using the cosmid in fluorescence in situ hybridization of human metaphase chromosomes, we localized the CBLc gene to band 13.2 of chromosome 19. We show that the 19q12.2-13.3 region where CBLc is located shows paralogy with two other regions of the human genome, 3q22-q27 and 11q22-q24 where CBLb and CBLa are located, respectively. Genes from several other families are located in these regions.

International audience; Hepatitis B virus (HBV) DNA is found chromosomally integrated into the genome of the majority of hepatocellular carcinomas (HCC) arising in chronic HBV carriers suggesting that, in some instances, viral sequences may be directly responsible for oncogenic conversion. In an attempt to clarify the oncogenic potential of integrated HBV sequences, we performed an extensive analysis of two single integrations present in HCC which developed in non-cirrhotic livers from HBsAg-positive Korean patients. In both cases, integrated viral sequences were characterized by a duplicated-inverted configuration involving the flanking cellular sequences, a pattern consistently found in many amplicons isolated from mammalian cells. Integration sites are characterized by an AT-rich content and the presence of topoisomerase I and II cleavage target sequences as well as other recombination-prone motifs. The chromosomal locations of the integration sites were determined as 8q13 and 10q22 in the human genome, two regions known to harbour genes involved in tumorigenesis. The cis-activating potential of the integrations in their original configuration was also investigated in a transient transfection assay in HepG2 cells. Integrated sequences, rather than activating heterologous promoters, show either no activity or a weak tendency to inhibit activation of neighbouring reporter genes. The implications of our findings for the understanding of primary liver cancer development are discussed.

Prader-Willi syndrome (PWS) is a neurogenetic disorder that results from the absence of a normal paternal contribution to the 15q11–13 region1–3. The clinical manifestations of PWS are a transient severe hypotonia in the newborn period, with mental retardation, hypogonadism and obesity observed later in development4. Five transcripts with exclusive expression from the paternal allele have been isolated, but none of these has been shown to be involved in PWS5,6. In this study, we report the isolation and characterization of NDN, a new human imprinted gene. NDN is exclusively expressed from the paternal allele in the tissues analysed and is located in the PWS region. It encodes a putative protein homologous to the mouse brain-specific NECDIN protein7, NDN; as in mouse, expression in brain is restricted to post-mitotic neurons. NDN displays several characteristics of an imprinted locus, including allelic DNA methylation and asynchronous DNA replication. A complete lack of NDN expression in PWS brain and f ibroblasts indicates that the gene is expressed exclusively from the paternal allele in these tissues and suggests a possible role of this new gene in PWS.

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Poly(ADP-ribose) polymerase (PARP) has been suggested to play a regulatory role in vivo, in DNA replication and/or DNA repair based mainly on its capacity to bind to DNA strand breaks. This interaction is modulated through auto poly(ADP-ribosylation). However, the biological function of PARP may also involve interactions with proteins such as topoisomerase I or DNA polymerase alpha, which may or may not be themselves ADP-ribosylated. Using the yeast two-hybrid method search for other proteins interacting with PARP, we have isolated a full-length cDNA clone coding for a protein of 158 amino acid residues. This amino acid sequence is 66 and 56% identical to yeast ubiquitin-conjugating enzymes Hus5 and Ubc9 of Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. Moreover, we have demonstrated that the expressed protein complements a S. cerevisiae yeast strain deficient for Ubc9. The protein encoded by the isolated cDNA is thus a new human counterpart of the ubiquitin-conjugating enzyme family and has been called hUbc9. The hubc9 gene locus has been assigned to the chromosomal location 16p13.2-p13.3. By means of two-hybrid analysis it was discovered that hUbc9 interacts with the automodification domain of PARP. This interaction was further confirmed using GST (glutathione-S-transferase) tagged fusion proteins: (i) in vivo, by transfecting cos7 cells with hUbc9 cloned in an eukaryotic expression vector, and (ii) in vitro, by mixing purified PARP with hUbc9 purified and expressed in bacteria. The possible significance and function of this interaction is discussed while taking into account the possible intracellular role of hUbc9.

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Using a rat histidine decarboxylase (HDC) cDNA probe, we have mapped the HDC gene by in situ hybridization to the ql5–q2l region of human chromosom e15 and to the E5-G region of murine chromosome 2. These localizations strengthen a syntenic group conserved between human chromosome 15 and mouse chromosome 2. The localization of the HDC gene on the human chromosome 15 map shows that it is not included within the Prader-Willi Syndrome region (PWCR).

International audience; A eDNA clone (HUT2) sharing 61.1% and 89.9% sequence identity with the human erythroid (HUTll) and the rabbit (UT2) urea transporters, respectively, was isolated by homology cloning from a human kidney library. HUT2 transcripts were restricted to the kidney and the HUT2 polypeptide was not immunoprecipitated with blood group Kidd-related antibodies (anti-Jk3) in coupled transcription-translation assays. Functional expression studies in Xenopus oocytes demonstrated that HUT2-mediated urea transport was not inhibited by p-chloromercuribenzene sulfonate (pCMBS) which, however, inhibited the urea flux mediated by HUTll. These findings demonstrate that at least two distinct urea transporters are present in human tissues. By in situ hybridiza-tion, the gene encoding HUT2 has been assigned to chromosome 18q12.l-q21-1, as found previously for the Kidd/urea transporter HUT11, suggesting that both genes evolved from duplication of a common ancestor.

Using the mouse delta-opioid receptor cDNA as a probe, we have isolated genomic clones encoding the human mu- and kappa-opioid receptor genes. Their organization appears similar to that of the human delta receptor gene, with exon-intron boundaries located after putative transmembrane domains 1 and 4. The kappa gene was mapped at position q11-12 in human chromosome 8. A full-length cDNA encoding the human kappa-opioid receptor has been isolated. The cloned receptor expressed in COS cells presents a typical kappa 1 pharmacological profile and is negatively coupled to adenylate cyclase. The expression of kappa-opioid receptor mRNA in human brain, as estimated by reverse transcription-polymerase chain reaction, is consistent with the involvement of kappa-opioid receptors in pain perception, neuroendocrine physiology, affective behavior, and cognition. In situ hybridization studies performed on human fetal spinal cord demonstrate the presence of the transcript specifically in lamina II of the dorsal horn. Some divergences in structural, pharmacological, and anatomical properties are noted between the cloned human and rodent receptors.

The gene encoding the urea transporter of human erythrocytes (HUT11 clone) has been cloned recently (Olives, B., Neau, P., Bailly, P., Hediger, M. A., Rousselet, G., Cartron, J. P., and Ripoche, P. (1994) J. Biol. Chem. 269, 31649-31652). Now, this gene has been assigned to chromosome 18q12-q21 by in situ hybridization, as also found for the Kidd (Jk) blood group locus. In coupled transcription-translation assays, the HUT11 cDNA directed the synthesis of a 36-kDa protein which was immunoprecipitated by a human anti-Jk3 antibody produced by immunized Jk(a-b-) donors whose red cells lack Kidd antigens. The anti-Jk3 antibody also immunoprecipitated a protein material of 46-60 kDa from all red cell membranes, except those from Jk(a-b-) cells. After N-glycanase digestion the 46-60-kDa component was reduced to 36 kDa. A rabbit antibody raised against the predicted NH2-terminal amino-acids of the HUT11 protein reacted on immunoblots with a 46-60-kDa component present in all human erythrocytes except those from Jk(a-b-) individuals. Jk(a-b-) red cells lack the Kidd/urea transport protein and have a selective defect of the urea transport capacity, but a normal water permeability and aquaporin-associated Colton blood group antigens. These findings indicate that the erythrocyte urea transporter is encoded by the Kidd locus and may have implications for the biology of urea transporters and their tissue-specific regulation.

LIM-homeodomain proteins are important in cell lineage specification and possibly mediate transcriptional processes in eukaryotes. During the screening of a mouse pituitary cDNA library, we isolated a partial cDNA coding for a novel gene product that exhibited a predicted amino-terminal sequence similar to the homeobox of LIM-homeodomain-containing proteins. Reverse transcriptase-polymerase chain reactions (RT-PCR) performed on mouse pituitary mRNA using degenerate oligonucleotides based on the conserved LIM-domain sequences, allowed the extension of the 5' end of the sequence. The composite 2.2-kb cDNA structure predicts a 400-amino-acid-long novel mouse (m) protein, called mLIM-3. This name was chosen since within the 59-amino-acid homeodomain, it exhibits 97% sequence identity to a recently reported Xenopus homologue xLIM-3. The gene coding for mLIM-3 maps to the murine chromosome 2, most probably within the 2B band. Based on sequence characteristics, we suggest that LIM-3 belongs to a distinct subfamily of LIM-containing homeoproteins. Ontogeny studies using in situ hybridization demonstrated that mLIM-3 transcripts can be detected on embryonic day 11 (e11) in the primordium of the hypophysis. Following a maximum between e12 and e14, lower levels persisted into adulthood, where mLIM-3 was expressed primarily in the anterior and intermediate lobes of the pituitary. These results were confirmed by Northern blot analysis in adult mice which revealed a 2.4-kb pituitary mRNA transcript. mLIM-3 transcripts were also detected in pituitary cell lines such as the somatotrophs GH3 and GH4C1, the gonadotroph alpha T3-1, and the corticotroph AtT-20 cells, but not in 20 other cell lines derived from peripheral, endocrine, and neural tissues. Starting from e11, we also observed a transient expression of mLIM-3 in the ventral part of the spinal cord, pons, and medulla oblongata, reaching a maximum at e13 and from p7 onward, the expression of this transcript is no longer detectable. mLIM-3 is also expressed in the pineal gland with high levels observed at e20. These data suggest a potential role for mLIM-3 in the transcriptional regulation of certain genes during morphogenesis and/or maintenance of the differentiated state of the pituitary, motor neurons, and pineal gland.

In situ hybridization experiments were performed using complementary DNA probes to determine the location on murine chromosomes of the three genes of the ryanodine receptor/Ca2+ channel family: RYR1, RYR2, and RYR3. The skeletal ryanodine receptor, or type 1 (Ryr1), maps to bands 7A2-7A3; the cardiac ryanodine receptor, or type 2 (Ryr2), to 13A1-13A2; and type 3 (Ryr3) to 2E5-2F3.

International audience; Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is a multifunctional protein containing two enzymes that act sequentially to catalyze the alpha-amidation of neuroendocrine peptides. Southern blot analysis of human placental DNA demonstrated that PAM is encoded by a single gene. The chromosomal localization of the PAM gene was established using in situ hybridization. A 2.2-kb human PAM cDNA hybridized to human metaphase chromosomes revealed a significant clustering of silver grains over chromosome 5 bands q14-q21. The gene encoding another enzyme important in the post-translational processing of neuroendocrine precursors, prohormone convertase 1 (PC1), is localized in the same region (5q15-q21).

Genomic clones containing the gene for the glutathione peroxidase-like androgen-regulated murine epididymal protein of 24 kilodaltons (arMEP24) were isolated. A 9-kilobase DNA fragment was sequenced and found to contain the entire coding region of the gene, which is divided into five exons. The exact sizes and boundaries of the exon blocks were deduced by comparison with the cDNA sequence. One major and four weak transcription initiation sites in the epididymis were localized by primer extension. The promoter of the gene does not contain a conventional TATA box immediately up-stream of the start site; rather, the sequence TATCA occurs at residue -35. Two CAAT boxes in opposite orientation and two putative binding sites for the transcription factor Sp1 were identified up-stream of the TATA-like box. To localize the cis-acting sequences responsible for androgen regulation of expression, fragments of the arMEP24 gene promoter region were cloned in front of the luciferase (LUC) reporter gene and cotransfected with an androgen receptor expression vector into CV-1 cells in a transient assay. LUC activities of CV-1 cells grown in the presence of various concentrations of 5 alpha-dihydrotestosterone were compared to LUC activities of untreated controls. The DNA fragment containing up to 200 nucleotides up-stream from the major transcription start site was sufficient for the full promoter activity, but not for the responsiveness to androgen induction. Depending on the 5 alpha-dihydrotestosterone concentration, a 2- to 4-fold induction of LUC activity was found if a -1797 to -167 arMEP24 gene fragment was used linked to the reporter gene driven by either the homologous promoter or the heterologous thymidine kinase promoter. Two or three copies of the imperfect palindromic sequence TGTTGAgagAGAACA, found at position -896 to -882 in the gene and resembling the consensus steroid hormone-responsive element, are able to confer androgen regulation to the thymidine kinase promoter independently of their orientation. These findings support evidence that transcriptional regulation of the arMEP24 gene occurs via the sequence TGTTGAgagAGAACA. Homologies found in the sequence up-stream of the promoter with several putative binding sites for erythroid-specific trans-acting regulatory proteins are discussed. Finally, the arMEP24 gene is located by in situ hybridization in the [A2-A4] region of mouse chromosome 13.

We have characterized cDNA clones that encode a newly discovered collagenous polypeptide. A 4-kilobase (kb) cDNA clone was initially isolated by screening a human fibroblast cDNA library with a probe encoding the collagenous domain of the human alpha 3(VI) collagen. Subsequent screening of another fibroblast cDNA library yielded overlapping clones having a total length of 5.4 kb, which contained an open reading frame of 1603 amino acids including a 21-amino acid signal peptide. The predicted polypeptide consists of 10 collagenous domains 15-422 amino acids long, which were interspersed with 11 noncollagenous (NC) domains. Except for a large N-terminal NC11 domain of 312 residues, most of the NC domains were short (11-39 residues) and cysteine-rich. The overall structural arrangement differed significantly from other known collagen chains. Further analysis indicated that the deduced polypeptide exhibited several structural features characteristically seen in members of the fibril-associated collagen, types IX, XII, and XIV. In addition, the cysteine-rich motifs in the NC domains resembled those found in the cuticle collagen of Caenorhabditis elegans. Northern blot analyses showed hybridization of the cDNA to a 5.5-kb mRNA in human fibroblasts and keratinocytes. The gene was localized by in situ hybridization to band p34-35 of human chromosome 1. The data clearly support the conclusion that the cDNA encodes a collagen chain that has not been previously described. We suggest that the cDNA clones encode the alpha 1 chain of type XVI collagen.

Interstitial deletion of the short arm of chromosome 17 was detected in three patients. They all had a similar phenotype with mental retardation, behavioural problems, facial dysmorphism, brachycephaly, a broad face with a flat midface, and short and broad hands. All three cases were ascertained over a six month period by two neuropaediatricians aware of this specific anomaly, which suggests that this microdeletion is not particularly rare. Comparison of the clinical and cytogenetic findings in a total of 24 patients allows a new contiguous gene syndrome to be defined that only high resolution analysis can detect. In two cases, molecular analysis confirmed the cytogenetic results. The Charcot-Marie-Tooth type Ia gene has recently been localised to the 17p11.2 sub-band.

International audience; The human T cell receptor gamma locus (TCRG) has previously been localized on chromosome 7 at band 7p15. In situ hybridization of a TCRG-specific probe allowed us to map the locus at 7p14----p15. These data confirm the previous localization and are in agreement with the molecular characterization of an inversion of chromosome 7, inv(7) (p14q35) which involves the TCRG locus.

cDNA clones containing sequences coding for the murine neural cell adhesion molecule (N-CAM) were used in Southern hybridizations on human genomic DNA and demonstrated approximately 90% homology between human and murine NCAM genes. In situ hybridization with one of these clones was performed on human metaphase chromosomes and allowed the localization of the human NCAM gene to band q23 of chromosome 11. The genes for two other cell surface molecules believed to be involved in cell-cell interactions, Thy-1 and the delta chain of the T3-T cell receptor complex, have recently been localized to the same region of chromosome 11 in man. Moreover, this region of the human chromosome 11 appears to be syntenic to a region of murine chromosome 9 that also contains the staggerer locus: staggerer mice show abnormal neurological features which may be related to abnormalities in the conversion of the embryonic to the adult forms of the N-CAM molecule.

Amiloride-sensitive Na+ channels belonging to the recently discovered NaC/DEG family of genes have been found in several human tissues including epithelia and central and peripheral neurons. We describe here the molecular cloning of a cDNA encoding a novel human amiloride-sensitive Na+ channel subunit that is principally expressed in the small intestine and has been called hINaC ( h uman i ntestine Na + c hannel). This protein is similar to the recently identified rodent channel BLINaC and is relatively close to the acid sensing ion channels (ASICs) (79 and 29% amino acid identity, respectively). ASICs are activated by extracellular protons and probably participate in sensory neurons to nociception linked to tissue acidosis. hINaC is not activated by lowering the external pH but gain-of-function mutations can be introduced and reveal when expressed in Xenopus oocytes, an important Na+ channel activity which is blocked by amiloride (IC50=0.5 μM). These results suggest the existence of a still unknown physiological activator for hINaC (e.g. an extracellular ligand). The presence of this new amiloride-sensitive Na+ channel in human small intestine probably has interesting physiological as well as physiopathological implications that remain to be clarified. The large activation of this channel by point mutations may be associated with a degenerin-like behavior as previously observed for channels expressed in nematode mechanosensitive neurons. The hINaC gene has been mapped on the 4q31.3–q32 region of the human genome.

International audience; The human hap retinoic acid receptor RAR beta has been localized by in situ hybridization to the p24 band of chromosome 3.

A 1.8 kb human cDNA probe for angiotensinogen (renin substrate) was used to determine the chromosomal location of the angiotensinogen gene by in situ hybridization. The results show that human chromosome region 1q4 contains the angiotensinogen gene. The human renin gene has also recently been assigned to the same band of chromosome 1. Thus, the angiotensinogen and renin genes are located in the same region of chromosome 1.

We describe the isolation and characterization of cDNA clones encoding human leukosialin, a major sialoglycoprotein of human leukocytes. Leukosialin is very closely related or identical to the sialophorin molecule, which is involved in T-cell proliferation and whose expression is altered in Wiskott-Aldrich syndrome (WAS), an X chromosome-linked immunodeficiency disease. Using a rabbit anti-serum to leukosialin, a cDNA clone was isolated from a lambda gt11 cDNA library constructed from human peripheral blood cells. This lambda gt11 clone was used to isolate longer cDNA clones that correspond to the entire coding sequence of leukosialin. DNA sequence analysis reveals three domains in the predicted mature protein. The extracellular domain is enriched for Ser, Thr, and Pro and contains four contiguous 18-amino acid repeats. The transmembrane and intracellular domains of the human leukosialin molecule are highly homologous to the rat W3/13 molecule. RNA gel blot analysis reveals two polyadenylylated species of 2.3 and 8 kilobases. Southern blot analysis suggests that human leukosialin is a single-copy gene. Analysis of monochromosomal cell hybrids indicates that the leukosialin gene is not X chromosome linked and in situ hybridization shows leukosialin is located on chromosome 16. These findings demonstrate that the primary mutation in WAS is not a defect in the structural gene for leukosialin.