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The HED (hidrotic ectodermal dysplasia) or Clouston syndrome gene (named ED2) has been mapped to the pericentromeric region of chromosome 13 (13q11) to a 2.4-cM interval flanked by markers D13S1828 and D13S1830. We have developed a BAC/PAC-based contig map of this region. This contig, comprising 23 clones and spanning 1.5 Mb, was established by mapping of 27 BAC/PAC end-derived STSs, 11 known polymorphic markers, 2 previously mapped genes, and 14 ESTs. The genomic clone overlaps were confirmed by restriction fragment fingerprint analysis. This contig provides the basis for genomic sequencing and gene identification in the ED2 critical region. Of the 14 ESTs mapped to the contig, 6 show homology to human genes and 8 appear to be novel. Expression patterns of the genes/ESTs were tested by Northern blot and RT-PCR. Full characterization of some of these genes, as well as the novel ESTs, will be useful in assessing their involvement in the HED/Clouston syndrome.

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Hidrotic ectodermal dysplasia (HED) or Clouston syndrome is a rare autosomal dominant disorder characterized by nail dystrophy, alopecia and palmoplantar hyperkeratosis, which maps to chromosome 13q11-q12.1. We confirmed linkage of HED to this region in a large French family. To define the critical region for HED, detailed haplotypes were constructed with new pericentromeric polymorphic markers. A recombination event in the family indicates that the HED locus maps centromeric to D13S1832. Our French family does not share a common haplotype with other pedigrees previously published (particularly French-Canadian), indicating that the mutations in these families are likely to be of different origin.

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The 5q- syndrome is a myelodysplastic syndrome with specific hematological features and a good prognosis. Using molecular mapping techniques, we have previously defined the critical region of gene loss of the 5q- chromosome in the 5q- syndrome as the approximately 5-Mb region at 5q31-q33 flanked by the genes for FGF1 and IL12B. This region is completely represented by a series of overlapping YACs, and we are currently generating a transcription map with the aim of identifying the tumor-suppressor gene associated with the development of the 5q- syndrome. In this study two techniques have been used: first, the screening of full-length cDNA libraries with radiolabeled YACs and second, the mapping of chromosome 5-specific expressed sequence tags (ESTs) to a YAC contig. A 1-Mb YAC contig encompassing the CSF1R gene has been used to screen a fetal brain cDNA library, and this has resulted in the identification of two genes comprising one known gene previously localized to the region (ADRB2) and one known gene previously unlocalized. Six of 135 chromosome 5-specific ESTs were localized by PCR screening to the YAC contig mapping to the critical region of the 5q- syndrome. IMAGE cDNA clones for each of the six ESTs have been obtained. These seven (excluding ADRB2) newly assigned cDNA clones were subjected to further analysis. The expression patterns of each of the cDNA clones have been established in a range of human tissues, including bone marrow. Six of seven cDNAs are expressed in human bone marrow. Six of seven cDNAs have no known homology to any deposited human sequences, and one (C29) is dihydropyrimidinase-related protein-3, a member of a novel gene family. Genomic localization and expression patterns would suggest that these newly assigned cDNAs represent potential candidate genes for the 5q- syndrome.

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We have sequenced the cDNA for the 23 kDa subunit of the human mitochondrial respiratory complex I. The deduced protein consists of 210 amino acids (Mr = 23705 Da) with a 34 amino acid N terminus presumably acting as a presequence for mitochondrial import. The predicted mature protein (Mr = 20290 Da) is 92% identical to the bovine mitochondrial subunit and 72% to the Rhodobacter capsulatus NUOI counterpart. Two clusters of four cysteine residues are conserved among these proteins. The gene (NDUFS8) coding for the human subunit has been mapped to chromosome 11q13.

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We describe the cloning of a human gene, named ChemR1, encoding a new putative chemokine receptor sharing 48% identity with CC-chemokine receptor (CCR)4 and 44% identity with CCR1. It displays four extracellular cysteines that are conserved among all other chemokine receptors. ChemR1 transcripts were detected by Northern blotting in the T lymphoblastic cell lines Jurkat and MOLT-4, but not in the pre-B lymphoblastic cell line JM-1. ChemR1 receptor transcripts were also detected by reverse transcription and polymerase chain reaction analysis in unstimulated CD4+ and CD8+ T cells and polymorphonuclear cells prepared from peripheral blood. The chromosomal localization was performed by radiation hybrid mapping and testing of a panel of yeast artificial chromosome clones. This allowed the assignment of the ChemR1 receptor gene to the p21.3-24 region of human chromosome 3, in close proximity with the functionally characterized CCR. Future work is required to identify the ligand(s) of this new chemokine receptor and to define its role in the recruitment of white blood cell populations.

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The five human CC-chemokine receptors functionally characterized to date were mapped by using a radiation hybrid panel and YAC contigs. The genes encoding CC-CKR1, CC-CKR2, CC-CKR3, and CC-CKR5 (designated respectively CMKBR1, CMKBR2, CMKBR3, and CMKBR5 in the Genome Data Bank) were found to be clustered in the 3p21.3 region of chromosome 3, between the AFM362WB9 and the WI-6983 markers. The four genes fall within a total distance of about 350 kb. The fifth gene (CMKBR4, encoding the CC-CKR4 receptor) was located more distally (3p24) on the same chromosome, between the FB18G7 and the D3S1768 markers. These localizations were confirmed by mapping the genes into the YAC contigs covering these regions. The clustering of chemokine receptor genes suggests a relatively recent expansion of the gene family by gene duplication. Deletions and duplications of the 3p21 region have been described in neoplastic disorders of the hematopoietic lineage, suggesting a potential link with the CC-chemokine receptor gene family.

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Nociceptin (orphanin FQ), the newly discovered natural agonist of opioid receptor-like (ORL1) receptor, is a neuropeptide that is endowed with pronociceptive activity in vivo. Nociceptin is derived from a larger precursor, prepronociceptin (PPNOC), whose human, mouse, and rat genes we have now isolated. The PPNOC gene is highly conserved in the three species and displays organizational features that are strikingly similar to those of the genes of preproenkephalin, preprodynorphin, and preproopiomelanocortin, the precursors to endogenous opioid peptides, suggesting the four genes belong to the same family-i.e., have a common evolutionary origin. The PPNOC gene encodes a single copy of nociceptin as well as of other peptides whose sequence is strictly conserved across murine and human species; hence it is likely to be neurophysiologically significant. Northern blot analysis shows that the PPNOC gene is predominantly transcribed in the central nervous system (brain and spinal cord) and, albeit weakly, in the ovary, the sole peripheral organ expressing the gene. By using a radiation hybrid cell line panel, the PPNOC gene was mapped to the short arm of human chromosome 8 (8p21), between sequence-tagged site markers WI-5833 and WI-1172, in close proximity of the locus encoding the neurofilament light chain NEFL. Analysis of yeast artificial chromosome clones belonging to the WC8.4 contig covering the 8p21 region did not allow to detect the presence of the gene on these yeast artificial chromosomes, suggesting a gap in the coverage within this contig.

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A set of 1091 human skeletal muscle cDNA clone inserts representing more than 800 human gene transcripts were spotted as PCR products at high density on nylon membranes. Replicas of the filters were hybridized in stringent conditions with 33P-radiolabeled cDNA probes transcribed from skeletal muscle poly(A)+ RNA. Hybridization signals were collected on phosphor screens and processed using a software specifically adapted for this application to identify and quantitate each spot. Parameters likely to influence the hybridization signal intensity were assessed to eliminate artifacts. Each clone was assigned to one of four intensity classes reflecting the steady-state level of transcription of the corresponding gene in skeletal muscle. Differential expression of specific gene transcripts was detected using complex cDNA probes derived from nine different tissues, allowing assessment of their tissue specificity. This made it possible to identify 48 novel gene transcripts (including 7 homologous or related to known sequences) with a muscle-restricted pattern of expression. These results were validated through the analysis of known muscle-specific transcripts and by Northern analysis of a subset of the novel gene transcripts. All these genes have been registered in the Genexpress Index, such that sequence, map, and expression data can be used to decipher their role in the physiology and pathology of human muscles.

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A yeast artificial chromosome library containing 33,000 clones with an average insert size of one megabase of human genomic DNA was extensively analysed by several different procedures for detecting overlaps and positional information. We developed an analysis strategy that resulted, after confirmatory tests, in a YAC contig map reliably covering about 75% of the human genome in 225 contigs having an average size of about ten megabases.

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The pimeloyl-CoA synthase from Bacillus sphaericus has been purified to homogeneity from an overproducing strain of Escherichia coli. The purification yielded milligram quantities of the synthase with a specific activity of 1 unit/mg of protein. Analysis of the products showed that this enzyme catalysed the transformation of pimelate into pimeloyl-CoA with concomitant hydrolysis of ATP to AMP. Using a continuous spectrophotometric assay, we have examined the catalytic properties of the pure enzyme. The pH profile under Vmax. conditions showed a maximum around 8.5. Apparent Km values for pimelate, CoASH, ATP.Mg2- and Mg2+ were respectively 145 microM, 33 microM, 170 microM and 2.3 mM. The enzyme was inhibited by Mg2+ above 10 mM. This acid-CoA ligase exhibited a very sharp substrate specificity, e.g. neither GTP nor pimelate analogues (di- or mono-carboxylic acids) were processed. The bivalent metal ion requirement was also investigated: Mn2+ (73%) and Co2+ (32%) but not Ca2+ could replace Mg2+. The enzyme was inhibited by metal chelators such as 1,10-phenanthroline and EDTA. The synthase was a homodimer with a 28,000-M(r) subunit. N-Terminal sequencing definitely proved that this enzyme was encoded by the bioW gene. A careful study of pimelate uptake by B. sphaericus, E. coli and Pseudomonas dentrificans showed that this metabolite crossed the membrane of these microorganisms by passive diffusion, ruling out the involvement of the bioX gene product as pimelate carrier.

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A continuous array of overlapping clones covering the entire human chromosome 21q was constructed from human yeast artificial chromosome libraries using sequence-tagged sites as landmarks specifically detected by polymerase chain reaction. The yeast artificial chromosome contiguous unit starts with pericentromeric and ends with subtelomeric loci of 21q. The resulting order of sequence-tagged sites is consistent with other physical and genetic mapping data. This set of overlapping clones will promote our knowledge of the structure of this chromosome and the function of its genes.

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A new approach for the isolation of chromosome-specific subsets from a human genomic yeast artificial chromosome (YAC) library is described. It is based on the hybridization with an Alu polymerase chain reaction (PCR) probe. We screened a 1.5 genome equivalent YAC library of megabase insert size with Alu PCR products amplified from hybrid cell lines containing human chromosome 21, and identified a subset of 63 clones representative of this chromosome. The majority of clones were assigned to chromosome 21 by the presence of specific STSs and in situ hybridization. Twenty-nine of 36 STSs that we tested were detected in the subset, and a contig spanning 20 centimorgans in the genetic map and containing 8 STSs in 4 YACs was identified. The proposed approach can greatly speed efforts to construct physical maps of the human genome.

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A class of alleles at the VNTR (variable number of tandem repeat) locus in the 5' region of the insulin gene (INS) on chromosome 11p is associated with increased risk of insulin-dependent diabetes mellitus (IDDM), but family studies have failed to demonstrate linkage. INS is thought to contribute to IDDM susceptibility but this view has been difficult to reconcile with the lack of linkage evidence. We thus investigated polymorphisms of INS and neighbouring loci in random diabetics, IDDM multiplex families and controls. HLA-DR4-positive diabetics showed an increased risk associated with common variants at polymorphic sites in a 19-kilobase segment spanned by the 5' INS VNTR and the third intron of the gene for insulin-like growth factor II (IGF2). As INS is the major candidate gene from this region, diabetic and control sequence were compared to identify all INS polymorphisms that could contribute to disease susceptibility. In multiplex families the IDDM-associated alleles were transmitted preferentially to HLA-DR4-positive diabetic offspring from heterozygous parents. The effect was strongest in paternal meioses, suggesting a possible role for maternal imprinting. Our results strongly support the existence of a gene or genes affecting HLA-DR4 IDDM susceptibility which is located in a 19-kilobase region of INS-IGF2. Our results also suggest new ways to map susceptibility loci in other common diseases.

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