The genexpress IMAGE knowledge base of the human muscle transcriptome: a resource of structural, functional, and positional candidate genes for muscle physiology and pathologies.

Sequence, gene mapping, and expression data corresponding to 910 genes transcribed in human skeletal muscle have been integrated to form the muscle module of the Genexpress IMAGE Knowledge Base. Based on cDNA array hybridization, a set of 14 transcripts preferentially or specifically expressed in muscle have been selected and characterized in more detail: Their pattern of expression was confirmed by Northern blot analysis; their structure was further characterized by full-insert cDNA sequencing and cDNA extension; the map location of the corresponding genes was refined by radiation hybrid mapping. Five of the 14 selected genes appear as interesting positional and functional candidate genes to study in relation with muscle physiology and/or specific orphan muscular pathologies. One example is discussed in more detail. The expression profiling data and the associated Genexpress Index2 entries for the 910 genes and the detailed characterization of the 14 selected transcripts are available from a dedicated Web server at. The database has been organized to provide the users with a working space where they can find curated, annotated, integrated data for their genes of interest. Different navigation routes to exploit the resource are discussed.

Reverse transcription in the presence of dideoxynucleotides to increase the sensitivity of expression monitoring with cDNA arrays.

Complex cDNA targets conforming to the nomenclature proposed by Duggan et al. (Nat. Genet. 21 [1 Suppl.]: 10-14) (where the immobilized nucleic acid is called "probe" and free nucleic acid is called "target") were prepared by priming reverse transcription of mRNA with oligo-(dT)-primers or random oligonucleotide primers in the presence or absence of dideoxynucleotides. These targets were then hybridized to high-density filters spotted with 1339 cDNA clone inserts from a skeletal muscle library. The addition of dideoxynucleotides was found to significantly improve the efficiency and reproducibility of the reverse transcription reaction, and consequently, to improve detection of the least abundant transcripts in expression profiling experiments.

Fucosyltransferase genes are dispersed in the genome: FUT7 is located on 9q34.3 distal to D9S1830.

Synthesis of A, B, H, Lewis and related histo-blood group antigens is catalyzed by different fucosyltransferases. Enzymatic acceptor specificity and tissue expression permit the definition of 2 types of alpha-2-fucosyltransferases and 5 types of alpha-3-fucosyltransferases encoded by specific genes registered as FUT1 to FUT7. We have previously assigned FUT4 to 11q21, the cluster FUT1-FUT2 to 19q13.3 and the cluster FUT6-FUT3-FUT5 to 19p13.3. The last gene cloned (FUT7) encodes an alpha-3-fucosyltransferase expressed in leukocytes which synthesizes the sialyl Le antigen, a selectin ligand. We have localized this gene by PCR assay using somatic cell hybrids, which retain rearrangements of chromosome 9 characterized in respect with the genetic microsatellite map, and then by screening a cosmid library. We assign FUT7 to chromosome band 9q34.3 telomeric to D9S1830 and close to the genes ABC2 and C8G.

Physical mapping of 49 microsatellite markers on chromosome 19 and correlation with the genetic linkage map.

We have regionally localized 49 microsatellite markers developed by Généthon using a panel of previously characterized somatic cell hybrids that retain fragments from chromosome 19. The tight correlation observed between the physical and the genetic orders of the microsatellites provide cytogenetic anchorages to the genetic map data. We propose a position for the centromere just above D19S415, from the study of two hybrids, each of which retains one of the two derivatives of a balanced translocation t(1;19)(q11;q11). Microsatellites, which can be identified by a standard PCR protocol, are useful tools for the localization of disease genes and for the establishment of YAC or cosmid contigs. These markers can also judiciously be used for the characterization of new hybrid cell line panels. We report such a characterization of 11 clones, 8 of which were obtained by irradiation-fusion. Using the whole hybrid panel, we were able to define the order of 12 pairs of genetically colocalized microsatellites. As examples of gene mapping by the combined use of microsatellites and hybrid cell lines, we regionally assigned the PVS locus between the 19q13.2 markers D19S417 and D19S423 and confirmed the locations of fucosyltransferase loci FUT1, FUT2, and FUT5.

Regional assignment of 68 new human gene transcripts on chromosome 11.

We have tested 80 expressed sequence-tagged site (eSTS) markers assigned to human chromosome 11 by the Genexpress program on a panel of somatic cell hybrids containing parts of this chromosome, characterized by cytogenetic data, reference markers, and with respect to the Généthon microsatellite genetic map. Sixty-eight new gene transcripts have been assigned to 25 subregions, one of which was newly defined by five of the eSTS markers. The markers are distributed on the short and long arms in agreement with their physical length. The genic map thus obtained has been integrated with the cytogenetic, genetic, and disease maps. Two eSTS markers have been further mapped with respect to a yeast artificial chromosome (YAC) contig close to the brain-derived neurotrophic factor (BDNF) gene and thus provide potential candidate genes for the mental retardation phenotype of WAGR (Wilms' tumor, aniridia, genitourinary abnormalities and mental retardation) syndrome. Altogether, the 68 new gene transcripts localized here represent more than a threefold increase in the number of unknown regionalized genes that could reveal potential candidate genes for the numerous orphan pathologies associated with chromosome 11.

Relative positions of two clusters of human alpha-L-fucosyltransferases in 19q (FUT1-FUT2) and 19p (FUT6-FUT3-FUT5) within the microsatellite genetic map of chromosome 19.

Five on the seven cloned human fucosyltransferase genes have been mapped to two clusters, one on 19q and the other on 19p. Comparative DNA sequence analysis showed the Généthon microsatellite D19S596 lies 2.2 kb downstream of the coding region of FUT1, indicating that the cluster comprising the closely linked FUT1 and FUT2 genes is located 4 cM distal to D19S412 (lod score 13.7) and 9 cM proximal to D19S571 (lod score 11.7). Polymorphic markers of FUT3, FUT5, and FUT6 were used for linkage analysis with 14 Généthon microsatellites in Indonesian families. These three loci constitute a cluster on 19p, located between the Généthon microsatellites D19S216 and D19S567, which are known to be only 1 cM distant from each other. Two cross-overs, one between FUT6 and FUT3 and the other between FUT3 and FUT5, suggest the gene order 19pter-D19S216-FUT6-FUT3-FUT5-D19S567++ +-cen. Comparison of genetic and physical maps suggests that the FUT6-FUT3-FUT5 cluster is located on 19p13.3 and the FUT1-FUT2 cluster on 19q13.3. FUT6, FUT3 and FUT5 genes share more than 85% homology and encode three similar, but distinct alpha(1,3) fucosyltransferases. FUT1 and FUT2 share about 70% homology and encode two distinct alpha(1,2)fucosyltransferases. No sequence homology was found between the genes of the two clusters. The members of each of these two clusters have probably emerged by duplication and divergent evolution of two unrelated ancestor genes.