Assignment of the chicken NOV gene (alias CCN3) to chromosome 2q34-->q36: Conserved and compared synteny between avian, mouse and human.
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Animals↗
Biomedical subjects
C Auffray
Publications and source records attributed to C Auffray.
Automatic quantitation of hybridization signals on cDNA arrays.
Large-scale hybridization of simple or complex cDNA probes to cDNA clones arrayed on high-density filters is a method frequently used to determine systematically the expression profiles of thousands of genes. Hybridization signal intensities, which reflect the level of transcription of the corresponding genes, are captured on phosphor screens with an imaging system. We describe a high-throughput system, Xdots-Reader, that performs automatic detection and quantitation of each signal on hundreds of images. Reproducibility of spot detection and quantitation within filters and between filters has been assessed in analysis of more than 850000 hybridization signals on 436 filters. The automatic analysis success was greater than 97%, with 424 of the 436 tested filters fully analyzed without any human intervention. XdotsReader is available from the Software Library at www.BioTechniques.com or at http://www. ami. univ-evry. fr/approximately tahi/XDotsReader. It runs on SUN workstations under UNIX (SunOS or Solaris) and on PC under LINUX. No particular hardware is required, and the software is compatible with any other software. It supports the main standard image formats.
Algorithms↗
At least one class I gene in restriction fragment pattern-Y (Rfp-Y), the second MHC gene cluster in the chicken, is transcribed, polymorphic, and shows divergent specialization in antigen binding region.
MHC genes in the chicken are arranged into two genetically independent clusters located on the same chromosome. These are the classical B: system and restriction fragment pattern-Y (Rfp-Y), a second cluster of MHC genes identified recently through DNA hybridization. Because small numbers of MHC class I and class II genes are present in both B: and Rfp-Y, the two clusters might be the result of duplication of an entire chromosomal segment. We subcloned, sequenced, and analyzed the expression of two class I loci mapping to Rfp-Y to determine whether Rfp-Y should be considered either as a second, classical MHC or as a region containing specialized MHC-like genes, such as class Ib genes. The Rfp-Y genes are highly similar to each other (93%) and to classical class Ia genes (73% with chicken B: class I; 49% with HLA-A). One locus is disrupted and unexpressed. The other, YFV, is widely transcribed and polymorphic. Mature YFV protein associated with beta(2)m arrives on the surface of chicken B (RP9) lymphoma cells expressing YFV as an epitope-tagged transgene. Substitutions in the YFV Ag-binding region (ABR) occur at four of the eight highly conserved residues that are essential for binding of peptide-Ag in the class Ia molecules. Therefore, it is unlikely that Ag is bound in the YFV ABR in the manner typical of class Ia molecules. This ABR specialization indicates that even though YFV is polymorphic and widely transcribed, it is, in fact, a class Ib gene, and Rfp-Y is a region containing MHC genes of specialized function.
Alleles↗
Identification of altered gene expression in skeletal muscles from Duchenne muscular dystrophy patients.
Mutations in the dystrophin gene lead to dystrophin deficiency, which is the cause of Duchenne muscular dystrophy (DMD). This important discovery more than 10 years ago opened a new field for very productive investigations. However, the exact functions of dystrophin are still not fully understood and the complex process leading to subsequent muscle fiber necrosis has not been clearly described; hence there has not yet been any marked improvement in patient treatment. To decipher the molecular mechanisms induced by a lack of dystrophin, we started identifying genes whose expression is altered in DMD skeletal muscles. The approach was based on differential screening of a human muscle cDNA array. Nine genes were found to be up- or downregulated. Our results indicate expression alterations in mitochondrial genes, titin, a muscle transcription factor and three novel genes. First characterizations of these novel genes indicated that two of them have striated muscle tissue specificity.
Adolescent↗
Refined localization of twenty-one genes in subregion p13.1 of human chromosome 1.
In this report, we describe a refinement of the human transcript map of chromosome 1p13.1, a subregion undergoing many aberrations in various types of human cancers. Publicly available genetic linkage, radiation hybrid and physical maps, as well as cytogenetic and sequence data were used to establish the relative order and orientation of ten known intragenic markers. The complete sequence of genomic clones of the region, available at the Sanger Centre, provided the tool for further studies performed by BLAST analysis against all cDNA sequences registered in the Genexpress Index2. This allowed us to assign to subband 1p13.1 nine of the ten known genes, an additional member of the gene family of one of these genes and eleven new transcripts. The remaining known gene and one additional new transcript map at the 1p13.1 and 1p13.2 boundary. The corresponding genes may be responsible for disorders related to this region. The resulting transcript map of 1p13.1 is presented in the printed article with additional data available on a dedicated Web site at the address http://idefix.upr420.vjf.cnrs.fr/CARTO.
Chromosome Mapping↗
Global analysis of gene expression in cells of the immune system II. Cell-free translation products and high-density filter hybridization data.
We have developed an experimental system for linking information on cell-free transcription and translation products from cDNA clones with data obtained from hybridization signals from complex probes. The work described in this paper consists of two distinct processes, one being the construction of a system of clonal addresses and the other the identification of expressed genes involved in the studied processes. We describe the use of this system to identify genes involved in thymus development. Complex probes from fetal thymuses (GD15, 17 and newborn) of Balb/c mice were used to identify genes, which are up- or downregulated during the process of differentiation. The full set of information is available in the Clone-base of the Basel Institute for Immunology and will be retrievable from the website of the collaborating laboratories.
Animals↗
DNA arrays: methods and applications: report on HUGO Meeting, Tartu, Estonia, 23-26 May, 1999.
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Alleles↗
The chicken B locus is a minimal essential major histocompatibility complex.
Here we report the sequence of the region that determines rapid allograft rejection in chickens, the chicken major histocompatibility complex (MHC). This 92-kilobase region of the B locus contains only 19 genes, making the chicken MHC roughly 20-fold smaller than the human MHC. Virtually all the genes have counterparts in the human MHC, defining a minimal essential set of MHC genes conserved over 200 million years of divergence between birds and mammals. They are organized differently, with the class III region genes located outside the class II and class I region genes. The absence of proteasome genes is unexpected and might explain unusual peptide-binding specificities of chicken class I molecules. The presence of putative natural killer receptor gene(s) is unprecedented and might explain the importance of the B locus in the response to the herpes virus responsible for Marek's diseases. The small size and simplicity of the chicken MHC allows co-evolution of genes as haplotypes over considerable periods of time, and makes it possible to study the striking MHC-determined pathogen-specific disease resistance at the molecular level.
Amino Acid Sequence↗
Identification of the Tapasin gene in the chicken major histocompatibility complex.
The Tapasin molecule plays a role in the assembly of major histocompatibility complex (Mhc) class I molecules in the endoplasmic reticulum, by mediating the interaction of class I-beta2-microglobulin dimers with TAP. We report here the identification of the Tapasin gene in the chicken Mhc (B complex). This gene is located at the centromeric end of the complex, between the class II B-LBI and B-LBII genes. Like its human counterpart it comprises 8 exons, but features a significantly reduced intron size as compared to the human gene. Chicken Tapasin codes for a transmembrane protein with a probable endoplasmic reticulum retention signal. Exons IV and V, and possibly exon III, code for separate domains that are related to the immunoglobulin (Ig) superfamily (this relationship was so far unrecognized for human Tapasin domain IV which has lost its two cysteines). Two different cDNAs corresponding to the Tapasin gene were isolated, possibly related to alternative splicing events; the Ig-like domain encoded by exon IV is missing in one of the cDNAs, suggesting either that this domain is not necessary for the protein to perform its function, or that the two alternatively spliced cDNAs are translated into two functionally different forms of the protein.
Amino Acid Sequence↗
Exclusion of muscle specific actinin-associated LIM protein (ALP) gene from 4q35 facioscapulohumeral muscular dystrophy (FSHD) candidate genes.
Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disorder for which no candidate gene has yet been identified. The gene corresponding to one of the novel human cDNAs that we cloned on the basis of a muscle restricted expression pattern [Piétu G, Alibert O, Guichard B, et al. Genome Res 1996;6:492-503] was mapped in the region of the FSHD1A genetic locus, i.e. one of the loci involved in this muscular dystrophy. The corresponding encoded protein contains a PDZ and a LIM domain, two protein-protein interaction domains, and was very recently shown to bind alpha-actinin-2 and was named ALP (actinin-associated LIM protein) [Xia H, Winokur S, Kuo W, Altherr M, Bredt D. J Cell Biol 1997;139:507-515]. We raised a specific polyclonal anti-ALP serum against an ALP recombinant polypeptide to evaluate the size, level of expression and subcellular localization of ALP in three patients, clearly diagnosed with FSHD disease. Quantitative or qualitative alterations of ALP expression have not been detected in any of them, thus prompting us to exclude ALP as a FSHD gene candidate.
Actinin↗
Concerted evolution of two Mhc class II B loci in pheasants and domestic chickens.
The major histocompatibility complex (Mhc) of the ring-necked pheasant contains two polymorphic Mhc class II B genes. We show here, by screening of a cDNA library and RT-PCR from RNA, that both of these loci, Phco-DAB1 and Phco-DAB2, normally are transcribed in the spleen. They differ mainly in the 3' untranslated (UT) region, with the transcript lengths, not including the poly(A) tails, being 1,100 nt for DAB1 and 955 nt for DAB2. These two loci are orthologous to the B-LBI and B-LBII loci of the domestic chicken, respectively. DAB1 and DAB2 therefore seem to have evolved from a duplication before the split of the evolutionary lineages leading to the pheasant and the domestic chicken ca. 20 MYA. This is the first report of an orthologous relationship between avian Mhc genes. Yet, the third exons of DAB1 and DAB2 were identical in all available sequences and differed at 10 positions from the exon 3 sequences of B-LBI/B-LBII. The species-specific exon 3 suggests that DAB1 and DAB2 are subject to concerted evolution, i.e., interlocus genetic exchange. The exon 2 sequences show characteristic polymorphism, with hypervariable segments occurring in different combinations in different alleles. Given the divergence in the 3'UT region, the finding of the same exon 2 sequence at both the DAB1 and the DAB2 loci in one of the pheasant haplotypes also suggests that interlocus genetic exchange does occur. Accordingly, the exon 2 sequences tended to cluster irrespective of locus in the phylogenetic analyses. Genetic exchange simultaneously involving both exon 2 and exon 3 may be facilitated by the short length of the intervening intron (< 100 bp) in pheasants and domestic chickens compared with, e.g., humans (about 3 kb).
3' Untranslated Regions↗
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.
Animals↗
The Genexpress IMAGE knowledge base of the human brain transcriptome: a prototype integrated resource for functional and computational genomics.
Expression profiles of 5058 human gene transcripts represented by an array of 7451 clones from the first IMAGE Consortium cDNA library from infant brain have been collected by semiquantitative hybridization of the array with complex probes derived by reverse transcription of mRNA from brain and five other human tissues. Twenty-one percent of the clones corresponded to transcripts that could be classified in general categories of low, moderate, or high abundance. These expression profiles were integrated with cDNA clone and sequence clustering and gene mapping information from an upgraded version of the Genexpress Index. For seven gene transcripts found to be transcribed preferentially or specifically in brain, the expression profiles were confirmed by Northern blot analyses of mRNA from eight adult and four fetal tissues, and 15 distinct regions of brain. In four instances, further documentation of the sites of expression was obtained by in situ hybridization of rat-brain tissue sections. A systematic effort was undertaken to further integrate available cytogenetic, genetic, physical, and genic map informations through radiation-hybrid mapping to provide a unique validated map location for each of these genes in relation to the disease map. The resulting Genexpress IMAGE Knowledge Base is illustrated by five examples presented in the printed article with additional data available on a dedicated Web site at the address http://idefix.upr420.vjf.cnrs.fr/EXPR++ +/ welcome.html.
Brain Chemistry↗
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.
Base Sequence↗
Molecular cloning and functional expression of a novel human gene encoding two 41-43 kDa skeletal muscle internal membrane proteins.
Systematic analysis of gene transcript repertoires prepared from libraries made with various specific human tissues permitted isolation of many partially sequenced cDNA clones. A few of these represented novel genes with limited or no similarity to known genes from humans or other species. The present study set out to isolate and sequence the full-length cDNA corresponding to one of these novel human transcripts, and identify the corresponding protein product at the subcellular level. Current sequence analyses have revealed that the protein contains a hydrophobic N-terminal segment and an internal leucine-zipper motif. Numerous sites of putative post-translational modifications, such as N-linked glycosylation, myristoylation and phosphorylation sites, were also identified. Using one monoclonal antibody raised against a recombinant fragment, two different 41-43 kDa proteins were detected in human skeletal muscle, heart and placenta homogenates at various ratios. Both immunodetected protein products of the novel human gene were distributed in the transverse tubules and/or near the junctional sarcoplasmic reticulum within skeletal muscle cells. Both proteins had physical properties believed to be attributable to integral membrane components. Finally, the GENX-3414 gene was chromosomally localized at position 4q24-q25.
Adult↗
A physical map of 30,000 human genes.
A map of 30,181 human gene-based markers was assembled and integrated with the current genetic map by radiation hybrid mapping. The new gene map contains nearly twice as many genes as the previous release, includes most genes that encode proteins of known function, and is twofold to threefold more accurate than the previous version. A redesigned, more informative and functional World Wide Web site (www.ncbi.nlm.nih.gov/genemap) provides the mapping information and associated data and annotations. This resource constitutes an important infrastructure and tool for the study of complex genetic traits, the positional cloning of disease genes, the cross-referencing of mammalian genomes, and validated human transcribed sequences for large-scale studies of gene expression.
Animals↗
Identification of 16 chicken microchromosomes by molecular markers using two-colour fluorescence in situ hybridization (FISH).
A feature of avian karyotypes is the presence of microchromosomes. As a typical avian genome, the chicken karyotype (2n = 78) consists of nine pairs of macrochromosomes, including the W and Z sexual chromosomes, and 30 pairs of indistinguishable microchromosomes usually ordered arbitrarily by decreasing size. Despite their reduced size, microchromosomes represent one-third of the genome and have a high gene density. So as to provide a tool to identify them, we developed a set of large insert-containing clones to be used as tags in two-colour fluorescence in situ hybridization experiments. Seventeen clones, six of which contain a microsatellite sequence and two others the fatty acid synthase gene or genes from the major histocompatibility complex, all presenting a strong hybridization signal, were selected for this purpose and enabled us to identify 16 different microchromosomes. The ability to recognize individual microchromosomes will be of great value for cytogenetic gene mapping, assignation of linkage groups from genetic maps and other studies on avian genome structure.
Animals↗
Integration of chicken cytogenetic and genetic maps: 18 new polymorphic markers isolated from BAC and PAC clones.
As an approach to integrate the chicken genetic and cytogenetic maps, bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) clones were localized by fluorescence in situ hybridization (FISH) on chromosomes and by genetic mapping on the East Lansing and Compton reference families. Some of the clones used in this study were previously selected for the presence of potentially polymorphic (CA)n repeats and a microsatellite marker was developed when possible for genetic mapping. For other clones, a single strand conformational polymorphism (SSCP) was developed and used for this purpose. Between the two approaches, 18 markers linking the cytogenetic and genetic maps, seven on macrochromosomes and 11 on microchromosomes, were generated. Our results enabled the assignment and orientation of a linkage group to chromosome 3, together with the assignment of linkage groups to eight different microchromosomes, a fraction of the genome lacking mapping data and for which the degree of coverage by the genetic map was not well estimated previously.
Animals↗