Characterization of EHD4, an EH domain-containing protein expressed in the extracellular matrix.
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To identify proteins that promote assembly of type VI collagen tetramers or stabilize type VI collagen filaments, a two-hybrid screen of a human placenta library was used and a new extracellular protein discovered. The cDNA sequence of the new protein encodes 541 amino acid residues. This cDNA sequence is identical to EHD4, a recently described member of the EH domain family of proteins. Two mRNAs of 4.4 and 3.0 kilobases were present in human skin fibroblasts and most tissues tested but were most prevalent in the heart. The chromosomal localization of the gene for this new protein was determined to be at 15q14-q15. Three polyclonal peptide antibodies were made against synthetic EHD4 peptides. The affinity-purified antibodies were used in immunofluorescent staining of developing limbs and matrices produced by human skin fibroblasts and mouse NIH3T3 fibroblasts in culture. Embryonic rat limb cartilage was strongly stained throughout development, and cultured fibroblasts deposited an extracellular filamentous network containing EHD4. In non-denaturing extracts of fetal bovine cartilage and in human skin fibroblast culture media, two components of approximately 220 and 158 kDa were observed, which, after reduction, migrated as a 56-kDa component on SDS-polyacrylamide gel electrophoresis. EHD4 is the first extracellular matrix protein described that contains an EH domain. (+info)
Duplicate mitf genes in zebrafish: complementary expression and conservation of melanogenic potential.
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Mutations in the zebrafish nacre/mitfa gene, expressed in all embryonic melanogenic cells, perturb only neural crest melanocytes, suggesting redundancy of mitfa with another gene in the zebrafish retinal pigment epithelium (RPE). Here, we describe a second zebrafish mitf gene, mitfb, which may fulfill this role. The proteins encoded by the two zebrafish mitf genes appear homologous to distinct isoforms generated by alternately spliced mRNAs of the single mammalian Mitf gene, suggesting specialization of the two zebrafish genes following a duplication event. Consistent with this hypothesis, expression of mitfa and mitfb is partially overlapping. mitfb is coexpressed with mitfa in the RPE at an appropriate time to compensate for loss of mitfa function in the nacre mutant but is not expressed in neural crest melanoblasts. Additionally, mitfb is expressed in the epiphysis and olfactory bulb where mitfa is not, and where Mitf expression has not previously been reported in other species. mitfb, but not a zebrafish ortholog of the closely related gene tfe3, can rescue neural crest melanophore development in nacre/mitfa mutant embryos when expressed via the mitfa promoter. These data suggest that mitfa and mitfb together may recapitulate the expression and functions of a single ancestral Mitf gene, and that mitfb may serve additional novel functions. (+info)
A dominant gene for developmental dyslexia on chromosome 3.
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Developmental dyslexia is a neurofunctional disorder characterised by an unexpected difficulty in learning to read and write despite adequate intelligence, motivation, and education. Previous studies have suggested mostly quantitative susceptibility loci for dyslexia on chromosomes 1, 2, 6, and 15, but no genes have been identified yet. We studied a large pedigree, ascertained from 140 families considered, segregating pronounced dyslexia in an autosomal dominant fashion. Affected status and the subtype of dyslexia were determined by neuropsychological tests. A genome scan with 320 markers showed a novel dominant locus linked to dyslexia in the pericentromeric region of chromosome 3 with a multipoint lod score of 3.84. Nineteen out of 21 affected pedigree members shared this region identical by descent (corrected p<0.001). Previously implicated genomic regions showed no evidence for linkage. Sequencing of two positional candidate genes, 5HT1F and DRD3, did not support their role in dyslexia. The new locus on chromosome 3 is associated with deficits in all three essential components involved in the reading process, namely phonological awareness, rapid naming, and verbal short term memory. (+info)
Partitioning of tissue expression accompanies multiple duplications of the Na+/K+ ATPase alpha subunit gene.
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Vertebrate genomes contain multiple copies of related genes that arose through gene duplication. In the past it has been proposed that these duplicated genes were retained because of acquisition of novel beneficial functions. A more recent model, the duplication-degeneration-complementation hypothesis (DDC), posits that the functions of a single gene may become separately allocated among the duplicated genes, rendering both duplicates essential. Thus far, empirical evidence for this model has been limited to the engrailed and sox family of developmental regulators, and it has been unclear whether it may also apply to ubiquitously expressed genes with essential functions for cell survival. Here we describe the cloning of three zebrafish alpha subunits of the Na(+),K(+)-ATPase and a comprehensive evolutionary analysis of this gene family. The predicted amino acid sequences are extremely well conserved among vertebrates. The evolutionary relationships and the map positions of these genes and of other alpha-like sequences indicate that both tandem and ploidy duplications contributed to the expansion of this gene family in the teleost lineage. The duplications are accompanied by acquisition of clear functional specialization, consistent with the DDC model of genome evolution. (+info)
High-resolution BAC-based map of the central portion of mouse chromosome 5.
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The current strategy for sequencing the mouse genome involves the combination of a whole-genome shotgun approach with clone-based sequencing. High-resolution physical maps will provide a foundation for assembling contiguous segments of sequence. We have established a bacterial artificial chromosome (BAC)-based map of a 5-Mb region on mouse Chromosome 5, encompassing three gene families: receptor tyrosine kinases (PdgfraKit-Kdr), nonreceptor protein-tyrosine type kinases (Tec-Txk), and type-A receptors for the neurotransmitter GABA (Gabra2, Gabrb1, Gabrg1, and Gabra4). The construction of a BAC contig was initiated by hybridization screening the C57BL/6J (RPCI-23) BAC library, using known genes and sequence tagged sites (STSs). Additional overlapping clones were identified by searching the database of available restriction fingerprints for the RPCI-23 and RPCI-24 libraries. This effort resulted in the selection of >600 BAC clones, 251 kb of BAC-end sequences, and the placement of 40 known and/or predicted genes within this 5-Mb region. We use this high-resolution map to illustrate the integration of the BAC fingerprint map with a radiation-hybrid map via assembled expressed sequence tags (ESTs). From annotation of three representative BAC clones we demonstrate that up to 98% of the draft sequence for each contig could be ordered and oriented using known genes, BAC ends, consensus sequences for transcript assemblies, and comparisons with orthologous human sequence. For functional studies, annotation of sequence fragments as they are assembled into 50-200-kb stretches will be remarkably valuable. (+info)
Chromosome-specific single-locus FISH probes allow anchorage of an 1800-marker integrated radiation-hybrid/linkage map of the domestic dog genome to all chromosomes.
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We present here the first fully integrated, comprehensive map of the canine genome, incorporating detailed cytogenetic, radiation hybrid (RH), and meiotic information. We have mapped a collection of 266 chromosome-specific cosmid clones, each containing a microsatellite marker, to all 38 canine autosomes by fluorescence in situ hybridization (FISH). A 1500-marker RH map, comprising 1078 microsatellites, 320 dog gene markers, and 102 chromosome-specific markers, has been constructed using the RHDF5000-2 whole-genome radiation hybrid panel. Meiotic linkage analysis was performed, with at least one microsatellite marker from each dog autosome on a panel of reference families, allowing one meiotic linkage group to be anchored to all 38 dog autosomes. We present a karyotype in which each chromosome is identified by one meiotic linkage group and one or more RH groups. This updated integrated map, containing a total of 1800 markers, covers >90% of the dog genome. Positional selection of anchor clones enabled us, for the first time, to orientate nearly all of the integrated groups on each chromosome and to evaluate the extent of individual chromosome coverage in the integrated genome map. Finally, the inclusion of 320 dog genes into this integrated map enhances existing comparative mapping data between human and dog, and the 1000 mapped microsatellite markers constitute an invaluable tool with which to perform genome scanning studies on pedigrees of interest. (+info)
Mutations in the fukutin-related protein gene (FKRP) cause a form of congenital muscular dystrophy with secondary laminin alpha2 deficiency and abnormal glycosylation of alpha-dystroglycan.
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The congenital muscular dystrophies (CMD) are a heterogeneous group of autosomal recessive disorders presenting in infancy with muscle weakness, contractures, and dystrophic changes on skeletal-muscle biopsy. Structural brain defects, with or without mental retardation, are additional features of several CMD syndromes. Approximately 40% of patients with CMD have a primary deficiency (MDC1A) of the laminin alpha2 chain of merosin (laminin-2) due to mutations in the LAMA2 gene. In addition, a secondary deficiency of laminin alpha2 is apparent in some CMD syndromes, including MDC1B, which is mapped to chromosome 1q42, and both muscle-eye-brain disease (MEB) and Fukuyama CMD (FCMD), two forms with severe brain involvement. The FCMD gene encodes a protein of unknown function, fukutin, though sequence analysis predicts it to be a phosphoryl-ligand transferase. Here we identify the gene for a new member of the fukutin protein family (fukutin related protein [FKRP]), mapping to human chromosome 19q13.3. We report the genomic organization of the FKRP gene and its pattern of tissue expression. Mutations in the FKRP gene have been identified in seven families with CMD characterized by disease onset in the first weeks of life and a severe phenotype with inability to walk, muscle hypertrophy, marked elevation of serum creatine kinase, and normal brain structure and function. Affected individuals had a secondary deficiency of laminin alpha2 expression. In addition, they had both a marked decrease in immunostaining of muscle alpha-dystroglycan and a reduction in its molecular weight on western blot analysis. We suggest these abnormalities of alpha-dystroglycan are caused by its defective glycosylation and are integral to the pathology seen in MDC1C. (+info)
Inactivation of human SRBC, located within the 11p15.5-p15.4 tumor suppressor region, in breast and lung cancers.
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A cDNA clone encoding human SRBC [serum deprivation response factor (sdr)-related gene product that binds to c-kinase] was isolated in a yeast two-hybrid screening, with amino acids 1-304 of BRCA1 as the probe. The human SRBC gene (hSRBC) was mapped to chromosome region 11p15.5-p15.4, close to marker D11S1323, at which frequent loss of heterozygosity (LOH) has been observed in sporadic breast, lung, ovarian, and other types of adult cancers as well as childhood tumors. hSRBC-coding region mutations including frame shift and truncation mutations were detected in a few ovarian and lung cancer cell lines. More significantly, the expression of hSRBC protein was down-regulated in a large fraction [30 (70%) of 43] of breast, lung, and ovarian cancer cell lines, whereas strong expression of hSRBC protein was detected in normal mammary and lung epithelial cells. The down-regulation of hSRBC expression in cancer cells was associated with hypermethylation of CpG dinucleotides in its promoter region, and 3 (60%) of 5 primary breast tumors and 11 (79%) of 14 primary lung tumors were also found to be hypermethylated. Treatment of breast cancer MCF7 cells with 5'azacytidine and Trichostatin A resulted in expression of hSRBC, confirming DNA methylation as the mode of inactivation. Our results suggest that epigenetic or mutational inactivation of hSRBC may contribute to the pathogenesis of several types of human cancers, marking hSRBC as a candidate tumor suppressor gene. (+info)