A high-resolution HAPPY map of Dictyostelium discoideum chromosome 6.
We have made a high-resolution HAPPY map of chromosome 6 of Dictyostelium discoideum consisting of 300 sequence-tagged sites with an average spacing of 14 kb along the approximately 4-Mb chromosome. The majority of the marker sequences were derived from randomly chosen clones from four different chromosome 6-enriched plasmid libraries or from subclones of YACs previously mapped to chromosome 6. The map appears to span the entire chromosome, although marker density is greater in some regions than in others and is lowest within the telomeric region. Our map largely supports previous gene-based maps of this chromosome but reveals a number of errors in the physical map. In addition, we find that a high proportion of the plasmid sequences derived from gel-enriched chromosome 6 (and that form the basis of a chromosome-specific sequencing project) originates from other chromosomes. (+info)
The detection of osmotic stimuli is essential for all organisms, yet few osmoreceptive proteins are known, none of them in vertebrates. By employing a candidate-gene approach based on genes encoding members of the TRP superfamily of ion channels, we cloned cDNAs encoding the vanilloid receptor-related osmotically activated channel (VR-OAC) from the rat, mouse, human, and chicken. This novel cation-selective channel is gated by exposure to hypotonicity within the physiological range. In the central nervous system, the channel is expressed in neurons of the circumventricular organs, neurosensory cells responsive to systemic osmotic pressure. The channel also occurs in other neurosensory cells, including inner-ear hair cells, sensory neurons, and Merkel cells. (+info)
Restricted species tropism of maedi-visna virus strain EV-1 is not due to limited receptor distribution.
The distribution of receptors for maedi-visna virus (MVV) was studied using co-cultivation assays for virus fusion and PCR-based assays to detect the formation of virus-specific reverse transcription products after virus entry. Receptors were present on cell lines from human, monkey, mouse, chicken, quail, hamster and ovine sources. Thus, the distribution of the receptor for MVV is more similar to that of the amphotropic type C retroviruses than to that of other lentiviruses. The receptor was sensitive to proteolysis by papain, but was resistant to trypsin. Chinese hamster ovary (CHO) and lung cells (V79 TOR) did not express functional receptors for MVV. The receptor was mapped to either chromosome 2 or 4 of the mouse using somatic cell hybrids. This allowed several candidates (e.g. MHC-II, CXCR4) that have been proposed for the MVV receptor to be excluded. (+info)
Mapping dispersed repetitive loci using semi-specific PCR cloning and somatic cell hybrid mapping.
A simple and effective method based upon semi-specific PCR followed by cloning has been developed. Chromosomal mapping of the generated fragment on a somatic cell hybrid panel identifies the chromosomal position, and yields a unique sequence tag for the site. Using this method, the chromosomal location of one porcine endogenous retrovirus (PERV) was determined. The porcine genomic sequences were first amplified by PCR using a PERV-specific primer and a porcine short interspersed nuclear element (SINE)-specific primer. PCR products were cloned, and those sequences that contained PERV plus flanking regions were selected using a second round of PCR and cloning. Sequences flanking the PERV were determined and a PERV-B was physically mapped on porcine chromosome 17 using a somatic hybrid panel. The general utility of the method was subsequently demonstrated by locating PERVs in the genome of PERV infected human 293 cells. This method obviates the need for individual library construction or linker/adaptor ligation, and can be used to quickly locate individual sites of moderately repeated, dispersed DNA sequences in any genome. (+info)
Identification, characterization, and mapping of expressed sequence tags from an embryonic zebrafish heart cDNA library.
The generation of expressed sequence tags (ESTs) has proven to be a rapid and economical approach by which to identify and characterize expressed genes. We generated 5102 ESTs from a 3-d-old embryonic zebrafish heart cDNA library. Of these, 57.6% matched to known genes, 14.2% matched only to other ESTs, and 27.8% showed no match to any ESTs or known genes. Clustering of all ESTs identified 359 unique clusters comprising 1771 ESTs, whereas the remaining 3331 ESTs did not cluster. This estimates the number of unique genes identified in the data set to be approximately 3690. A total of 1242 unique known genes were used to analyze the gene expression patterns in the zebrafish embryonic heart. These were categorized into seven categories on the basis of gene function. The largest class of genes represented those involved in gene/protein expression (25.9% of known transcripts). This class was followed by genes involved in metabolism (18.7%), cell structure/motility (16.4%), cell signaling and communication (9.6%), cell/organism defense (7.1%), and cell division (4.4%). Unclassified genes constituted the remaining 17.91%. Radiation hybrid mapping was performed for 102 ESTs and comparison of map positions between zebrafish and human identified new synteny groups. Continued comparative analysis will be useful in defining the boundaries of conserved chromosome segments between zebrafish and humans, which will facilitate the transfer of genetic information between the two organisms and improve our understanding of vertebrate evolution. (+info)
Conservation of Mhc class III region synteny between zebrafish and human as determined by radiation hybrid mapping.
In the HLA, H2, and other mammalian MHC:, the class I and II loci are separated by the so-called class III region comprised of approximately 60 genes that are functionally and evolutionarily unrelated to the class I/II genes. To explore the origin of this island of unrelated loci in the middle of the MHC: 19 homologues of HLA class III genes, we identified 19 homologues of HLA class III genes as well as 21 additional non-class I/II HLA homologues in the zebrafish and mapped them by testing a panel of 94 zebrafish-hamster radiation hybrid cell lines. Six of the HLA class III and eight of the flanking homologues were found to be linked to the zebrafish class I (but not class II) loci in linkage group 19. The remaining homologous loci were found to be scattered over 14 zebrafish linkage groups. The linkage group 19 contains at least 25 genes (not counting the class I loci) that are also syntenic on human chromosome 6. This gene assembly presumably represents the pre-MHC: that existed before the class I/II genes arose. The pre-MHC: may not have contained the complement and other class III genes involved in immune response. (+info)
RHdb: the Radiation Hybrid database.
Since July 1995, the European Bioinformatics Institute (EBI) has maintained RHdb (http://www.ebi.ac.uk/RHdb), a public database for radiation hybrid data. Radiation hybrid mapping is an important technique for determining high resolution maps. RHdb is also served by CORBA servers. The EBI is an Outstation of the European Molecular Biology Laboratory (EMBL). (+info)
A novel syndrome affecting multiple mitochondrial functions, located by microcell-mediated transfer to chromosome 2p14-2p13.
We have studied cultured skin fibroblasts from three siblings and one unrelated individual, all of whom had fatal mitochondrial disease manifesting soon after birth. After incubation with 1 mM glucose, these four cell strains exhibited lactate/pyruvate ratios that were six times greater than those of controls. On further analysis, enzymatic activities of the pyruvate dehydrogenase complex, the 2-oxoglutarate dehydrogenase complex, NADH cytochrome c reductase, succinate dehydrogenase, and succinate cytochrome c reductase were severely deficient. In two of the siblings the enzymatic activity of cytochrome oxidase was mildly decreased (by approximately 50%). Metabolite analysis performed on urine samples taken from these patients revealed high levels of glycine, leucine, valine, and isoleucine, indicating abnormalities of both the glycine-cleavage system and branched-chain alpha-ketoacid dehydrogenase. In contrast, the activities of fibroblast pyruvate carboxylase, mitochondrial aconitase, and citrate synthase were normal. Immunoblot analysis of selected complex III subunits (core 1, cyt c(1), and iron-sulfur protein) and of the pyruvate dehydrogenase complex subunits revealed no visible changes in the levels of all examined proteins, decreasing the possibility that an import and/or assembly factor is involved. To elucidate the underlying molecular defect, analysis of microcell-mediated chromosome-fusion was performed between the present study's fibroblasts (recipients) and a panel of A9 mouse:human hybrids (donors) developed by Cuthbert et al. (1995). Complementation was observed between the recipient cells from both families and the mouse:human hybrid clone carrying human chromosome 2. These results indicate that the underlying defect in our patients is under the control of a nuclear gene, the locus of which is on chromosome 2. A 5-cM interval has been identified as potentially containing the critical region for the unknown gene. This interval maps to region 2p14-2p13. (+info)