Radiation Hybrid Panel Mapping Research Articles

Tandem duplication of thefabp1bgene and subsequent divergence of the tissue-specific distribution offabp1b.1andfabp1b.2transcripts in zebrafish (Danio rerio)

We describe a fatty acid-binding protein 1 (fabp1b.2) gene and its tissue-specific expression in zebrafish embryos and adults. The 3.5 kb zebrafish fabp1b.2 gene is the paralog of the previously described zebrafish fabp1a and fabp1b genes. Using the LN54 radiation hybrid mapping panel, we assigned the zebrafish fabp1b.2 gene to linkage group 8, the same linkage group to which fabp1b.1 was mapped. fabp1b.1 and fabp1b.2 appear to have arisen by a tandem duplication event. Whole-mount in situ hybridization of a riboprobe to embryos and larvae detected fabp1b.2 transcripts in the diencephalon and as spots in the periphery of the yolk sac. In adult zebrafish, in situ hybridization revealed fabp1b.2 transcripts in the anterior intestine and skin, and reverse transcription PCR (RT-PCR) detected fabp1b.2 transcripts in the intestine, brain, heart, ovary, skin, and eye. By contrast, fabp1b.1 transcripts were detected by RT-PCR in the liver, intestine, heart, testis, ovary, and gills. The tissue-specific distribution of transcripts for the tandemly duplicated fabp1b.1 and fabp1b.2 genes in adult tissues and during development suggests that the duplicated fabp1b genes of zebrafish have acquired additional functions compared with the ancestral fabp1 gene, i.e., by neofunctionalization. Furthermore, these functions were subsequently divided between fabp1b.1 and fabp1b.2 owing to subfunctionalization.

Assignment of CCR7 gene to chicken chromosome 27 by radiation hybrid panel mapping

The protein encoded by CC chemokine receptor 7 (CCR7) is a member of the G protein-coupled receptor family. This receptor was identified as a gene induced by the Epstein-Barr virus (EBV), and is thought to be a mediator of EBV effects on B lymphocytes. This receptor is expressed in various lymphoid tissues and activates B and T lymphocytes. It has been shown to control the migration of memory T cells to inflamed tissues, as well as stimulate dendritic cell maturation. To map the CCR7 gene in chicken chromosome, a 6 000 rads chicken-hamster radiation hybrid panel (ChickRH6) was used. PCR of samples from ChickRH6 revealed that the location of CCR7 gene is linked to the maker SEQ0347 (6 cR away) with LOD score of 16.6 and that the marker SEQ0347 is located on chromosome 27 at 27 cR of RH (radiation hydrid) map. We compared the corresponding human mRNA sequence with the predicted coding sequence of chicken CCR7 gene, and found that the assembled contig shared a high percentage of similarity with that of the human gene.

Assignment of Dicer Gene to Chicken Chromosome 5 by Radiation Hybrid Panel Mapping

Assignment of Dicer Gene to Chicken Chromosome 5 by Radiation Hybrid Panel Mapping

Assignment1 of the oviductal glycoprotein 1 gene (OVGP1) to porcine chromosome 4q22→q23 by radiation hybrid panel mapping

Assignment1 of the oviductal glycoprotein 1 gene (OVGP1) to porcine chromosome 4q22→q23 by radiation hybrid panel mapping

Assignment1 of the bovine PTGFR and HSD3B1 genes to bovine chromosome 3 with somatic and radiation hybrid panel mapping

Sao Paulo State Univ, UNESP, Dept Biol IBILCE, Lab Comparat Genom, BR-15054000 Sao Jose do Rio Preto, SP, Brazil

Assignment1 of the subunit C of succinate dehydrogenase complex (SDHC) gene to bovine chromosome 2 with somatic and radiation hybrid panel mapping

Sao Paulo State Univ, UNESP, IBILCE, Dept Biol,Lab Comparat Genom, BR-15054000 Sao Jose do rio Preto, SP, Brazil

Assignment of Signal Transducer and Activator of Transcription 5A (STAT5A) gene to porcine chromosome 12p13→p11 by radiation hybrid panel mapping

Assignment of Signal Transducer and Activator of Transcription 5A (STAT5A) gene to porcine chromosome 12p13→p11 by radiation hybrid panel mapping

Assignment of cellular retinoic acid-binding protein 1 (CRABP1) and 2 (CRABP2) to porcine chromosome 7q12→q23 and 4q21→q23 by somatic cell and radiation hybrid panel mapping

CRABPs (cellular retinoic acid binding proteins) belong to members of a superfamily of lipid-binding proteins (Mansfield et al., 1998) that are thought to act by maintaining tolerable concentrations of intracellular RA (retinoic acid), as modulators of RA catabolism (Gorry et al., 1994) and as intracellular transporters for RA from the cytoplasm to the nuclear receptor (Flagiello et al., 1997). Based on their significance roles in binding lipids, they are thought to be candidate genes for meat quality related traits in domestic animals. The mapping of the porcine CRABP1 and CRABP2 genes are one step towards further investigation on their possible roles in meat quality traits in pig.

An alternative to radiation hybrid mapping for large-scale genome analysis in barley

The presence of a monosomic gametocidal chromosome (GC) in a barley chromosome addition line of common wheat generates structural aberrations in the barley chromosome as well as in the wheat chromosomes of gametes lacking the GC. A collection of structurally aberrant barley chromosomes is analogous to a panel of radiation hybrid (RH) mapping and is valuable for high-throughput physical mapping. We developed 90 common wheat lines (GC lines) containing aberrant barley 7H chromosomes induced by a gametocidal chromosome, 2C. DNAs isolated from these GC lines provided a panel of 7H chromosomal fragments in a wheat genetic background, comparable with RH mapping panels in mammals. We used this 7H GC panel and the methodology for RH mapping to physically map PCR-based barley markers, SSRs and AFLPs, onto chromosome 7H, relying on polymorphism between the 7H chromosome and the wheat genome. We call this method GC mapping. This study describes a novel adaptation and combination of methods of inducing chromosomal rearrangements to produce physical maps of markers. The advantages of the presented method are similar to RH mapping in that non-polymorphic markers can be used and the mapping panels can be relatively easily obtained. In addition, mapping results are cumulative when using the same mapping set with new markers. The GC lines will be available from the National Bioresources Project-KOMUGI ( http://www.nbrp.jp/index.jsp ).

Assignment of protein kinase, AMP-activated,beta 2 non-catalytic subunit (PRKAB2) gene to porcine chromosome 4q21→ 23 by somatic cell and radiation hybrid panel mapping

Assignment of protein kinase, AMP-activated,beta 2 non-catalytic subunit (PRKAB2) gene to porcine chromosome 4q21→ 23 by somatic cell and radiation hybrid panel mapping

Assignment of the porcine FUS1 gene to SSC13q21→q22 by somatic cell and radiation hybrid panel mapping

FUS1 is a candidate tumor suppressor gene organized in a 370 kb region which contains a set of 19 genes. The human FUS1 genomic sequence spans 3.3 kb containing 3 exons (NM_007275; Lerman and Minna, 2000). Overexpression of the FUS1 gene leads to G1 arrest and growth inhibition of lung cancer cells (Kondo et al., 2001). The human and the murine FUS1 genes were assigned to chromosome HSA3p21.3 and MMU9F1, respectively. The sequence characterization of a porcine PAC clone revealed the identical gene cluster for pigs and human with respect to genes FUS1–HYAL2–HYAL1– FUS2–HYAL3. The porcine HYAL3 gene has recently been mapped to SSC13q21→q22 by fluorescence in situ hybridization (FISH) and radiation hybrid panel analysis while the somatic cell hybrid panel analysis resulted in the localization of the gene to SSC13q42→ (1/2q46) (Gatphayak et al., 2003). Considering the gene order in the cluster, the latter assignment is in accordance with the mapping of the porcine HYAL1 gene – which was provisionally called LUCA1 (for LUng CAncer) – to SSC13q23→ (1/2)q41 by Lahbib-Mansais et al. (1999) also using the somatic cell hybrid panel. Here we report the mapping of the porcine FUS1 gene by somatic cell and radiation hybrid panel and provide enhanced knowledge about the localization of the gene cluster. Methods and materials

The zebrafish mutant gene chardonnay (cdy) encodes divalent metal transporter 1 (DMT1)

Iron is an essential nutrient required for the function of all cells, most notably for the production of hemoglobin in red blood cells. Defects in the mechanisms of iron absorption, storage, or utilization can lead to disorders of iron-limited erythropoiesis or iron overload. In an effort to further understand these processes, we have used the zebrafish as a genetic system to study vertebrate iron metabolism. Here we characterized the phenotype of chardonnay (cdy), a zebrafish mutant with hypochromic, microcytic anemia, and positioned the mutant gene on linkage group 11. The cdy gene was isolated by a functional genomics approach in which we used a combination of expression studies, sequence analyses, and radiation hybrid panel mapping. We identified erythroid-specific genes using a whole embryo mRNA in situ hybridization screen and placed these genes on the zebrafish genomic map. One of these genes encoded the iron transporter divalent metal transporter 1 (DMT1) and colocalized with the cdy gene. We identified a nonsense mutation in the cdy allele and demonstrated that, whereas wild-type zebrafish DMT1 protein can transport iron, the truncated protein expressed in cdy mutants is not functional. Our studies further demonstrate the conservation of iron metabolism in vertebrates and suggest the existence of an alternative pathway of intestinal and red blood cell iron uptake.

The Functional Significance of Absence: The Chromosomal Segment Harboring Tp53 Is Absent from the T55 Rat Radiation Hybrid Mapping Panel

The T55 rat radiation hybrid (RH) mapping panel has been reported to retain the entire rat genome at retention frequencies between 22% and 37%. However, we found that a small segment of rat chromosome 10 harboring at least four different genes, including Tp53, was completely absent from the panel (retention frequency = 0%). Two other markers located in the vicinity exhibited much reduced retention (2–6%). RH clones are generated by transferring highly fragmented DNA into a recipient cell. There might be a strong selection against the transfer and retention of chromosome segments harboring an intact Tp53, as the action of this gene might prevent proliferation and establishment of the RH clone. Our finding further suggests that unexpected low retention or absence of chromosome segments in an RH panel may represent indications that the segments harbor genes with important functions in cell proliferation control.

Cloning, Expression, and Mapping of a Gene That Is Upregulated in Adipose Tissue of Mice Deficient in Bombesin Receptor Subtype-3

To identify novel obesity-related genes in adipose tissue, differential display was performed using bombesin receptor subtype-3 (BRS-3)-deficient mice. These mice exhibit mild late-onset obesity. We report that a gene, Urb, is upregulated in these mice. Full-length Urb cDNA is approximately 3 kb long and comprises an open reading frame of 949 amino acid residues. Interestingly, Urb mRNA expression in brown adipose tissue of BRS-3-deficient mice is fourfold higher than that in wild-type controls. Enhanced Urb mRNA expression was also observed in brain, digestive tissues, kidney, and lung. Within the brain, Urb mRNA is detected in the dorsal endopiriform nucleus and choroid plexus. A T31 radiation hybrid mapping panel revealed that the Urb gene maps to mouse chromosome 16. Collectively, these findings suggest that Urb may have a unique function in the regulation of body weight and energy metabolism.

Assignment of neuronal pentraxin I (NPTX1) gene to porcine chromosome 12pter by somatic cell and radiation hybrid panel mapping

The neuronal pentraxin I belongs to a family of functionally diverse proteins sharing some structural features like the formation of pentrameric complexes (Goodman et al., 1996). The NPTX1 gene is expressed in neurons and the protein was shown to bind the presynaptic toxin taipoxin. Therefore it is supposed to play a role in mediating synaptic uptake. The mouse and human neuronal pentraxin I genes were assigned to chromosomes 11E and 17q25 respectively. The mapping of the NPTX1 gene to the human chromosome regions for RusselSilver syndrome and campomelic dysplasia-1 makes it a potential candidate for genetic disorders (Omeis et al., 1996). The gene including the 5)and 3)-UTR is highly conserved among different species. The mapping of the porcine gene will enable further investigations on its possible role in inherited diseases.

A radiation hybrid mapping panel for the rhesus macaque.

The genomes of nonhuman primates have recently become highly visible candidates for full genome analysis, as they provide powerful models of human disease and a better understanding of the evolution of the human genome. We describe the creation of a 5000 rad radiation hybrid (RH) mapping panel for the rhesus macaque. Duplicate genotypes of 84 microsatellite and coding gene sequence tagged sites from six macaque chromosomes produced an estimated whole genome retention frequency of 0.33. To test the mapping ability of the panel, we constructed RH maps for macaque chromosomes 7 and 9 and compared them to orthologous locus orders in existing human and baboon maps derived from different methodologies. Concordant marker order between all three species maps suggests that the current panel represents a powerful mapping resource for generating high-density comparative maps of the rhesus macaque and other species genomes.

Evolutionary Breakpoints on Human Chromosome 21

Segments of the long arm of human chromosome 21 are conserved, centromere to telomere, in mouse chromosomes 16, 17, and 10. There have been 28 genes identified in human chromosome 21 between TMPRSS2, whose orthologue is the most distal gene mapped to mouse chromosome 16, and PDXK, whose orthologue is the most proximal gene mapped to mouse chromosome 10. Only 6 of these 28 genes have been mapped in mouse, and all are located on chromosome 17. To better define the chromosome 17 segment and the 16 to 17 transition, we used a combination of mouse radiation hybrid panel mapping and physical mapping by mouse: human genomic sequence comparison. We have determined the mouse chromosomal location of an additional 12 genes, predicted the location of 7 more,and defined the endpoints of the mouse chromosome 17 region. The mouse chromosome 16/chromosome 17 evolutionary breakpoint is between human genes ZNF295 and UMODL1, showing there are seven genes in the chromosome 16 segment distal to Tmprss2. The chromosome 17/chromosome 10 breakpoint seems to have involved a duplication of the gene PDXK, which on chromosome 21 lies immediately distal to the KIAA0179 gene. These data suggest that there may be as few as 21 functional genes in the mouse chromosome 17 segment. This information is important for defining existing and constructing more complete mouse models of Down syndrome.

Immune-type receptor genes in zebrafish share genetic and functional properties with genes encoded by the mammalian leukocyte receptor cluster.

An extensive, highly diversified multigene family of novel immune-type receptor (nitr) genes has been defined in Danio rerio (zebrafish). The genes are predicted to encode type I transmembrane glycoproteins consisting of extracellular variable (V) and V-like C2 (V/C2) domains, a transmembrane region and a cytoplasmic tail. All of the genes examined encode immunoreceptor tyrosine-based inhibition motifs in the cytoplasmic tail. Radiation hybrid panel mapping and analysis of a deletion mutant line (b240) indicate that a minimum of approximately 40 nitr genes are contiguous in the genome and span approximately 0.6 Mb near the top of zebrafish linkage group 7. One flanking region of the nitr gene complex shares conserved synteny with a region of mouse chromosome 7, which shares conserved synteny with human 19q13.3-q13.4 that encodes the leukocyte receptor cluster. Antibody-induced crosslinking of Nitrs that have been introduced into a human natural killer cell line inhibits the phosphorylation of mitogen-activated protein kinase that is triggered by natural killer-sensitive tumor target cells. Nitrs likely represent intermediates in the evolution of the leukocyte receptor cluster.

Cloning, Expression, and Mapping of a Mouse Gene, Uchl4, Highly Homologous to Human and Mouse Uchl3

Ubiquitin carboxyl-terminal hydrolases (UCHs) are implicated in the proteolytic processing of polymeric ubiquitin. We have isolated a novel mouse gene for ubiquitin carboxyl-terminal hydrolase isozyme L4. The gene named Uchl4 encodes a novel member of the family of ubiquitin carboxyl-terminal hydrolases (UCHs) whose predicted amino acid sequence shows 95% identity to mouse UCH-L3 and 94% identity to human UCH-L3. Genomic structure, chromosome localization, and expression pattern of Uchl3 and Uchl4 were characterized in the mouse. Both Uchl3 and Uchl4 were expressed in various tissues examined; however, expression level was quite lower in Uchl4. While Uchl3 consists of at least 9 exons spanning about 12 kb, Uchl4 was an intronless gene with a size of about 2 kb. By PCR-based analysis with T31 radiation hybrid mapping panel, Uchl3 and Uchl4 were mapped on mouse chromosome 9 and 14, respectively.

CDNA and Protein Sequence, Genomic Organization, and Analysis of cis Regulatory Elements of Mouse and Human PLVAP Genes

PV-1 is a novel protein component of the endothelial fenestral and stomatal diaphragms. PV-1 cDNA and protein sequences are highly conserved across species. The conserved extracellular domain features found in rat, mouse, and human PV-1 protein are four N-glycosylation sites, two coiled-coil domains, a proline-rich region, and even cysteine spacing. No consensus site in the intracellular domain was found. Northern blotting of mouse and human tissues is in agreement with and expands those performed in rat and correlated well with the postulated presence of PV-1 in the endothelial diaphragms. The genomic organization of the human and mouse genes (HGMW-approved symbol PLVAP) has been determined, and the analysis of their 5′ flanking regions has found a highly conserved classical TATA-driven promoter that shows several transcription factor consensus binding sites. Radiation hybrid panel mapping has localized the human and mouse PLVAP genes to chromosomes 19p13.2 and 8B3–C1, respectively.