A comparative radiation hybrid map of bovine chromosome 18 and homologous chromosomes in human and mice

A comprehensive radiation hybrid (RH) map and a high resolution comparative map of Bos taurus (BTA) chromosome 18 were constructed, composed of 103 markers and 76 markers, respectively, by using a cattle-hamster somatic hybrid cell panel and a 5,000 rad whole-genome radiation hybrid (WGRH) panel. These maps include 65 new assignments (56 genes, 3 expressed-sequence tags, 6 microsatellites) and integrate 38 markers from the first generation WGRH(5,000) map of BTA18. Fifty-nine assignments of coding sequences were supported by somatic hybrid cell mapping to markers on BTA18. The total length of the comprehensive map was 1666 cR(5,000). Break-point positions within the chromosome were refined and a new telomeric RH linkage group was established. Conserved synteny between cattle, human, and mouse was found for 76 genes of BTA18 and human chromosomes (HSA) 16 and 19 and for 34 cattle genes and mouse chromosomes (MMU) 7 and 8. The new RH map is potentially useful for the identification of candidate genes for economically important traits, contributes to the expansion of the existing BTA18 gene map, and provides new information about the chromosome evolution in cattle, humans, and mice.     

Evidence for the evolutionary origin of human chromosome 21 from comparative gene mapping in the cow and mouse.

To determine the extent of conservation between bovine syntenic group U10, human chromosome 21 (HSA 21), and mouse chromosome 16 (MMU 16), 11 genes were physically mapped by segregation analysis in a bovine-hamster hybrid somatic cell panel. The genes chosen for study span MMU 16 and represent virtually the entire q arm of HSA 21. Because the somatostatin gene (SST), an HSA 3/MMU 16 locus, was previously shown to be in U10, the transferrin gene (TF), an HSA 3/MMU 9 marker, was also mapped to determine whether U10 contains any HSA 3 genes not represented on MMU 16. With the exception of the protamine gene PRM1 (HSA 16/MMU 16), all of the genes studied were syntenic on bovine U10. Thus, all homologous loci from HSA 21 that have been studied in the cow are on a single chromosome. The bovine homolog of HSA 21 also carries several HSA 3 genes, two of which have homologous loci on MMU 16. The syntenic association of genes from the q arm of HSA 3 with HSA 21 genes in two mammalian species, the mouse and the cow, indicates that HSA 21 may have that contained genes now residing on HSA 3. Additionally, the syntenic association of TF with SST in the cow permits the prediction that the rhodopsin gene (RHO) is proximal to TF on HSA 3q.     

A high-resolution whole-genome cattle-human comparative map reveals details of mammalian chromosome evolution

Approximately 3,000 cattle bacterial artificial chromosome (BAC)-end sequences were added to the Illinois-Texas 5,000-rad RH (RH, radiation hybrid) map. The BAC-end sequences selected for mapping are approximately 1 Mbp apart on the human chromosomes as determined by blastn analysis. The map has 3,484 ordered markers, of which 3,204 are anchored in the human genome. Two hundred-and-one homologous synteny blocks (HSBs) were identified, of which 27 are previously undiscovered, 79 are extended, 26 were formed by previously unrecognized breakpoints in 18 previously defined HSBs, and 23 are the result of fusions. The comparative coverage relative to the human genome is approximately 91%, or 97% of the theoretical maximum. The positions of 64% of all cattle centromeres and telomeres were reassigned relative to their positions on the previous map, thus facilitating a more detailed comparative analysis of centromere and telomere evolution. As an example of the utility of the high-resolution map, 22 cattle BAC fingerprint contigs were directly anchored to cattle chromosome 19 [Bos taurus, (BTA) 19]. The order of markers on the cattle RH and fingerprint maps of BTA19 and the sequence-based map of human chromosome 17 [Homo sapiens, (HSA) 17] were found to be highly consistent, with only two minor ordering discrepancies between the RH map and fingerprint contigs. The high-resolution Illinois-Texas 5,000-rad RH and comparative maps will facilitate identification of candidate genes for economically important traits, the phylogenomic analysis of mammalian chromosomes, proofing of the BAC fingerprint map and, ultimately, aid the assembly of cattle whole-genome sequence.     

Exceptional conservation of horse-human gene order on X chromosome revealed by high-resolution radiation hybrid mapping

Development of a dense map of the horse genome is key to efforts aimed at identifying genes controlling health, reproduction, and performance. We herein report a high-resolution gene map of the horse (Equus caballus) X chromosome (ECAX) generated by developing and typing 116 gene-specific and 12 short tandem repeat markers on the 5,000-rad horse x hamster whole-genome radiation hybrid panel and mapping 29 gene loci by fluorescence in situ hybridization. The human X chromosome sequence was used as a template to select genes at 1-Mb intervals to develop equine orthologs. Coupled with our previous data, the new map comprises a total of 175 markers (139 genes and 36 short tandem repeats, of which 53 are fluorescence in situ hybridization mapped) distributed on average at approximately 880-kb intervals along the chromosome. This is the densest and most uniformly distributed chromosomal map presently available in any mammalian species other than humans and rodents. Comparison of the horse and human X chromosome maps shows remarkable conservation of gene order along the entire span of the chromosomes, including the location of the centromere. An overview of the status of the horse map in relation to mouse, livestock, and companion animal species is also provided. The map will be instrumental for analysis of X linked health and fertility traits in horses by facilitating identification of targeted chromosomal regions for isolation of polymorphic markers, building bacterial artificial chromosome contigs, or sequencing.     

Fine linkage mapping of the blood pressure quantitative trait locus region on rat chromosome 1.

To narrow the area known to contain the blood pressure quantitative trait locus (QTL) on rat chromosome 1, we constructed a fine linkage map covering the blood pressure OTL region on the chromosome using 22 genetic markers informative for stroke-prone spontaneously hypertensive rats of the Izumo colony (SHRSP/Izm) and Wistar-Kyoto rats of the Izumo colony (WKY/Izm). Linkage mapping was done by genotyping 626 backcrossed rats from matings between SHRSP/Izm and WKY/Izm. Nineteen genetic markers informative for the two strains were selected from public databases. Two markers were newly isolated by screening a rat genomic library. One marker was mapped using a restriction endonuclease polymorphism. The region between DlWox29 and D1Smu11 was covered with 22 informative markers placed every 0.6 cM on average. In addition, 6 physiological candidates for a hypertension gene were mapped in this region either by linkage or by radiation hybrid (RH) mapping. This information should be essential for the construction and analysis of congenic strains for this QTL region.     

Comparative mapping of the human 22q11 chromosomal region and the orthologous region in mice reveals complex changes in gene organization

The region of human chromosome 22q11 is prone to rearrangements. The resulting chromosomal abnormalities are involved in Velo-cardio-facial and DiGeorge syndromes (VCFS and DGS) (deletions), “cat eye” syndrome (duplications), and certain types of tumors (translocations). As a prelude to the development of mouse models for VCFS/DGS by generating targeted deletions in the mouse genome, we examined the organization of genes from human chromosome 22q11 in the mouse. Using genetic linkage analysis and detailed physical mapping, we show that genes from a relatively small region of human 22q11 are distributed on three mouse chromosomes (MMU6, MMU10, and MMU16). Furthermore, although the region corresponding to about 2.5 megabases of the VCFS/DGS critical region is located on mouse chromosome 16, the relative organization of the region is quite different from that in humans. Our results show that the instability of the 22q11 region is not restricted to humans but may have been present throughout evolution. The results also underscore the importance of detailed comparative mapping of genes in mice and humans as a prerequisite for the development of mouse models of human diseases involving chromosomal rearrangements.     

Genetic fine-structure mapping in human chromosome 11 by use of repetitive DNA sequences.

A method is described for mapping of the DNA fragments of a human chromosome produced by restriction enzyme treatment of the total DNA from a hybrid cell containing a single human chromosome. The method involves production and selection of somatic cell mutants containing deletions of the human chromosome and application of a hybridization probe consisting of an individual member copy of a repetitive human DNA family. A linear map has been constructed of 19 marker DNA fragments and 5 immunological and biochemical markers on human chromosome 11, selected as a model chromosome for these studies. This approach appears to be widely applicable, is independent of cytogenetic analysis, promises to be capable of revealing the existence of rearrangements as well as deletions, appears to be amenable to further increase in resolving power, and offers potential application in various human genetic problems.     

Fine structure mapping of a gene-rich region of wheat carrying Ph1, a suppressor of crossing over between homoeologous chromosomes

The wheat gene-rich region (GRR) 5L0.5 contains many important genes, including Ph1, the principal regulator of chromosome pairing. Comparative marker analysis identified 32 genes for the GRR controlling important agronomic traits. Detailed characterization of this region was accomplished by first physically localizing 213 wheat group 5L-specific markers, using group 5 nulli-tetrasomics, three Ph1 gene deletion/insertion mutants, and nine terminal deletion lines with their breakpoints around the 5L0.5 region. The Ph1 gene was localized to a much smaller region within the GRR (Ph1 gene region). Of the 61 markers that mapped in the four subregions of the GRR, 9 mapped in the Ph1 gene region. High stringency sequence comparison (e < 1 x10(-25)) of 157 group 5L-specific wheat ESTs identified orthologs for 80% sequences in rice and 71% in Arabidopsis. Rice orthologs were present on all rice chromosomes, although most (34%) were on rice chromosome 9 (R9). No single collinear region was identified in Arabidopsis even for a smaller region, such as the Ph1 gene region. Seven of the nine Ph1 gene region markers mapped within a 450-kb region on R9 with the same gene order. Detailed domain/motif analysis of the 91 putative genes present in the 450-kb region identified 26 candidates for the Ph1 gene, including genes involved in chromatin reorganization, microtubule attachment, acetyltransferases, methyltransferases, DNA binding, and meiosis/anther specific proteins. Five of these genes shared common domains/motifs with the meiosis specific genes Zip1, Scp1, Cor1, RAD50, RAD51, and RAD57. Wheat and Arabidopsis homologs for these rice genes were identified.     

Linkage mapping of the human CSF2 and IL3 genes.

Interleukin 3 (encoded by the IL3 gene) and granulocyte-macrophage colony-stimulating factor (encoded by the CSF2 gene) are small secreted polypeptides that bind to specific cell surface receptors and regulate the growth, gene expression, and differentiation of many of the hematopoietic cell lineages, particularly nonlymphoid cells. The IL3 and CSF2 genes have been cloned and mapped to human chromosome bands 5q23-31. Only 10 kilobases of DNA separates the two genes, suggesting that they have a common origin and/or regulation. We have cloned 70 kilobases of genomic DNA that includes the IL3 and CSF2 genes, as well as flanking sequences, and report a physical map of this region. Several unique-sequence DNA segments have been identified in this region, and one of these fragments detects two restriction fragment length polymorphisms in DNA from unrelated Caucasians. Segregation of these DNA polymorphisms was followed in the Centre Etudé du Polymorphisme Humaine (CEPH) panel of 40 large three-generation pedigrees, and linkage was detected with 17 genetic markers previously typed in these families. Multipoint linkage analysis permits the placement of the region containing the IL3 and CSF2 structural genes on the recombination-genetic linkage map of chromosome 5q and thereby allows the role of these genes in leukemogenesis to be more critically examined.     

Physical mapping of autonomic/sympathetic candidate genetic loci for hypertension in the human genome: a somatic cell radiation hybrid library approach

Allelic variation at multiple genetic loci may contribute to hypertension. Since autonomic/sympathetic dysfunction may play an early, pathogenic, heritable role in hypertension, we evaluated candidate loci likely to contribute to such dysfunction, including catecholamine biosynthetic enzymes, catecholamine transporters, neuropeptides, and adrenergic receptors. Since chromosomal locations and physical map positions of many of these loci had not yet been identified, we used the GeneBridge4 human/hamster radiation (somatic cell) hybrid library panel (resolution approximately 1 to approximately 1.5 Mb), along with specifically designed oligonucleotide primers and PCR (200-400 bp products) to position these loci in the human genome. Primers were designed from sequences outside the coding regions (3'-flanking or intronic segments) to avoid cross-species (hamster) amplification. Chromosomal positions were assigned in cR (centi-Ray) units ( approximately 270 Kbp/cR(3000) for GeneBridge 4). A total of 13 loci were newly assigned chromosomal positions; of particular interest was a cluster of adrenergic candidate loci on chromosome 5q (including ADRB2, ADRA1A, DRD1, GPRK6, and NPY6R), a region harbouring linkage peaks for blood pressure. Such physical map positions will enable more precise selection of polymorphic microsatellite and single nucleotide polymorphism markers at these loci, to aid in linkage and association studies of autonomic/sympathetic dysfunction in human hypertension.     

Radiation hybrid mapping of the zebrafish genome

The zebrafish is an excellent genetic system for the study of vertebrate development and disease. In an effort to provide a rapid and robust tool for zebrafish gene mapping, a panel of radiation hybrids (RH) was produced by fusion of irradiated zebrafish AB9 cells with mouse B78 cells. The overall retention of zebrafish sequences in the 93 RH cell lines that constitute the LN54 panel is 22%. Characterization of the LN54 panel with 849 simple sequence length polymorphism markers, 84 cloned genes and 122 expressed sequence tags allowed the production of an RH map whose total size was 11,501 centiRays. From this value, we estimated the average breakpoint frequency of the LN54 RH panel to correspond to 1 centiRay = 148 kilobase. Placement of a group of 235 unbiased markers on the RH map suggests that the map generated for the LN54 panel, at present, covers 88% of the zebrafish genome. Comparison of marker positions in RH and meiotic maps indicated a 96% concordance. Mapping expressed sequence tags and cloned genes by using the LN54 panel should prove to be a valuable method for the identification of candidate genes for specific mutations in zebrafish.     

Comparative gene mapping: order of loci on the X chromosome is different in mice and humans.

For comparative studies we have used the somatic cell hybridization approach to regionally map genes on the mouse X chromosome. Fibroblasts from a mouse with the balanced reciprocal translocation T(XD;16B5)16H were fused with a Chinese hamster cell line (V79/380-6) deficient in activity of the enzyme hypoxanthine phosphoribosyltransferase (HPRT). Interpecific cell hybrids were initially selected for retention of the mouse translocation chromosome carrying the Hprt gene. Subsequently, hybrid clones were counterselected to force segregation of this chromosome. Selected and counterselected hybrid clones were analyzed for their chromosome content by trypsin/Giemsa banding and for expression of the mouse forms of the X-linked enzymes HPRT and alpha-galactosidase (GALA) by isoelectric focusing. The results indicate that the breakpoint on the mouse X chromosome (in band XD) has separated the genes for HPRT (Hprt) and for GALA (Ags). Hprt is proximal to the breakpoint in region Xcen-XD and Ags is distal in region XD-Xter. The gene order in the mouse (centromere-Hprt-Ags) is therefore inverted when compared to the order of the homologous loci on the long arm of the human X (centromere-GALA-HPRT).     

Chromosomal locations of the human and mouse genes for precursors of epidermal growth factor and the beta subunit of nerve growth factor.

DNA probes for pre-pro-epidermal growth factor (EGF) and the precursor of the beta subunit of nerve growth factor (NGF) were used to chromosomally map human and mouse EGF and NGF genes in panels of human-mouse and mouse-Chinese hamster somatic cell hybrids. The EGF and NGF genes were mapped to human chromosomes 4 and 1, respectively, by using human-mouse cell hybrids. A combination of regional mapping using a chromosome 1 translocation and comparative gene mapping suggests that the human NGF gene is in the p21-p22.1 region of chromosome 1. In mouse-Chinese hamster cell hybrids, both genes were assigned to mouse chromosome 3. A knowledge of the chromosomal assignment of these genes should help in our understanding of their regulation and role in development and disease.     

CpG island clones from a deletion encompassing the gene for adenomatous polyposis coli.

Adenomatous polyposis coli (APC), a dominantly inherited disorder, has been mapped to chromosome 5q15-q21 by family linkage studies. Cells from patients with deletions in this region, in one case associated with polyposis in a family, have been used to construct human hamster hybrid cell lines that retain either the normal or deleted chromosome 5. These lines have been used to identify markers from the region of the polyposis gene obtained by cloning the ends of 0.5- to 2-megabase BssHII fragments purified by pulsed-field gel electrophoresis. Three markers are described that map within the deletions and must therefore be close to the APC gene.     

Centromere mapping and orientation of the molecular linkage map of rice (Oryza sativa L.).

Rice has become a model cereal plant for molecular genetic research. Rice has the most comprehensive molecular linkage maps with more than 2000 DNA markers and shows synteny and colinearity with the maps of other cereal crops. Until now, however, no information was available about the positions of centromeres and arm locations of markers on the molecular linkage map. Secondary and telotrisomics were used to assign restriction fragment length polymorphism markers to specific chromosome arms and thereby to map the positions of centromeres. More than 170 restriction fragment length polymorphism markers were assigned to specific chromosome arms through gene dosage analysis using the secondary and telotrisomics and the centromere positions were mapped on all 12 linkage groups. The orientations of seven linkage groups were reversed to fit the "short arm on top" convention and the corrected map is presented.     

Universal mapping probes and the origin of human chromosome 3.

Universal mapping probes (UMPs) are defined as short segments of human DNA that are useful for physical and genetic mapping in a wide variety of mammals. The most useful UMPs contain a conserved DNA sequence immediately adjoined to a highly polymorphic CA repeat. The conserved region determines physical gene location, whereas the CA repeat facilitates genetic mapping. Both the CA repeat and its neighboring sequence are highly conserved in evolution. This permits molecular, cytogenetic, and genetic mapping of UMPs throughout mammalia. UMPs are significant because they make genetic information cumulative among well-studied species and because they transfer such information from "map rich" organisms to those that are "map poor." As a demonstration of the utility of UMPs, comparative maps between human chromosome 3 (HSA3) and the rat genome have been constructed. HSA3 is defined by at least 12 syntenic clusters located on seven different rat chromosomes. These data, together with previous comparative mapping information between human, mouse, and bovine genomes, allow us to propose a distinct evolutionary pathway that connects HSA3 with the chromosomes of rodents, artiodactyls, and primates. The model predicts a parsimonious phylogenetic tree, is readily testable, and will be of considerable use for determining the pathways of mammalian evolution.     

Restriction fragment length polymorphism linkage map for Arabidopsis thaliana

We have constructed a restriction fragment length polymorphism linkage map for the nuclear genome of the flowering plant Arabidopsis thaliana. The map, containing 90 randomly distributed molecular markers, is physically very dense; greater than 50% of the genome is within 1.9 centimorgans, or approximately 270 kilobase pairs, of the mapped DNA fragments. The map was based on the meiotic segregation of markers in two different crosses. The restriction fragment length polymorphism linkage groups were integrated with the five classically mapped linkage groups by virtue of mapped mutations included in these crosses. Markers consist of both cloned Arabidopsis genes and random low-copy-number genomic DNA clones that are able to detect polymorphisms with the restriction enzymes EcoRI, Bgl II, and/or Xba I. These cloned markers can serve as starting points for chromosome walking, allowing for the isolation of Arabidopsis genes of known map location. The restriction fragment length polymorphism map also can associate clones of unknown gene function with mutant phenotypes, and vice versa.     

Human genes involved in cholesterol metabolism: chromosomal mapping of the loci for the low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl-coenzyme A reductase with cDNA probes.

Cellular cholesterol metabolism is regulated primarily through the coordinate expression of two proteins, the low density lipoprotein (LDL) receptor and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (EC 1.1.1.34). We have used cDNA probes for the human genes encoding these proteins to determine the precise chromosomal location of the two loci. By in situ hybridization we have regionally mapped the LDL receptor gene, LDLR, to the short arm of chromosome 19 in bands p13.1-p13.3. This result concurs with and extends a previous study in which LDLR was mapped to chromosome 19 by screening somatic cell hybrids with a species-specific monoclonal antibody. We have assigned the HMG-CoA reductase gene, HMGCR, to chromosome 5 by Southern blotting of DNA from a somatic cell hybrid panel and to bands 5q13.3-q14 by in situ hybridizations of the cDNA probe to human metaphase cells with normal and rearranged chromosomes.