B. P. Chowdhary

Comparative analysis of the cattle and human genomes: detection of ZOO-FISH and gene mapping-based chromosomal homologies

Comparative chromosome painting with individual human chromosome-specific libraries (CSLs) on cattle metaphase chromosomes delineated 46 homologous chromosomal segments between the two species. Continuous arrangement of these segments on individual cattle chromosomes demonstrates a nearly complete coverage of the bovine karyotype and shows physical boundaries of bovine chromosomal segments homologous to individual human chromosomes. Alignment of the available comparative gene mapping data with the homologous segments strongly supports the detected gross homologies between the karyotypes of the two species. In addition to cattle, four human CSLs were hybridized to sheep metaphase chromosomes also, to further verify the known karyotype homology within the Bovidae. Besides its application to karyotype evolution research, the comparative knowledge provides for rapid expansion of the much needed Type I locus-based bovine gene map.

Pigs with the dominant white coat color phenotype carry a duplication of the KIT gene encoding the mast/stem cell growth factor receptor.

Comparative mapping data suggested that the dominant white coat color in pigs may be due to a mutation in KIT which encodes the mast/stem cell growth factor receptor. We report here that dominant white pigs lack melanocytes in the skin, as would be anticipated for a KIT mutation. We found a complete association between the dominant white mutation and a duplication of the KIT gene, or part of it, in samples of unrelated pigs representing six different breeds. The duplication was revealed by single strand conformation polymorphism (SSCP) analysis and subsequent sequence analysis howing that white pigs transmitted two nonallelic KIT sequences. Quantitative Southern blot and quantitative PCR analysis, as well as fluorescence in situ hybridization (FISH) analysis, confirmed the presence of a gene duplication in white pigs. FISH analyses showed that KIT and the very closely linked gene encoding the platelet-derived growth factor receptor (PDG-FRA) are both located on the short arm of Chromosome (Chr) 8 at band 8pl2. The result revealed an extremely low rate of recombination in the centromeric region of this chromosome, since the closely linked (0.5 cM) serum albumin (ALB) locus has previously been in situ mapped to the long arm (8ql2). Pig Chr 8 shares extensive conserved synteny with human Chr 4, but the gene order is rearranged.

A comparative map of the porcine and human genomes demonstrates ZOO-FISH and gene mapping-based chromosomal homologies

ZOO-FISH with chromosome-specific DNA libraries (CSLs) from individual flow-sorted human chromosomes was applied on porcine metaphase chromosomes to establish segment homology between the pig and human karyotypes. Forty-seven porcine chromosomal segments corresponding to all human chromosomes except the Y were delineated, resulting in a nearly complete coverage of the porcine karyotype. The syntenic segments detected were further confirmed by the gene mapping information available in the two species. A map demarcating physical boundaries of human homologies on individual pig chromosomes is complemented with a detail survey of the physical and genetic linkage mapping data in the two species. The resultant map, thus, provides a comprehensive and updated comparative status of the human and porcine genomes.

Zoo-FISH delineates conserved chromosomal segments in horse and man

Human chromosome specific libraries (CSLs) were individually applied to equine metaphase chromosomes using the fluorescencein situ hybridization (FISH) technique. All CSLs, except Y, showed painting signals on one or several horse chromosomes. In total 43 conserved chromosoma segments were painted. Homoeology could not, however, be detected for some segments of the equine genome. This is most likely related to the very weak signals displayed by some libraries, rather than to the absence of similarity with the human genome. In spite of divergence from the human genome, dated 70–80 million years ago, a fairly high degree of synteny conservation was observed. In seven cases, whole chromosome synteny was detected between the two species. The comparative painting results agreed completely with the limited gene mapping data available in horses, and also enabled us provisionally to assign one linkage group (U2) and one syntenic group (NP, MPI, IDH2) to specific equine chromosomes. Chromosomal assignments of three other syntenic groups are also proposed. The findings of this study will be of significant use in the expansion of the hitherto poorly developed equine gene map.

Assignment of the porcine calcium release channel gene, a candidate for the malignant hyperthermia locus, to the 6p11----q21 segment of chromosome 6.

Several studies point to the possibility that malignant hyperthermia (MH) in pigs is caused by a defect in the calcium release channel (CRC) of skeletal muscle sarcoplasmic reticulum. The locus for MH is closely linked to the glucosephosphate isomerase (GPI) locus, near the centromere of chromosome 6. We demonstrate synteny of the genes for CRC and GPI using somatic cell hybrid lines, and assign the CRC gene to chromosome 6p11----q21 by in situ hybridization.

The PiGMaP consortium cytogenetic map of the domestic pig (Sus scrofa domestica)

llNRA, Laboratoire de Grnrtique Cellulaire, BP27, 31326 Castanet-Tolosan, France 2Department of Functional Morphology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands 3Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Uppsala, Sweden 4Division of Anatomy, Department of Anatomy and Physiology, The Royal Veterinary and Agricultural University, Copenhagen, Denmark 5Division of Animal Genetics, Department of Animal Science and Animal Health, The Royal Veterinary and Agricultural University, Copenhagen, Denmark 6Department of Clinical Genetics, University of Ulm, Ulm, Germany 7Laboratoire de Cytomrtrie, CEA, Fontenay-aux Roses, France

Linkage and comparative mapping of the locus controlling susceptibility towards E. coli F4ab/ac diarrhoea in pigs

In 1995, Edfors-Lilja and coworkers mapped the locus for the E. coli K88ab (F4ab) and K88ac (F4ac) intestinal receptor to pig chromosome 13 (SSC13). Using the same family material we have refined the map position to a region between the microsatellite markers Sw207 and Sw225. Primers from these markers were used to screen a pig BAC library and the positive clones were used for fluorescent in situ hybridization (FISH) analysis. The results of the FISH analysis helped to propose a candidate gene region in the SSC13q41→q44 interval. Shotgun sequencing of the FISH-mapped BAC clones revealed that the candidate region contains an evolutionary breakpoint between human and pig. In order to further characterise the rearrangements between SSC13 and human chromosome 3 (HSA3), detailed gene mapping of SSC13 was carried out. Based on this mapping data we have constructed a detailed comparative map between SSC13 and HSA3. Two candidate regions on human chromosome 3 have been identified that are likely to harbour the human homologue of the gene responsible for susceptibility towards E. coli F4ab/ac diarrhoea in pigs.

Comparison of horse Chromosome 3 with donkey and human chromosomes by cross-species painting and heterologous FISH mapping

Abstract. The melanocortin 1 receptor (MC1R), mast/stem cell growth factor receptor (KIT), and platelet-derived growth factor receptor α (PDGFRA) are loci that all belong to equine linkage group 2 (LG2). Of these, KIT was fluorescent in situ hybridization (FISH) mapped to ECA3q21 with equine cDNA and heterologous porcine BAC probes, while MC1R was localized to ECA3p12 and PDGFRA to ECA3q21 with heterologous porcine BAC probes. A three-step comparison between ECA3 and donkey chromosomes was carried out. First, microdissected ECA3 painting probe was used on donkey chromosomes, which showed disruption of the equine synteny. Next, human (HSA) Chromosomes (Chrs) 16q and 4 specific paints, known to be homologous to ECA3p and 3q, respectively, were applied to detect homologous chromosomal segment(s) in donkey. Finally, four genes (MC1R, ALB, PDGFRA, KIT) and two equine microsatellite markers (SGCV18 and SGCV33) located on ECA3 were FISH mapped to donkey chromosomes. The findings refined the cross species painting homology results and added six new markers to the nascent donkey gene map. The hypothesis that Tobiano coat color in horses may be associated with a chromosomal inversion involving genes within LG2 was tested by G-banding-based cytogenetic analysis and ordering of four loci—KIT, PDGFRA, albumin (ALB), and MC1R—in Tobiano and non-tobiano (homozygous as well as heterozygous) horses. However, no difference either in banding patterns or location/relative order of the genes was observed in the three classes. The study highlights successful FISH mapping of BAC probes across evolutionarily diverged species, viz., pig and horse/donkey, and represents the first use of large-sized individual clones across distantly related farm animals.

Cosmid-derived markers anchoring the bovine genetic map to the physical map.

The mapping strategy for the bovine genome described in this paper uses large insert clones as a tool for physical mapping and as a source of highly polymorphic microsatellites for genetic typing, and was one objective of the BovMap Project funded by the European Union (UE). Eight-three cosmid and phage clones were characterized and used to physically anchor the linkage groups defining all the bovine autosomes and the X Chromosome (Chr). By combining physical and genetic mapping, clones described in this paper have led to the identification of the linkage groups corresponding to Chr 9, 12, 16, and 25. In addition, anchored loci from this study were used to orient the linkage groups corresponding to Chr 3, 7, 8, 9, 13, 16, 18, 19, and 28 as identified in previously published maps. Comparison of the estimated size of the physical and linkage maps suggests that the genetic length of the bovine genome may be around 4000 cM.

Precise localization of the genes for glucose phosphate isomerase (GPI), calcium release channel (CRC), hormone-sensitive lipase (LIPE), and growth hormone (GH) in pigs, using nonradioactive in situ hybridization

Fluorescence in situ hybridization (FISH) was applied, using genomic DNA clones, to precisely localize the genes for GPI, CRC, LIPE, and GH on pig chromosomes. The porcine CRC gene was localized to band 6q12 using both genomic and cDNA clones. The GPI and LIPE genes, which are closely linked to the CRC gene, were also mapped to the same band (6q12), using genomic lambda clones. The mapping data are a refinement of earlier findings, wherein radioactive in situ hybridization was used and the assignments included both the short and long arms. Results of the present study clearly exclude the short arm as the location for the three genes. Further, using a genomic cosmid clone, the GH gene was mapped to band 12p14. Compared to the earlier assignments, which included almost the entire short arm of the chromosome due to the use of radioactive in situ hybridization, the present FISH findings provide a band-specific localization for the gene. A modified, simpler version of the posthybridization trypsin/EDTA banding method is also presented.