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Among the advanced karyotype analysis approaches embraced by animal cytogenetics during the past decade, chromosome paintinghas had the greatest impact. Generation of chromosome specific paints is considered pivotal to his development. Additionally, ability to use these paints across species (referred to as Zoo-FISHor comparative painting) is undisputedly the most important breakthrough that has contributed to our ability to compare karyotypes of a wide range of evolutionarily highly diverged chromosome painting, and makes them aware of the tools/resources available to carry out this research in a variety of animal species. An overview of the current status of comparative chromosome painting results across closely as well as distantly related species is presented. Findings from different studies show how some segmental syntenies are more conserved as compared to others. The comparisons provide insight into the likely constitution of a vertebrate/mammalian ancestral karyotype and help understand some of the intricacies about karyotype evolution. Importance of comparative painting in setting the stage for rapid development of gene maps in a number of economically important species is elaborated.

Neuropeptide Y (NPY) receptors mediate a variety of physiological responses including feeding and vasoconstriction. To investigate the evolutionary events that have generated this receptor family, we have sequenced and determined the chromosomal localizations of all five presently known mammalian NPY receptor subtype genes in the domestic pig, Sus scrofa (SSC). The orthologs of the Y(1) and Y(2) subtypes display high amino acid sequence identities between pig, human, and mouse (92%-94%), whereas the Y(4), Y(5), and y(6) subtypes display lower identities (76%-87%). The lower identity of Y(5) is due to high sequence divergence in the large third intracellular loop. The NPY1R, NPY2R, and NPY5R receptor genes were localized to SSC8, the NPY4R to SSC14, and NPY6R to SSC2. Our comparisons strongly suggest that the tight cluster of NPY1R, NPY2R, and NPY5R on human chromosome 4 (HSA4) represents the ancestral configuration, whereas the porcine cluster has been split by two inversions on SSC8. These 3 genes, along with adjacent genes from 14 other gene families, form a cluster on HSA4 with extensive similarities to a cluster on HSA5, where NPY6R and >13 other paralogs reside, as well as another large cluster on HSA10 that includes NPY4R. Thus, these gene families have expanded through large-scale duplications. The sequence comparisons show that the NPY receptor triplet NPY1R-NPY2R-NPY5R existed before these large-scale duplications.

The terminal part of the long arm of the bovine X chromosome (bands Xq41--q43) was microdissected. The DNA thus obtained was PCR amplified, labelled and used as painting probe on cattle, sheep, goat and buffalo chromosomes. In cattle, as expected, distinct hybridization signals were observed on bands Xq41--q43. In sheep and goat, the painting signals were observed on the proximal part of the long arm of the X chromosome, adjacent to the centromere (Xp12--q12). In buffalo, however, the terminal part of the X chromosome involving bands q44--q47 was painted. The findings contribute towards developing a better understanding of the comparative organization of the X chromosome in the four bovidae species. Proposed models of evolutionary rearrangements within the X chromosome of the four species are examined in light of the results obtained.

Four lambda clones (λG11, λC4, λG14 and λ G17) from the porcine MHC class III region were labelled with either biotin-14-dATP or digoxigenin-11-dUTP and hybridized in two different combinations to DNA fibres. The latter were prepared from agarose-embedded porcine peripheral lymphocytes lysed with proteinase K, then spread and fixed on poly-L-lysine-coated slides. Hybridization signals thus obtained confirm the order of the clones previously reported using pulsed-field gel electrophoresis (PFGE). Measurements of probe sizes, gap distances between probes and total length of DNA encompassing the probes were made. Three different methods, namely relative length, Watson—Crick standard and probe size standard-based conversions, were used to estimate the parameters in kilobases. These methods of data conversion were compared with each other and with the available PFGE data, and their utility and accuracy were evaluated.

Although gene maps for a variety of evolutionarily diverged mammalian species have expanded rapidly during the past few years, until recently it has been difficult to precisely define chromosomal segments that are homologous between species. A solution to this problem has come from the development of Zoo-FISH, also known as cross-species chromosome painting. The use of Zoo-FISH to identify regions of chromosomal homology has allowed the transfer of information from map-rich species such as human and mouse to a wide variety of other species. From a Zoo-FISH analysis spanning four mammalian orders (Primates, Artiodactyla, Carnivora, and Perissodactyla), and involving eight species (human, pig, cattle, Indian muntjac, cat, American mink, harbor seal, and horse), three distinct classes of synteny conservation have been designated: (1) conservation of whole chromosome synteny, (2) conservation of large chromosomal blocks, and (3) conservation of neighboring segment combinations. This analysis has also made it possible to identify a set of chromosome segments (based on human chromosome equivalents) that probably made up the karyotype of the common ancestor of the four orders. This approach provides a basis for developing a picture of the ancestral mammalian karyotype, but a full understanding will depend on studies encompassing more diverse combinations of mammalian orders.

The porcine genes encoding the immunoglobulin gamma heavy chain (IGHG), cAMP-dependent protein kinase catalytic beta subunit (PRKACB), and transition protein 2 (TNP2) were mapped to Chromosomes (Chrs) 7 q25–q26, 6q31–q33, and 3p13-cent, respectively, by in situ hybridization. Localization of the IGHG gene confirms the assignment of linkage group III to Chr 7. Our results show that the IGHG locus in pigs, similar to the situation in other mammalian species, viz. humans, mouse, cattle, and river buffaloes, is located on the terminal region of the chromosome. The assignment of the PRKACB gene extends the homology observed between porcine Chr 6q and human Chr 1p. Mapping of the TNP2 gene provides the first marker assigned to the p arm of Chr 3 in pigs. The present study contributes to the development of the physical gene map in pigs and also bears significance in terms of comparative gene mapping.

Fluorescence in situhybridization (FISH) technique was applied to localize seven clones derived from a porcine (SSC) intestinal directionally cloned cDNA library. The size of the clones ranged from 1.1 to 1.3 kb. Three of the clones corresponded to histidyl-tRNA synthetase (HARS), immunoglobulin alpha (IGA) and lysozyme (LYZ) and mapped to SSC2q28–q29, 7q2.6 and 5p11 respectively. The available human—pig comparative painting data and sequence homology comparisons assisted in a tentative identification of the other three clones as glutathione-S-transferase (GST), glutathione-S-transferase mu (GSTM1) and immunoglobulin lambda gene cluster (IGL>1w0,4,14, 5q2.4 and 14q22–q23 respectively. The remaining clone representing an EST mapped to 1p24–p25. These localizations contribute to the transcript map in pig and are significant as comparative markers. Difficulties associated with the mapping of small sequences using FISH are discussed.

Genetic variation at the porcine submaxillary gland mucin (MUC) locus was revealed by RFLP analysis. A three-allele system was defined, and codominant inheritance was confirmed using a three-generation pedigree comprising 10 founder individuals, 26 F1 animals, and 200 F2 offspring. Linkage analysis was performed against a set of 56 genetic markers. The mucin locus was assigned to a previously reported linkage group comprising the markers interferon gamma (IFNG), diacylglycerol kinase (DAGK), and an anonymous microsatellite (S0092). Furthermore, the genes for mucin and interferon gamma were physically localized to porcine chromosome 5q2.3 and 5p1.2-q1.1 by fluorescence and radioactive in situ hybridization, respectively, thereby assigning four new markers to chromosome 5. This chromosome shares homologies with chromosome 5 in cattle and chromosome 12 in humans.

A better insight into the occurrence of microsatellites in a range of taxa may help to understand the evolution of simple repeats. Previous studies have found the relative abundance of several repeat motifs to differ among mammals, invertebrates, and plants. Absolute numbers of microsatellites also tend to correlate positively with genome size. We analyzed the occurrence, frequency, and distribution of microsatellites in birds, a taxon with one of the smallest known genome sizes among vertebrates. Dot-blot hybridization revealed that about half of 22 different di-, tri-, and tetranucleotide repeat motifs were clearly more common in human than in three species of birds: chicken, woodpecker, and swallow. For the remaining motifs no clear difference was found. From searching avian database sequences we estimated there to be 30,000-70,000 microsatellites longer than 20 bp in the avian genome. The number of (CA) > or = 10 would be around 7000-9000 and the number of (CA) > or = 14 about 3000. The calculated density of avian microsatellites (total, one every 20-39 kb; (CA) > or = 10, one every 136-150 kb) is much lower than that estimated for the human genome (one every 6 and 30 kb, respectively). This may be explained by the fact that the avian genome contains relatively less noncoding DNA than most mammals and that avian SINE/LINE elements do not terminate in poly(A) tails, which are known to provide a resource for the evolution of simple repeats in mammals. We found no association between microsatellites and SINEs in birds. Primed in situ labeling suggested fairly even distribution of (CA)n repeats over chicken macrochromosomes and intermediate chromosomes, whereas the microchromosomes, a large part of the Z and W chromosomes, and most telomeres and centromeres had very low concentrations of (CA)n microsatellites. The scarcity of microsatellites on the microchromosomes is compatible to these regions likely being unusually rich in coding sequences. The low microsatellite density in the genome in general and on the microchromosomes in particular imposes an obstacle for the development of marker-rich genetic maps of chicken and other birds, and for the localization of quantitative trait genes.

Although gene maps for a variety of evolutionarily diverged mammalian species have expanded rapidly during the past few years, until recently it has been difficult to precisely define chromosomal segments that are homologous between species. A solution to this problem has come from the development of Zoo-FISH, also known as cross-species chromosome painting. The use of Zoo-FISH to identify regions of chromosomal homology has allowed the transfer of information from map-rich species such as human and mouse to a wide variety of other species. From a Zoo-FISH analysis spanning four mammalian orders (Primates, Artiodactyla, Carnivora, and Perissodactyla), and involving eight species (human, pig, cattle, Indian muntjac, cat, American mink, harbor seal, and horse), three distinct classes of synteny conservation have been designated: (1) conservation of whole chromosome synteny, (2) conservation of large chromosomal blocks, and (3) conservation of neighboring segment combinations. This analysis has also made it possible to identify a set of chromosome segments (based on human chromosome equivalents) that probably made up the karyotype of the common ancestor of the four orders. This approach provides a basis for developing a picture of the ancestral mammalian karyotype, but a full understanding will depend on studies encompassing more diverse combinations of mammalian orders.

Three X chromosome-specific bovine cosmids were used for fluorescencein situ hybridization mapping on reindeer (Rangifer tarandus) chromosomes, to test whether such large genomic clones could be used for comparative mapping across distantly related species. All three cosmids showed distinct unique hybridization sites on the reindeer X. Comparative map locations of these cosmids, together with the relative C-banding and genome size data on the X chromosomes of the two species, provide preliminary indications that the short and long arms of bovine X correspond, respectively, to the long and short arms of the reindeer X. The study also demonstrates that cosmid clones can be used successfully for comparative mapping across species that diverged 35 million years ago.

PR-39 is a porcine 39-aa peptide antibiotic composed of 49% proline and 24% arginine, with an activity against Gram-negative bacteria comparable to that of tetracycline. In Escherichia coli, it inhibits DNA and protein synthesis. PR-39 was originally isolated from pig small intestine, but subsequent cDNA cloning showed that the gene is expressed in the bone marrow. The open reading frame of the clone showed that PR-39 is made as 173-aa precursor whose proregion belongs to the cathelin family. The PR39 gene, which is rather compact and spans only 1784 bp has now been sequenced. The coding information is split into four exons. The first exon contains the signal sequence of 29 residues and the first 37 residues of the cathelin propart. Exons 2 and 3 contain only cathelin information, while exon 4 codes for the four C-terminal cathelin residues and the mature PR-39 peptide extended by three residues. The sequenced upstream region (1183 bp) contains four potential recognition sites for NF-IL6 and three for APRF, transcription factors known to regulate genes for both cytokines and acute phase response factors. Genomic hybridizations revealed a fairly high level of restriction fragment length polymorphism and indicated that there are at least two copies of the PR39 gene in the pig genome. PR39 was mapped to pig chromosome 13 by linkage and in situ hybridization mapping. The gene for the human peptide antibiotic FALL-39 (also a member of the cathelin family) was mapped to human chromosome 3, which is homologous to pig chromosome 13.

Restriction fragment length polymorphisms are described for the genes coding for tripeptidyl peptidase II (TPP2) and tyrosinase related protein II (TYRP2) in pigs. A linkage group comprising these loci and the locus for blood group M (EAM) was established by two-point lod score analysis in a three-generation pedigree. Multipoint analysis indicated the linear order EAM-1.1-TYRP2-8.4-TPP2 (recombination distances are given as Kosambi cM). The linkage group was assigned to porcine chromosome 11—the first on this chromosome—throughin situ hybridization mapping of the TPP2 gene. TPP2 is the first gene localized on this chromosome usingin situ hybridization.

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.

Comparative mapping data suggested that the dominant white coat color in pigs may be due to a mutation in KITwhich 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 KITmutation. We found a complete association between the dominant white mutation and a duplication of the KITgene, 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 KITsequences. 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 KITand 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.

Chromosome microdissection is an important means to efficiently generate a large number of markers from a desired region of a genome. The present study was designed to initiate microdissection and amplification of DNA from whole chromosomes, aims, or bands of porcine chromosomes. The following pig (SSC) chromosomes/segments were scraped: SSC1p, SSC1q26–q2.13, SSC2q11-q14, SSC4q12-q25, SSC13, SSC13q12-q31, SSC13q32-q43, SSC13q32-q43, SSC15, and SSC16q21-q23. After amplification and PCR-labelling, the DNA from the dissected segments were painted back to normal metaphase chromosomes to test their identity. Microdissection of some of the segments (on SSC4, 13 and 15) coincides with the mapping of economically important traits. As a first step towards generation of markers, microcloning of amplilied product from SSC1p and SSC15 was carried out. The libraries were screened with a (GT)55 oligonucleotide probe. Future prospects of such a work in Farm animals are discussed.

Contrary to extensive banding similarities of the autosomes, there are variations in the morphology as well as banding patterns of the X chromosomes of various species of bovidae family. We used three bovine X chromosome specific cosmid clones for fluorescence in situ hybridization mapping on cattle, goat, sheep, and river buffalo chromosomes to identify homologous regions in the genomes of the four species. The three cosmids (cIOBT 314, 945, and 1489) mapped to well spaced locations on buffalo X, and both arms of cattle X chromosome. However, of the three probes, cosmids cIOBT 314 and 1489 showed no hybridization signal on any of the goat-sheep chromosomes. Incidentally, the two cosmids show correspondence to those regions of the buffalo X chromosome which are proposed to be lost during the structural reshuffle of the latter, leading to sheep/goat X chromosomes. The lack of hybridization signal of the two cosmids in sheep and goat, therefore, indicates that these DNA sequences are most likely absent in the two genomes and probably correspond to the segments lost during evolution of their X chromosomes.

The hospital records of 125 children, aged 14 years or less treated for abdominal tuberculosis (TB) at Jawahar Lal Nehru Medical College and Hospital, Ajmer, India between January 1987 and December 1996, were studied to analyse the various patterns of abdominal TB in children and to evaluate the role of various investigations in researching a diagnosis. Abdominal pain was the presenting symptom in 100 (80 per cent) patients. Detectable ascites was present in 55 (44 per cent) and visible peristalsis in 45 (36 per cent) cases. Laboratory investigations and radiographs were not conclusive in the majority of the patients. In 45 (36 per cent) patients in whom no tissue diagnosis was available, the diagnosis was based on a dramatic clinical response to anti-tuberculous chemotherapy. Fifty (40 per cent) patients were treated conservatively with anti-tuberculous drugs alone; the remaining patients underwent laparotomy for diagnosis and relief of bowel obstruction. There were no post-operative deaths and on follow-up, good recovery was observed in these patients. Abdominal TB in children is of a non-specific nature and defies diagnosis with non-invasive investigations; laparotomy is required for a definitive diagnosis in the majority of the patients. However, if a high index of suspicion is maintained in endemic areas, a therapeutic trial of anti-tuberculous chemotherapy is justified.

Historically, the mammalian Y chromosome has been considered degenerate and gene poor, with the primary or only function to initiate male sex determination and differentiation. These views started to change after the male specific region (MSY) of the human Y chromosome was sequenced in 2003, showing that MSY carries a number of coding genes and transcriptional units with functions in spermatogenesis and development. Since then, MSY sequence data have been obtained for the chimp, macaque, cattle, pig, dog, cat, and mouse—all showing that mammalian MSY carries functional genes but is also characterized by rapid gene expansions, turnover, and dynamic molecular processes that alter gene content and organization among species. Here we present the most comprehensive gene catalog of the horse MSY. The genes were mapped by sequencing and assembling 9.36 Mb of equine MSY using a tiling path of 94 BAC clones and a combination of short and long insert libraries on several next generation sequencing platforms. Genes were annotated using MAKER software, BLAST against reference genes and cDNAs, testis RNaseq transcripts, and the SNAP program to build most likely gene models. Gene expression profiles were determined by RT-PCR in a panel of nine adult tissues and nine tissues from 49- to 51-d-old equine embryos. Altogether, we identified 52 unique MSY genes and transcripts, of which 16 were novel. Thirty genes had one copy, while the copy numbers of 24 other genes ranged from two (TXLNGY) to 28 (ETY3), resulting in a total of 221 MSY genes and transcripts. This implies that the small equine MSY, with its 23.6 genes per Mb (considering all copies), is one of most gene-dense eutherian Y chromosomes studied to date. Among MSY genes and transcripts, 10 were horse- or equid-specific and Y-borne, 14 were of autosomal origin, and 30 had a gametologue in the X chromosome. Thus, horse MSY has also retained the highest number of ancestral X-Y genes among eutherian MSYs studied so far. The annotated horse MSY sequence was aligned with sequence assemblies of related equids, showing that the vast majority of the MSY sequence is substantially more conserved among equids than among primates. The expression pattern between donkey and horse was also surprisingly comparable. The horse MSY gene catalog establishes a basis to study the contribution of Y genes to stallion reproductive biology and provides new insight into Y chromosome evolution and function in mammals.

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.

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.