NEMO: a tool for analyzing gene and chromosome territory distributions from 3D-FISH experiments.

UNLABELLED: Three-dimensional fluorescence in situ hybridization (3D-FISH) is used to study the organization and the positioning of chromosomes or specific sequences such as genes or RNA in cell nuclei. Many different programs (commercial or free) allow image analysis for 3D-FISH experiments. One of the more efficient open-source programs for automatically processing 3D-FISH microscopy images is Smart 3D-FISH, an ImageJ plug-in designed to automatically analyze distances between genes. One of the drawbacks of Smart 3D-FISH is that it has a rather basic user interface and produces its results in various text and image files thus making the data post-processing step time consuming. We developed a new Smart 3D-FISH graphical user interface, NEMO, which provides all information in the same place so that results can be checked and validated efficiently. NEMO gives users the ability to drive their experiments analysis in either automatic, semi-automatic or manual detection mode. We also tuned Smart 3D-FISH to better analyze chromosome territories. AVAILABILITY: NEMO is a stand-alone Java application available for Windows and Linux platforms. The program is distributed under the creative commons licence and can be freely downloaded from https://www-lgc.toulouse.inra.fr/nemo

Chromosomal assignments for porcine genes encoding enzymes in hepatic metabolic pathways.

Increasing the number of mapped genes will facilitate (1) the identification of potential candidate genes for a trait of interest within quantitative trait loci regions and (2) comparative mapping. The metabolic activities of the liver are essential for providing fuel to peripheral organs, for regulation of amino acid, carbohydrate and lipid metabolism and for homoeostasis of vitamins, minerals and electrolytes. We aimed to identify and map genes coding for enzymes active in the liver by somatic cell genetics in order to contribute to the improvement of the porcine gene map. We mapped 28 genes of hepatic metabolic pathways including six genes whose locations could be confirmed and 22 new assignments. Localization information in human was available for all but one gene. In total 24 genes were assigned to in the expected chromosomal regions on the basis of the currently available information on the comparative human and pig map while for four genes our results suggest a new correspondence or extended regions of conservation between porcine and human chromosomes.

Isolation of porcine expressed sequence tags for the construction of a first genomic transcript map of the skeletal muscle in pig.

To identify genes with effects on meat quality and production traits we developed an adult porcine skeletal muscle cDNA library. After pre-screening this library with seven genes highly expressed in skeletal muscle, 385 non-hybridizing clones were sequenced from both ends to yield 510 expressed sequence tags (ESTs). Together with those ESTs previously generated from this library, we have produced 701 porcine skeletal muscle ESTs. These ESTs were grouped into 306 different cDNA species and compared with the human skeletal muscle transcriptional profiles obtained from different databases. Furthermore we mapped 107 of these cDNAs using a somatic cell hybrid panel with genes mapping over all the autosomes (except on chromosome 11) and on chromosome X. The mapping of these cDNAs contributed to the construction of a first genomic transcript map of the skeletal muscle tissue in pig.

Detection of quantitative trait loci for growth and fatness in pigs.

A quantitative trait locus (QTL) analysis of growth and fatness data from a three-generation experimental cross between Meishan (MS) and Large White (LW) pig breeds is presented. Six boars and 23 F1 sows, the progeny of six LW boars and six MS sows, produced 530 F2 males and 573 F2 females. Nine growth traits, i.e. body weight at birth and at 3, 10, 13, 17 and 22 weeks of age, average daily gain from birth to 3 weeks, from 3 to 10 weeks and from 10 to 22 weeks of age, as well as backfat thickness at 13, 17 and 22 weeks of age and at 40 and 60 kg live weight were analysed. Animals were typed for a total of 137 markers covering the entire porcine genome. Analyses were performed using two interval mapping methods: a line-cross (LC) regression method where founder lines were assumed to be fixed for different QTL alleles and a half-/full-sib (HFS) maximum likelihood method where allele substitution effects were estimated within each half-/full-sib family. Both methods revealed highly significant gene effects for growth on chromosomes 1, 4 and 7 and for backfat thickness on chromosomes 1, 4, 5, 7 and X, and significant gene effects on chromosome 6 for growth and backfat thickness. Suggestive QTLs were also revealed by both methods on chromosomes 2 and 3 for growth and 2 for backfat thickness. Significant gene effects were detected for growth on chromosomes 11, 13, 14, 16 and 18 and for backfat thickness on chromosome 8, 10, 13 and 14. LW alleles were associated with high growth rate and low backfat thickness, except for those of chromosome 7 and to a lesser extent early-growth alleles on chromosomes 1 and 2 and backfat thickness alleles on chromosome 6.

Construction of a high-resolution RH map of the human 2q35 region on TNG panel and comparison with a physical map of the porcine homologous region 15q25.

This article describes the construction of a high-resolution radiation hybrid map of Hsap 2q35 by using the TNG RH panel generated by irradiation with 50,000 rads. We were able to build a framework map of 1300 cR(50,000) including 34 markers ordered with odds higher than 1000:1. The comprehensive map includes 77 loci and describes a region of 3 Mb around the SLC11A1 gene. Because of the very small size of the fragments retained and a reduced retention frequency, it was difficult to build a high-resolution multi-point map of this region by using the TNG RH panel. Nevertheless, this study confirmed the very high potential of this RH panel for constructing a human, high-resolution physical map (2.3 kb/cR(50,000)). Moreover, human ESTs from Hsap 2q35 were hybridized with porcine BAC contigs to establish a porcine transcript map of the homologous region Sscr 15q25 (greater than 2.5 Mb). We identified 17 new genes in this porcine chromosomal region. We were able to compare the location of 26 genes mapped in both species. The gene order was similar except for two possible minor discrepancies in the Desmin sub-region, suggesting the existence of a porcine micro-region between TNP1 and IL8RB with an unknown origin.

In the QTL region surrounding porcine MHC, gene order is conserved with human genome.

On the porcine genome, the region surrounding the Major Histocompatibility Complex, also called Swine Leukocyte Antigens (SLA), is of particular interest not only owing to itq role in the control of immune response, but also because of its influence on many traits such as growth, fatness, and meat quality. To help in the identification of responsible genes, detailed comparative maps of the MHC region in mammalian species and powerful mapping tools allowing accurate ordering of genes and markers in this region are needed. In this report, we describe the use of the recently developed IMpRH radiation hybrid panel, to construct a higher density radiation hybrid map of swine Sscr 7p-q12, containing 23 additional loci. Our results show that the gene order is conserved between the two MHC-containing regions, even if an inversion is observed above the QTL region in the region containing DEK, SCA1, and EDN1 genes. The framework map produced shows that the IMpRH panel permits the ordering of genes and markers in the three MHC classes and would thus allow accurate localization of ESTs and candidate genes.

Comparative mapping between humans and pigs: localization of 58 anchorage markers (TOASTs) by use of porcine somatic cell and radiation hybrid panels.

To increase the number of Type I markers that are directly informative for comparative mapping, 58 anchorage markers, TOASTs (Traced Orthologous Amplified Sequence Tags), were mapped in pig. With specific consensus primers, 76 TOASTs were tested in pig: 50 were regionally localized in pig on a somatic cell hybrid panel (SCHP), and 51 were mapped on the whole genome, INRA/University of Minnesota porcine Radiation Hybrid panel (IMpRH). Comparison of marker positions on RH and cytogenetic maps indicated general concordance except for two chromosomal regions. For RH mapping, all markers, apart from one, were significantly linked (LOD > 4.8) to a marker of the first-generation radiation hybrid map. Localization of new markers on the initial map is necessary for drawing a framework map as shown for Chromosome Sscr 14. The addition of four TOASTs has enabled us to propose an improved map, using a threshold likelihood ratio of 1000/1. At the whole-genome level, this work significantly increased (by 50%) the number of precisely mapped genes on the porcine RH map and confirmed that the IMpRH panel is a valuable tool for high-resolution gene mapping in pig. Porcine PCR products were sequenced and compared with human sequences to verify their identity. Most of the localizations made it possible to either confirm or refine the previous comparative data between humans and pigs obtained through heterologous chromosomal painting or gene mapping. Moreover, the use of TOASTs in mapping studies appears to be a complement to other strategies using CATS, human ESTs, or heterologous FISH with BACs which had already been applied to improve the gene density of comparative genomic maps for mammals.

IMpRH server: an RH mapping server available on the Web.

SUMMARY: The INRA-Minnesota Porcine Radiation Hybrid (IMpRH) Server provides both a mapping tool (IMpRH mapping tool) and a database (IMpRH database) of officially submitted results. The mapping tool permits the mapping of a new marker relatively to markers previously mapped on the IMpRH panel. The IMpRH database is the official database for submission of new results and queries. The database not only permits the sharing of public data but also semi-private and private data.

A mutation in PRKAG3 associated with excess glycogen content in pig skeletal muscle.

A high proportion of purebred Hampshire pigs carries the dominant RN- mutation, which causes high glycogen content in skeletal muscle. The mutation has beneficial effects on meat content but detrimental effects on processing yield. Here, it is shown that the mutation is a nonconservative substitution (R200Q) in the PRKAG3 gene, which encodes a muscle-specific isoform of the regulatory gamma subunit of adenosine monophosphate-activated protein kinase (AMPK). Loss-of-function mutations in the homologous gene in yeast (SNF4) cause defects in glucose metabolism, including glycogen storage. Further analysis of the PRKAG3 signaling pathway may provide insights into muscle physiology as well as the pathogenesis of noninsulin-dependent diabetes mellitus in humans, a metabolic disorder associated with impaired glycogen synthesis.

Localization of 113 anchor loci in pigs: improvement of the comparative map for humans, pigs, and goats.

In total, 113 genes that have already been located in humans and goats were cytogenetically mapped in pigs. For this purpose, 165 gene-containing bacterial artificial chromosomes (BACs) isolated in goats were used in heterologous fluorescent in situ hybridization on porcine chromosomes. Among them, 113 (or 69%) gave clear and specific signals, and 52 did not work in heterologous conditions. These localizations are a significant contribution to development of the porcine gene map and also to the comparative map for humans and pigs. They allowed us to specify the information obtained by Zoo-FISH while taking the gene order into account; the number of conserved fragments detected for human and pig chromosomes reached 84. The average size of conserved fragments could be estimated at 33 cM. As these genes had already been mapped in goats, the comparison was extended to ruminants. The previous results obtained in this species, suggesting a correlation between human chromosome abnormalities and evolutionary breakpoints, were confirmed in pigs.

Comparative gene mapping workshop: progress in agriculturally important animals.

Following the successful Comparative Mapping Workshop held at Fraser Island, Australia in 1995, HUGO organized a second workshop of 41 invited participants, held at Toulouse, France on May 3 and 4, 1999. The aim of the conference was to focus on recent developments in genome mapping in a variety of vertebrate species, with particular emphasis on progress in farm animals (cattle, pigs, chickens, sheep, horses, goats, and deer). In addition, representatives from important experimental mammalian and vertebrate organisms (e.g. mice, rats, dogs, fugu, and marsupials) also participated in the meeting. After a rapid overview of developments in the construction and comparison of genome maps in a wide variety of species, discussion focused on how comparative genomics will play a vital role in the genetic dissection of multigenic traits and the characterization of agriculturally important loci in agricultural species. Acceleration of gene discovery with heterologous ESTs (Expressed Sequence Tags) or collections of ESTs was discussed. Recent developments in the construction of cDNA libraries and the efficiency of tools such as whole genome radiation hybrids (RH) and large fragment clone libraries (YACs and in particular BACs) were discussed. Proposed criteria to improve the identification of homologous genes between species and recommendations for nomenclatures were identified. Particular emphasis was placed on how the integration of biological databases could help the scientific community.

A first-generation porcine whole-genome radiation hybrid map.

A whole-genome radiation hybrid (WG-RH) panel was used to generate a first-generation radiation map of the porcine (Sus scrofa) genome. Over 900 Type I and II markers were used to amplify the INRA-University of Minnesota porcine Radiation Hybrid panel (IMpRH) comprised of 118 hybrid clones. Average marker retention frequency of 29.3% was calculated with 757 scorable markers. The RHMAP program established 128 linkage groups covering each chromosome (n = 19) at a lod >/= 4.8. Fewer than 10% of the markers (59) could not be placed within any linkage group at a lod score >/=4.8. Linkage group order for each chromosome was determined by incorporating linkage data from the swine genetic map as well as physical assignments. The current map has an estimated ratio of approximately 70 kb/cR and a maximum theoretical resolution of 145 kb. This initial map forms a template for establishing accurate YAC and BAC contigs and eventual positional cloning of genes associated with complex traits.

A radiation hybrid map of the RN region in pigs demonstrates conserved gene order compared with the human and mouse genomes.

We recently constructed a 7000-rad porcine whole-genome radiation hybrid (RH) panel with the primary objective of integrating linkage maps of microsatellites with evolutionary conserved genes into one ordered map. In order to evaluate the resolution of this RH panel, we have now constructed a radiation hybrid map of the Chromosome (Chr) 15q2.3-q2.6 region containing the RN gene. This gene has large effects on glycogen content in muscle and meat quality. Ten microsatellites covering a region of 55 centiMorgans and eight genes (AE3, FN1, IGFBP5, INHA, IRS1, PAX3, TNP1, and VIL1) were placed on the Sscr15 RH map. All the genes, except IRS1, were mapped on the RH map between microsatellites located in 15q2.5. The relative order of AE3 and INHA was inverted on the porcine physical map in comparison with the mouse linkage map. The order of other genes already mapped in the mouse (FN1, IGFBP5, TNP1, VIL1, INHA/AE3, and PAX3) was identical in pigs. We found no clear difference between the gene order on pig Chr 15 and human Chr 2q.