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The integration of genetic information in the cellular and nuclear environments is crucial for deciphering the way in which the genome functions under different physiological conditions. Experimental techniques of 3D nuclear mapping, a high-flow approach such as transcriptomic data analyses, and statistical methods for the development of co-expressed gene networks, can be combined to develop an integrated approach for depicting the regulation of gene expression. Our work focused more specifically on the mechanisms involved in the transcriptional regulation of genes expressed in muscle during late foetal development in pig. The data generated by a transcriptomic analysis carried out on muscle of foetuses from two extreme genetic lines for birth mortality are used to construct networks of differentially expressed and co-regulated genes. We developed an innovative co-expression networking approach coupling, by means of an iterative process, a new statistical method for graph inference with data of gene spatial co-localization (3D DNA FISH) to construct a robust network grouping co-expressed genes. This enabled us to highlight relevant biological processes related to foetal muscle maturity and to discover unexpected gene associations between IGF2, MYH3 and DLK1/MEG3 in the nuclear space, genes that are up-regulated at this stage of muscle development.

Changes in the nuclear positioning of specific genes, depending on their expression status, have been observed in a large diversity of physiological processes. However, gene position is poorly documented for immune cells which have been subjected to activation following bacterial infection. Using a pig model, we focused our study on monocyte-derived macrophages and neutrophils, as they are the first lines of defence against pathogens. We examined whether changes in gene expression due to LPS activation imply that genes have repositioned in the nuclear space. We first performed a transcriptomic analysis to identify the differentially expressed genes and then analysed the networks involved during lypopolysaccharide/interferon gamma activation in monocyte-derived macrophages. This allowed us to select four up-regulated (IL1β, IL8, CXCL10 and TNFα) and four down-regulated (VIM, LGALS3, TUBA3 and IGF2) genes. Their expression statuses were verified by quantitative real-time RT-PCR before studying their behaviour in the nuclear space during macrophage activation by means of 3D fluorescence in situ hybridization. No global correlation was found between gene activity and radial positioning. Only TNFα belonging to the highly transcribed MHC region on chromosome 7 became more peripherally localized in relation to the less decondensed state of its chromosome territory (CT) in activated macrophages. The analysis of gene positioning towards their CT revealed that IL8 increases significantly its tendency to be outside its CT during the activation process. In addition, the gene to CT edge distances increase for the three up-regulated genes (IL8, CXCL10 and TNFα) among the four analysed. Contrarily, the four down-regulated genes did not change their position. The analysis of gene behaviour towards their CT was extended to include neutrophils for three (TNFα, IL8 and IL1β) up- and two (IGF2 and TUBA3) down-regulated genes, and similar results were obtained. The analysis was completed by studying the four up-regulated genes in fibroblasts, not involved in immune response. Our data suggest that relocation in the nuclear space of genes that are differentially expressed in activated immune cells is gene and cell type specific but also closely linked to the entire up-regulation status of their chromosomal regions.

Neutrophils are essential components of the innate immune system due to their ability to kill and phagocytose invading microbes. They possess a lobulated nucleus and are capable of extensive and complex changes in response to bacterial stimulation. The aim of our study was to investigate whether the 3D nuclear organization of porcine neutrophils was modified upon stimulation. The organization of centromeres, telomeres, and chromosome territories (chromosomes 2, 3, 7, 8, 12, 13, and 17) was studied on structurally preserved nuclei using 3D fluorescence in situ hybridization, confocal microscopy, and image analysis. By differential labeling of centromeres of acrocentric and metacentric/submetacentric chromosomes, we showed that centromeres associated to form chromocenters but did so preferentially between chromosomes with the same morphology. Upon activation, some of these chromocenters dispersed. Telomeres were also found to form clusters, but their number remained unchanged in lipopolysaccharide-stimulated neutrophils. The analysis of the position of chromocenters and telomere clusters showed a more internal location of the latter compared to the former. The analysis of chromosome territories revealed that homologs were distributed randomly among lobes whatever the cell's status. The volume of these territories was not proportional to chromosome length, and some chromosomes (chr 3, 12, 13, and 17) were more prone to decondensation when neutrophils were stimulated. Thus, our study demonstrated that activation of neutrophils resulted in several modifications of their nuclear architecture: a decrease in the number of non-acrocentric chromocenters and the decondensation of several chromosomes.

This study reports a high-resolution comparative map between human chromosomes and porcine chromosomes 2 (SSC2) and 16 (SSC16), pointing out new homologies and evolutionary breakpoints. SSC2 is of particular interest because of the presence of several important QTLs. Among 226 porcine ESTs selected according to their expected localization, 151 were RH mapped and ordered on SSC2. This study confirmed the extensive conservation between SSC2 and HSA11 and HSA19 and refined the homology with HSA5 (three blocks defined). Furthermore the SSC2q pericentromeric region was shown to be homologous to another human chromosome (HSA1). A complex organization of these syntenies was demonstrated on SSC2q. Our strategy led us to improve also the SSC16 RH map by adding 45 markers. Two-color fluorescence in situ hybridization of markers representative of each synteny confirmed block order. Finally, 29 breakpoints were identified in both species, and porcine BACs containing two breakpoints were isolated.

We report on the construction of a high-resolution comparative map of porcine chromosome 17 (SSC17) focusing on evolutionary breakpoints with human chromosomes. The comparative map shows high homology with human chromosome 20 but suggests more limited homologies with other human chromosomes. SSC17 is of particular interest in studies of chromosomal organization due to the presence of QTLs that affect meat quality and carcass composition. A total of 158 pig ESTs available in databases or developed by the Sino-Danish Pig Genome Sequencing Consortium were mapped using the INRA-University of Minnesota porcine radiation hybrid panel. The high-resolution map was further anchored by fluorescence in situ hybridization. This study confirmed the extensive conservation between SSC17 and HSA20 and enabled the gene order to be determined. The homology of the SSC17 pericentromeric region was extended to other human chromosomes (HSA4, HSA8) and the chromosomal breakpoint boundaries were accurately defined. In total 15 breakpoints were identified.

In the frame of the European program GenetPig, we localized on the Pig map 105 coding sequences (type I markers) from different origins, using INRA-University of Minnesota porcine Radiation Hybrid Panel (IMpRH, 101 markers) and somatic cell hybrid panel (SCHP, 93 markers, of which only four were not also mapped using IMpRH). Thus, we contributed to the improvement of the porcine high-resolution map, and we complemented the integration between the RH and cytogenetic maps. IMpRH tools allowed us to map 101 new markers relatively to reference markers of the first generation radiation hybrid map. Ninety out of 101 markers are linked to an already mapped marker with a LOD score greater than 4.8. Seventy-eight markers were informative for comparative mapping. Comparison of marker positions on the RH map with those obtained on the cytogenetic map or those expected by Human-Pig comparative map data suggested to us to be cautious with markers linked with a LOD lower than 6. These results allowed us to specify chromosomal fragments well conserved between humans and pigs and also to suggest new correspondences (Sscr1-Hsap3, Sscr9-Hsap9, Sscr13-Hsap11, Sscr15-Hsap6) confirmed by FISH on pig chromosomes. We examined in more detail the comparative map between Hsap12 and Sscr5 considering gene order, which suggests that rearrangements have occurred within the conserved synteny.

In this study we examined homologies between 1,735 porcine microsatellites and human sequence. For 1,710 microsatellites we directly used the sequence flanking the repeat available in GenBank. For a set of 305 microsatellites, a BAC library was screened and end-sequencing provided 461 additional sequences. Altogether 2,171 porcine sequences were tentatively aligned with the sequence of the human genome using the fasta program. Human homologies were observed for 652 microsatellite loci and porcine chromosome assignments available for 623 microsatellites provide useful links in the human and pig comparative map. Moreover for 92 STS, a significant sequence similarity was detected using at least two sequences and in all cases corresponding human locations were consistent. The present study allowed the integration of anonymous markers and the porcine linkage map into the framework of the comparative data between human and porcine genomes (http://w3.toulouse.inra.fr/lgc/pig/msat/). Moreover all conserved syntenic segments were defined on human chromosomes., (Copyright 2003 S. Karger AG, Basel)

The GENETPIG database has been established for storing and disseminating the results of the European project: 'GENETPIG: identification of genes controlling economic traits in pig'. The partners of this project have mapped about 630 porcine and human ESTs onto the pig genome. The database collects the mapping results and links them to other sources of mapping data; this includes pig maps as well as available comparative mapping information. Functional annotation of the mapped ESTs is also given when a significant similarity to cognate genes was established. The database is accessible for consultation via the Internet at http://www.infobiogen.fr/services/Genetpig/.

A comparative map of human chromosome 12 (HSA 12) and pig chromosome 5 (SSC 5) was constructed using ten pig expressed sequence tags (ESTs). These ESTs were isolated from primary granulosa cell cultures by differential display (EST b10b), or from a granulosa cDNA library (VIIIE1, DRIM, N*9, RIIID2 and RVIC1) or from a small intestine cDNA library (ATPSB, ITGB7, MYH9, and STAT2). Also used were two Traced Orthologous Amplified Sequence Tags (TOASTs) (LALBA, TRA1), one microsatellite-associated gene (IGF1) and finally five human YACs selected for their cytogenetic position, with a view to increasing the number of informative markers for the comparison. Large-insert clones were obtained by screening a pig bacterial artificial chromosome (BAC) library with specific primers for each EST and TOAST and for IGF1. These BACs were used as probes for fluorescent in situ hybridisation (FISH) both on porcine and human metaphases. In addition, the human YACs were FISH mapped on pig chromosomes. This allowed us to refine and, in some cases, to correct the previous mapping obtained with a somatic cell hybrid panel. While these data confirm chromosome painting results showing that the distal part of SSC 5p arm is conserved on HSA 22, while the rest of the chromosome corresponds to HSA 12, they also demonstrate gene-order differences between human and pig. In addition, it was also possible to determine the position of the synteny breakpoint., (Copyright 2001 S. Karger AG, Basel)

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.

Large-scale sequencing of cDNAs from numerous tissues is currently being performed within the framework of the Human Genome Project. These expressed sequence tags (ESTs) are then mapped on a radiation hybrid panel to produce a high-resolution map of human genes. In this report, we estimate the efficiency of mapping these ESTs in the pig. A total of 344 human ESTs from Généthon were selected for amplification in other species by Zoo-PCR: 186 of these could be reproducibly amplified by use of pig DNA and the corresponding human primer pairs. One-hundred seven of these were tested on a porcine-rodent somatic cell hybrid panel, permitting regional localizations of 65 ESTs with agarose or single-strand conformation polymorphism analysis gels. The corresponding pig PCR products were sequenced: 60 ESTs matched significantly with the expected human sequences. Fifty-one of these localizations in the pig are in agreement with the comparative mapping data between humans and pigs based on heterologous chromosome painting. Seven ESTs that were localized in an unexpected region may indicate new chromosomal correspondences. This work significantly increases the number of genes mapped on the pig genome and demonstrates that this approach can be successfully applied to improve the gene density of mammalian genomic maps in chromosomal regions of interest, such as those in which QTL (Quantative Trait Loci) have been identified.

Intersexuality has been reported in pigs and investigations are in progress to identify and physically map the genes involved in sex-reversal. In this study we have mapped on porcine chromosomes seven genes which might be implicated in this developmental pathway. Four genes were mapped by radioactive in situ hybridization: AMH (Anti-Müllerian Hormone) and WT1 (Wilms' Tumor gene 1) were both mapped to pig chromosome 2 (SSC2) in the q14-->q21 and p14-->q11 regions, placental CYP19 (cytochrome P450, subfamily XIX) and FTZF1 (fushi tarazu factor [Drosophila] homolog 1, alias SF1 [steroidogenic factor 1]) to pig chromosome 1 (SSC1) in the q14-->q17 and q210-->q211 regions respectively. Four other genes were regionally located by PCR analysis on a cytogenetically characterized porcine somatic cell hybrid panel: a second CYP19 gene (expressed in embryo) was mapped to porcine 1q12-->q17, AHC (alias DAX1, adrenal hypoplasia congenital) to porcine Xp24, SOX2 and SOX9 (SRY sex determining region Y-box 2 and 9) to 13q23-->q41 and 12p13-->p11 respectively. These results are in global agreement with mapping data available in other mammalian species.

A highly significant genetic association has been found between some alleles of the swine Major Histocompatibility Complex SLA (Swine Leukocyte Antigen genetic complex) and the cytosolic malic enzymatic activity level in muscles. The aim of this study was to find out whether this genetic association was due to a close linkage of the SLA region and the gene coding for the enzyme. Since no swine cytosolic malic enzyme sequence (ME1) was available, we isolated several overlapping fragments that spanned the almost entire malic enzyme transcript both by screening of a swine cDNA library and by RT-PCR. The results indicated the existence of two transcripts of 2. 0 and 3.1 kb, which probably correspond to two alternative forms of one gene. The sequence of the transcript was highly similar to the other published mammalian cytosolic NADP+-dependent malic enzyme cDNA, especially within the four functional domains. Two major bands at 3.7 and 2.4 kb were detected on Northern blots containing the RNA from 25 tissues from fetuses and adult pigs. A high expression level was found in the adrenal gland, muscle, liver, and peripheral nerves. The analysis of malic enzyme RFLPs in five SLA informative families revealed an independent segregation of the ME1 gene from the SLA region. In situ hybridization results localized the cytosolic malic enzyme on the swine Chromosome (Chr) 1p1.2, except that the association between SLA and the malic enzyme activity level was due to a physical genetic linkage. Thus, the mechanisms underlying this association remain to be elucidated.

Recently two main genetic maps [Rohrer et al. Genetics 136, 231 (1994); Archibald et al. Mamm. Genome 6, 157 (1995)] and a cytogenetic map [Yerle et al. Mamm. Genome 6, 175 (1995)] for the porcine genome were reported. As only a very few micro-satellites are located on the cytogenetic map, it appears to be important to increase the relationships between the genetic and cytogenetic maps. This document describes the regional mapping of 100 genetic markers with a somatic cell hybrid panel. Among the markers, 91 correspond to new localizations. Our study enabled the localization of 14 new markers found on both maps, of 54 found on the USDA map, and of 23 found on the PiGMaP map. Now 21% and 43% of the markers on the USDA and PiGMaP linkage maps respectively are physically mapped. This new cytogenetic information was then integrated within the framework of each genetic map. The cytogenetic orientation of the USDA linkage maps for Chromosomes (Chrs) 3, 8, 9, and 16 and of PiGMaP for Chr 8 was determined. USDA and PiGMaP linkage maps are now oriented for all chromosomes, except for Chrs 17 and 18. Moreover, the linkage group "R" from the USDA linkage map was assigned to Chr 6.

A cDNA clone encoding the complete coding sequence for porcine alpha-1-antitrypsin (or alpha 1-protease inhibitor, PI) was isolated and its DNA sequence determined. The cDNA is assumed to encode alpha-1-antitrypsin on the basis of its sequence similarity to the corresponding cDNAs for human, baboon, rat, mouse, sheep and cow. The porcine cDNA clone was used in conjunction with BamHI, KpnI, MspI, SacI, TaqI and XbaI to develop restriction fragment length polymorphism-based genetic markers for linkage mapping in pigs. The cDNA has also been used to map the porcine PI locus to chromosome 7q2.4- > q2.6 by radioactive in situ hybridization. Thus, the PI locus has been added to the developing physical and genetic maps of the porcine genome.

Four homeobox genes that belong to the four homeobox gene clusters known in mammals have been regionally assigned to four distinct porcine chromosomes in conserved regions between human and pig. HOXA11, HOXB6, HOXC8, and HOXD4 genes were mapped by radioactive in situ hybridization to porcine Chromosomes (Chrs) 18q21-24 (with a secondary signal in 16q14-21), 12p11-12, 5p11-12, and 15q22-23 respectively. Besides, we have also revealed the presence of a porcine homeobox (pig Hbx24) which, although showing DNA sequence homology with a mouse gene of HOXB cluster, was located on porcine Chr 3 (3p14-13) outside the Hox clusters. To support the identity of the homeobox gene clusters analyzed and in the light of the high sequence similarity among homeobox genes, we also localized markers known to be mapped near each Hox cluster in human. In this way, four genes were also mapped in pig: GAPD (5q12-21), GAD1 (15q21-22), INHBA (18q24), and IGFBP3 (18q24). Mapping of HOXA11, INHBA, and IGFBP3 on pig Chr 18 constitutes the first assignments of genes on this small chromosome. These new localizations extend the information on the conservation of four human chromosomal regions in the pig genome.

A panel of 27 pig x rodent somatic cell hybrids was produced and characterized cytogenetically. The first step of this study consisted of hybridizing a SINE probe to GTG-banded metaphases of each hybrid clone in order to count and identify the normal pig chromosomes and to detect rearranged ones. The second step consisted of using the DNA of each clone as a probe after pIRS-PCR (porcine interspersed repetitive sequence-polymerase chain reaction) amplification to highly enrich it in pig sequences. These probes, hybridized to normal pig metaphase chromosomes, enabled the identification of the complete porcine complement in the hybrid lines. Whole chromosomes and fragments were characterized quickly and precisely, and results were compared. In addition to this cytogenetic characterization, molecular verification was also carried out by using primers specific to six microsatellites and to one gene previously mapped to pig chromosomes. The results obtained allow us to conclude that we have produced a panel that is informative for all porcine chromosomes. This panel constitutes a highly efficient tool to establish not only assignments of genes and markers but also regional localizations on pig chromosomes.

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are members of the glycoprotein hormone family and play essential roles in gametogenesis and sexual development of mammals. Fragments of the porcine genes coding for the beta subunits of FSH and LH were amplified by PCR and used as probes for radioactive in situ hybridization in order to map these loci in the pig. Primers were chosen on the genomic DNA nucleotide sequences of FSHB and LHB as published in GenBank. Fragments of 1,127 bp (FSHB) and 1,239 bp (LHB) were cloned and verified by sequencing. FSHB was localized to pig chromosome bands 2p1.6-->p1.2 and LHB to pig chromosome band 6q2.1. The localization of LHB to the so-called halothane region of chromosome 6 could be expected from comparative mapping data. For FSHB, no conclusions can be drawn in this respect since, up to the present, too few genes are located on porcine chromosome 2.

The genes coding for the insulin-like growth factor-1 receptor (IGF1R) and a porcine endothelium-derived 21-residue vasoconstrictor peptide (EDN) were localized in the pig by means of radioactive in situ hybridization. IGF1R was mapped to chromosome region 1q1.7-->q2.1 and EDN to 7p1.3-->p1.2. The results are discussed in relation to the position of their homologous loci in man.

The genes coding for the regulatory type I alpha subunit (PRKAR1A) and the catalytic beta subunit (PRKACB) of cAMP-dependent protein kinase and the genes for interleukin 1 alpha (IL1A) and interleukin 1 beta (IL1B) were localized in the pig by means of radioactive in situ hybridization. PRKAR1A was mapped to 12p1.4 and PRKARB to 6q3.1-->q3.3. The genes for IL1A and IL1B were both assigned to Chromosome (Chr) 3, in the region q1.2-->q1.3 and q1.1-->q1.4, respectively. The cDNA nucleotide sequences of these porcine genes were compared with those of human, mouse, and cattle. The location of the genes was discussed in relation to the position of their homologous loci in these mammalian species.

The nerve growth factor beta gene (NGFB) belongs to a conserved syntenic group on human chromosome 1 and mouse Chromosome 3. The objective of this study was the isolation of a part of the porcine NGFB gene and its use as a genetic and physical marker in the pig genome. On the basis of a nucleotide sequence comparison among different species, NGFB-specific primers were chosen to amplify the corresponding porcine sequence by PCR. A pig genomic DNA fragment of 763 bp was isolated, and its DNA sequence, containing the complete coding sequence of mature NGFB, was determined. It was demonstrated that pig NGFB is largely homologous with NGFB of other species (mouse, cattle, chicken) and especially with human NGFB; the isolated clone shows 91% nucleotide and 99% translated amino acid sequence identity to the human NGFB sequence. The porcine DNA clone allowed us to identify an RFLP marker for genetic mapping in pigs and was used to map the NGFB gene to pig chromosome region 4q1.6-->q2.3 by radioactive in situ hybridization. Previously, we had localized to the same chromosome another member of this syntenic group, the gene encoding alpha 1 Na+,K+ ATPase (ATP1A1). These results show that a portion of the homologous region between human chromosome 1 and mouse Chromosome 3 is also conserved on pig chromosome 4.

The gene encoding the porcine growth hormone (GH) has been localized to the q-arm of chromosome 12 using high-resolution R-banded chromosomes for in situ hybridization. We report here the localization of GH on the p-arm of this chromosome when using in situ hybridization on high-resolution G-banded chromosomes. Sequential Q- and R-banding show that this discrepancy is caused by a reversed orientation of chromosome 12 in the R-banded high-resolution karyotype published by Rønne et al. (1987) and the G-banded standard karyotype.

Two genes coding for Na+,K(+)-ATPase alpha and beta subunits are localized on pig chromosome 4, to the q1.6-->q2.3 and 1.3-->q2.1 regions, respectively, by radioactive in situ hybridization. According to nucleotide and amino acid sequence comparisons with different human isoforms of Na+,K(+)-ATPase, these pig alpha beta ATPase genes show strong homologies with human alpha 1 and beta subunit ATPase genes, respectively. These results are discussed with respect to comparative mapping data of conserved genes in mammalian species. We showed that the pig cDNA probes encoding ATPase alpha and beta genes reveal DNA polymorphism in Meishan and Large White pigs.

The genes coding for glucose regulated protein, 78kDal (GRP78), hormone-sensitive lipase (LIPE), plasminogen activator or urokinase (PLAU), and D-amino acid oxidase (DAO) were localized in the pig by radioactive in situ hybridization. GRP78 was mapped to 1q2.10-->q2.13 and LIPE was localized to chromosome 6cen-->q1.2. The genes for PLAU and DAO were both assigned to chromosome 14, in the region q2.4-->q2.6 and q2.1-->q2.3, respectively. The results are compared to mapping data in other mammalian species.

A review of the present status of the porcine gene map is given with references. A total of 84 loci have now been studied, and genes have been assigned to 17 chromosomes. Among them, six chromosomes are defined by only one marker. No loci have been attributed yet to three chromosomes.

The porcine gene for luteinizing hormone/choriogonadotropin receptor (LHCGR) was localized to chromosome 3q2.2----q2.3 using radioactive and nonradioactive in situ hybridization. A computer-assisted image-analysis system was developed which facilitated detection of the position of silver grains and fluorescent spots on the chromosomes after in situ hybridization. Compared with autoradiographic visualization, the nonisotopic procedure proved to be more rapid, precise, and highly specific; however, nonradiographic in situ hybridization was much less efficient than the autoradiographic technique for the detection of unique DNA sequences with small probes. From these results and published gene-mapping data, it was concluded that the synteny between LHCGR and MDH1 observed in man is conserved in the pig genome.