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Phenylalanine hydroxylase–deficient (PAH-deficient) phenylketonuria (PKU) results in systemic hyperphenylalaninemia, leading to neurotoxicity with severe developmental disabilities. Dietary phenylalanine (Phe) restriction prevents the most deleterious effects of hyperphenylalaninemia, but adherence to diet is poor in adult and adolescent patients, resulting in characteristic neurobehavioral phenotypes. Thus, an urgent need exists for new treatments. Additionally, rodent models of PKU do not adequately reflect neurocognitive phenotypes, and thus there is a need for improved animal models. To this end, we have developed PAH-null pigs. After selection of optimal CRISPR/Cas9 genome-editing reagents by using an in vitro cell model, zygote injection of 2 sgRNAs and Cas9 mRNA demonstrated deletions in preimplantation embryos, with embryo transfer to a surrogate leading to 2 founder animals. One pig was heterozygous for a PAH exon 6 deletion allele, while the other was compound heterozygous for deletions of exon 6 and of exons 6–7. The affected pig exhibited hyperphenylalaninemia (2000–5000 μM) that was treatable by dietary Phe restriction, consistent with classical PKU, along with juvenile growth retardation, hypopigmentation, ventriculomegaly, and decreased brain gray matter volume. In conclusion, we have established a large-animal preclinical model of PKU to investigate pathophysiology and to assess new therapeutic interventions., A phenylalanine hydroxylase–deficient pig model of phenylketonuria is established and characterized.

Correct pairing, synapsis and recombination between homologous chromosomes are essential for normal meiosis. All these events are strongly regulated, and our knowledge of the mechanisms involved in this regulation is increasing rapidly. Chromosomal rearrangements are known to disturb these processes. In the present paper, synapsis and recombination (number and distribution of MLH1 foci) were studied in three boars (Sus scrofa domestica) carrying different chromosomal rearrangements. One (T34he) was heterozygote for the t(3;4)(p1.3;q1.5) reciprocal translocation, one (T34ho) was homozygote for that translocation, while the third (T34Inv) was heterozygote for both the translocation and a pericentric inversion inv(4)(p1.4;q2.3). All three boars were normal for synapsis and sperm production. This particular situation allowed us to rigorously study the impact of rearrangements on recombination. Overall, the rearrangements induced only minor modifications of the number of MLH1 foci (per spermatocyte or per chromosome) and of the length of synaptonemal complexes for chromosomes 3 and 4. The distribution of MLH1 foci in T34he was comparable to that of the controls. Conversely, the distributions of MLH1 foci on chromosome 4 were strongly modified in boar T34Inv (lack of crossover in the heterosynaptic region of the quadrivalent, and crossover displaced to the chromosome extremities), and also in boar T34ho (two recombination peaks on the q-arms compared with one of higher magnitude in the controls). Analyses of boars T34he and T34Inv showed that the interference was propagated through the breakpoints. A different result was obtained for boar T34ho, in which the breakpoints (transition between SSC3 and SSC4 chromatin on the bivalents) seemed to alter the transmission of the interference signal. Our results suggest that the number of crossovers and crossover interference could be regulated by partially different mechanisms.

Chantier qualité GA; International audience; The occurrence of interchromosomal effects (ICE) in reciprocal translocation carriers still remains contradictory in the human literature. We used the pig as an animal model to investigate whether the structure of the reciprocal translocations as well as the size and/or type of the chromosomes not involved in the rearrangement may influence the occurrence and the extent of ICE. Analyses of chromosomal sperm content by fluorescence in situ hybridization (FISH) using whole-chromosome painting probes for 7 chromosomes (1, 10, 11, 13, 18, X and Y) were carried out on sperm samples of 2 boars with normal semen parameters carrying different balanced reciprocal translocations: 38, XY, t(3;15)(q27;q13) or 38, XY, t(12;14)(q13;q21). One fertile boar with normal karyotype was also studied as a control. Aneuploidy rates for the 7 chromosomes were estimated by scoring 10,000 to 20,000 spermatozoa for each probe combination. No significant ICE was found except for chromosome 1 in the case of the t(3;15) translocation. Even if statistically significant, this ICE remained very weak and should have very little impact on the reproductive performance of the carrier boar. The size and/or type of chromosomes not involved in the translocation do not seem to have a major influence on the occurrence of ICE. The structure of the translocation could play a role, but complementary studies should be carried out to confirm this assumption.

International audience; For the first time in the domestic pig, meiotic recombination along the 18 porcine autosomes was directly studied by immunolocalization of MLH1 protein. In total, 7,848 synaptonemal complexes from 436 spermatocytes were analyzed, and 13,969 recombination sites were mapped. Individual chromosomes for 113 of the 436 cells (representing 2,034 synaptonemal complexes) were identified by immunostaining and fluorescence in situ hybridization (FISH). The average total length of autosomal synaptonemal complexes per cell was 190.3 µm, with 32.0 recombination sites (crossovers), on average, per cell. The number of crossovers and the lengths of the autosomal synaptonemal complexes showed significant intra- (i.e. between cells) and inter-individual variations. The distributions of recombination sites within each chromosomal category were similar: crossovers in metacentric and submetacentric chromosomes were concentrated in the telomeric regions of the p- and q-arms, whereas two hotspots were located near the centromere and in the telomeric region of acrocentrics. Lack of MLH1 foci was mainly observed in the smaller chromosomes, particularly chromosome 18 (SSC18) and the sex chromosomes. All autosomes displayed positive interference, with a large variability between the chromosomes.

[Figure][1] Traces of evolutionary history appear in functional morphology and DNA sequences of living and extinct species. These remnants of the past can lead to insights into the relationships among extant groups of animals, the forces driving evolution, and the utility of animal models for studying human disease. We present below one evolutionary interpretation of the still-disputed hierarchy of surviving placental mammalian orders (excluding monotremes and marsupials), a synthesis of accumulated molecular and morphological inferences. The time scale is derived largely from molecular data; indicated fossil remains are much younger, raising controversies around the precise age of mammal ancestors. The tips of the phylogenetic tree depict genomes for 21 representative species from 11 orders, which are labeled on the vertical lines. These examples show genome-wide homology alignments assessed by comparative gene mapping or direct visualization of chromosome painting, in which fluorescently labeled, individual chromosomes are hybridized to chromosomes from distantly related species. Species were selected to maximize the number of orders represented and to illustrate patterns of genome conservation. The 24 human chromosomes are distinguished by 24 colors, and regions of human chromosome homology in other species are indicated by color and human chromosome number (for example, cat chromosome A1 contains gene stretches homologous to human chromosomes 5 and 13). Selected gene homologs in each species represent a subset of extensive comparative gene mapping data; thus genes listed may not reflect the extreme borders of the conserved segments. Gene orders have been determined in human, cat, pig, sheep, rat, mouse, and zebrafish; in other species the genes are listed in the order in which they appear in humans. The zebrafish map, including 25 linkage groups not yet assigned to the 25 chromosomes, is presented to illustrate the array of gene segments conserved for 450 million years. Further information and discussion of dissenting views of mammalian evolution can be found in this issue of Science and the associated web site at ![Figure][1] CREDITS Science Coordinators: Barbara R. Jasny and Pamela J. Hines Authors: Phylogenetic tree Stephen J. O'Brien, National Cancer Institute, Frederick, MD, USA; John F. Eisenberg, University of Florida, Gainesville, FL, USA; Michael Miyamoto, University of Florida, Gainesville, FL, USA; S. Blair Hedges, Pennsylvania State University, University Park, PA, USA; Sudhir Kumar, Arizona State University, Tempe, AZ, USA; Don E. Wilson, Smithsonian Institution, Washington, DC, USA. Genomic maps Stephen J. O'Brien, Marilyn Menotti-Raymond, William J. Murphy, William G. Nash, Leslie A. Lyons, Joan C. Menninger, Roscoe Stanyon, Johannes Wienberg, Neal G. Copeland, Nancy A. Jenkins, National Cancer Institute, Frederick, MD, USA; Joel Gellin, Martine Yerle, Institut National de la Recherche Agronomique, Castanet-Tolosan, France; Leif Andersson, Swedish University of Agricultural Sciences, Uppsala, Sweden; James Womack, Texas A & M University, College Station, TX, USA; Thomas Broad, AgResearch, Invermay, Mosgiel, New Zealand; John Postlethwait, University of Oregon, Eugene, OR, USA; Oleg Serov, Institute of Cytology and Genetics, Siberian Branch of the Academy of Sciences of Russia, Novosibirsk, Russia; Ernie Bailey, University of Kentucky, Lexington, KY, USA; Michael R. James, Wellcome Trust Centre for Human Genetics, Headington, UK; Takeshi K. Watanabe, Otsuka GEN Research Institute, Tokushima, Japan; Matthew J. Wakefield, Jennifer Marshall Graves, La Trobe University, Melbourne, Australia. Design and Art Direction: C. Faber Smith Graphics: Nathalie Cary Illustrations: Katharine Sutliff Production Assistance: Holly Bishop, Leslie Blizard, Inja Lin, Darcel Pugh Proofreading: Harry Jach [1]: pending:yes

Chantier qualité GA; International audience; Klinefelter’s syndrome (KS) is the most common sex chromosome abnormality identified in human males. This syndrome is generally associated with infertility. Men with KS may have a 47,XXY or a 46,XY/47,XXY karyotype. Studies carried out in humans and mice suggest that only XY cells are able to enter and complete meiosis. These cells could originate from the XY cells present in mosaic patients or from XXY cells that have lost one X chromosome. In pig, only 3 cases of pure 39,XXY have been reported until now, and no meiotic analysis was carried out. For the first time in pig species we report the analysis of a 38,XY/39,XXY boar and describe the origin of the supplementary X chromosome and the chromosomal constitutions of the germ and Sertoli cells.

Chantier qualité GA; International audience; Inversions are well-known structural chromosomal rearrangements in humans and pigs. Such rearrangements generally have no effect on the carriers’ phenotype. However, the presence of an inversion can lead to spermatogenesis impairments and to the production of unbalanced (recombinant) gametes, responsible for early miscarriages, stillbirth, or congenital abnormalities. Sperm samples from boars heterozygote for pericentric inv(2)(p1.1;q1.1), inv(2) (p1.1;q2.1), inv(1)(p2.1;q2.10), or inv(1)(p2.4;q2.9), as well as for paracentric inv(2)(q1.3;q2.5) or inv(1)(q1.2;q2.4) were analyzed using sperm FISH (fluorescent in situ hybridization on decondensed sperm heads) to determine the male meiotic segregation profiles of the rearrangements. Furthermore, the availability of sperm samples for 2 unrelated carriers of inv(2)(p1.1;q1.1) allowed us to check for the occurrence of inter-individual variability of the rates of unbalanced meiotic products for this rearrangement. The estimated proportions of recombinant gametes were very low for all the inversions studied (0.62%, 1.30%, 3.05%, 1.27%, 4.12% and 0.84%, respectively), albeit significantly higher than the control. The rearrangements should therefore have very little impact on the reproductive performance of the carriers. No difference was found between the 2 carriers of inv(2)(p1.1;q1.1), suggesting a lack of inter-individual variability for this rearrangement. Overall, no significant correlation was found between the sizes of the inverted fragments and the proportions of recombinant (unbalanced) gametes for the 6 inversions studied. This is in contradiction with most human results. Further studies (pairing and recombination analysis using immunostaining techniques) should be carried out to elucidate the origin of such an inter-species difference.

Chantier qualité spécifique "Auteurs Externes" département de Génétique animale : uniquement liaison auteur au référentiel HR-Access; International audience; Abstract. Heterozygous carriers of Robertsonian translocations generally have a normal phenotype but present reproductive failure. In cattle, the t(1; 29) Robertsonian translocation is very common and carriers show a 3–5% decrease in fertility. Some data suggest that female carriers have a higher decrease than male carriers but no direct studies of the chromosome content of oocytes from a t(1; 29) carrier cow have been performed so far. Four heterozygous carrier cows underwent hormonal stimulations and follicles punctions and about 800 oocytes were matured in vitro. Six hundred metaphase II preparations were obtained and analysed by fluorescent in situ hybridization with bovine chromosome 1 and 29 painting probes. Proportions of different kinds of oocytes were as-sessed: 74.11% (292/394) were normal and balanced, 4.06% (16/394) unbalanced and 21.83% (86/394) diploid. For all cows, the number of normal oocytes was not significantly different from the number of translocated oocytes but the diploidy and unbalanced rate were significantly different between them. As found in bulls, the meiotic segregation pattern in cows has shown a preponderance of alternate products. However, the frequency of unbalanced gametes determined in females (4.06%) was significantly higher than the frequency observed in males (2.76%). The divergence in the rate of diploid gametes (0.04% vs. 21.83%) is mainly explained by the difference between males and females.

A reciprocal translocation between the q arm of the Y chromosome and the q arm of chromosome 14 was identified in a young, phenotypically normal boar presenting azoospermia. Testicular biopsies were analyzed by classical histological and immunolocalization techniques, and by fluorescence in situ hybridization. Meiotic pairing analysis of 85 pachytene spreads showed the presence of an open structure corresponding to a quadrivalent formed by chromosomes 14, X, and the derivative chromosomes 14 and Y in 84.7% of the cases. In the remaining cases (15.3%), a ‘trivalent plus univalent’ configuration was observed. Immunolocalization of γH2AX revealed the presence of this modified histone in the chromatin domains of unsynapsed segments (centromeric region of chromosome 14) and spreading of the γH2AX signal from the XY body throughout chromosome 14 in 7.05% of the cells analyzed. The potential causes of the observed infertility, i.e. activation of meiotic checkpoints and/or silencing of genes necessary for the progression of meiosis, are discussed.

Whole-genome radiation hybrid (RH) panels have been constructed for several species, including cattle. RH panels have proven to be an extremely powerful tool to construct high-density maps, which is an essential step in the identification of genes controlling important traits, and they can be used to establish high-resolution comparative maps. Although bovine RH panels can be used with ovine markers to construct sheep RH maps based on bovine genome organization, only some (c. 50%) of the markers available in sheep can be successfully mapped in the bovine genome. So, with the development of genomics and genome sequencing projects, there is a need for a high-resolution RH panel in sheep to map ovine markers. Consequently, we have constructed a 12 000-rad ovine whole-genome RH panel. Two hundred and eight hybrid clones were produced, of which 90 were selected based on their retention frequency. The final panel had an average marker retention frequency of 31.8%. The resolution of this 12 000-rad panel (SheepRH) was estimated by constructing an RH framework map for a 23-Mb region of sheep chromosome 18 (OAR18) that contains a QTL for scrapie susceptibility.

The full-length cDNA of the porcine SMPX gene was obtained by the rapid amplification of cDNA ends (RACE). The nucleotide sequences and the predicted protein sequences share high sequence identity with both human and mouse. The promoter of SMPX was sequenced and then analyzed to find the promoter binding sites. The reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that SMPX has a high level of expression in heart and skeletal muscle, a very low expression in lung and spleen and no expression in liver, kidney, fat and brain. Moreover, SMPX has a differential expression level in skeletal muscle, the expression in 65-day embryos being higher than other stages. The porcine SMPX was mapped to SSCXp24 by using a somatic cell hybrid panel (SCHP) and was found closely linked to SW1903 using the radiation hybrid panel IMpRH. An A/G single nucleotide polymorphism (PCR-RFLP) in the 3'-untranslated region (3'-UTR) was detected in eight breeds. The analysis of allele frequency distribution showed that introduced pig breeds (Duroc and Large White) have a higher frequency of allele A while in the Chinese indigenous pig breeds (Qingping pig, Lantang pig, YushanBlack pig, Large Black-White pig, Small Meishan) have a higher frequencies of allele G. The association analysis using an experimental population (188 pigs), which included two cross-bred groups and three pure-blood groups, suggested that the SNP genotype was associated with intramuscular fat content.

In 1964, Gustavsson and Rockborn first described the 1/29 Robertsonian translocation in cattle. Since then, several studies have demonstrated the negative effect of this particular chromosomal rearrangement on the fertility of carrier animals. During the last decade, meiotic segregation patterns have been studied on human males carrying balanced translocations using FISH on decondensed sperm nuclei. In this work, we have applied the ‘Sperm-FISH’ technique to determine the chromosomal content of spermatozoa from two bulls heterozygous for the 1/29 translocation and one normal bull (control). 5425 and 2702 sperm nuclei were scored, respectively, for the two heterozygous bulls, using whole chromosome painting probes of chromosomes 1 and 29. Very similar proportions of normal (or balanced) spermatozoa resulting from alternate segregation were observed (97.42% and 96.78%). For both heterozygous bulls, the proportions of nullisomic and disomic spermatozoa did not follow the theoretical 1:1 ratio. Indeed, proportions of nullisomic spermatozoa were higher than those of disomic sperma tozoa (1.40% vs 0.09% (bull 1) and 1.29% vs 0.15% (bull 2) for BTA1, and 0.65% vs 0.40% (bull 1) and 1.11% vs 0.63% (bull 2) for BTA29). The average frequencies of disomic and diploid spermatozoa in the normal bull were 0.11% and 0.05%, respectively.

BACKGROUND: The comparison of male and female meiotic segregation patterns for individuals carrying identical reciprocal translocations has been rarely reported in mammalian species. The main comparative study involving males and females with comparable genetic background has been performed in the mouse. Swine is another relevant animal model species for meiotic studies. Here we present the segregation patterns determined for sows carrying one of the two following reciprocal translocations: 38, XX, rcp(3;15)(q27;q13), and 38, XX, rcp(12;14)(q13;q21). These segregation data were compared to those previously obtained for closely related boars carrying the same balanced chromosomal rearrangements. METHODS: Dual colour in situ hybridization of whole chromosome painting probes was carried out on metaphases of in vitro-matured oocytes II. Segregation results were obtained for 118 and 206 metaphases II respectively for the two translocations. RESULTS: Significant differences between sexes were demonstrated for both rearrangements. For instance, for the 3/15 translocation, the chromosomally unbalanced gametes were of different origin: preponderance of the adjacent-I segregation in the male (31.4%), and of the adjacent-II (14.3%) and 3:1 (14.3%) segregations in females. For the 12/14 translocation, the proportion of balanced gametes was greater in males than in females (75.9 and 59.4% respectively). CONCLUSION: This study is a new scientific contribution to compare the segregation patterns of male and female carriers of identical chromosomal rearrangements. The results obtained are consistent with those previously reported in mice. Hypotheses to interpret the observed differences between the two translocations, as well as between the male and female segregation patterns, are formulated and discussed.

The PA28 activator gamma-subunit encoded by the PSME3 gene is the third component of the PA28 activator complex, which is the 11S regulator of the 20S proteasome. The open reading frame (ORF) sequence of the porcine PSME3 gene encoding the proteasome activator gamma-subunits (or proteasome activator subunit 3) was determined. The deduced amino acid sequence shows 100% identity with the corresponding human and murine sequence. Two single nucleotide substitutions, one located in intron 5 (I5), the other one in exon 8 (E8), were detected using polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP). Analysis on allele frequencies of the two polymorphic sites determined in different pig breeds (Duroc, Tibet, Qingping, Meishan, Erhualian and Mingzhu) showed large differences between Duroc and Chinese indigenous pig breeds investigated. The PSME3 gene was physically assigned to SSC12p11 - (2/3) p13 in the vicinity of the GH gene. This result provides an additional type I marker to the GH linkage group on SSC12.

Chantier qualité spécifique "Auteurs Externes" département de Génétique animale : uniquement liaison auteur au référentiel HR-Access; International audience

The Suidae and the Dicotylidae (or Tayassuidae) are related mammalian families, both belonging to the artiodactyl suborder Suiformes, which diverged more than 37 million years ago. Cross-species chromosome painting was performed between the domestic pig (Sus scrofa; 2n = 38), a representative of the Suidae, and two species of the Dicotylidae: the collared peccary (Tayassu tajacu; 2n = 30) and the white-lipped peccary (T. pecari; 2n = 26). G-banded metaphase chromosomes of the two peccaries were hybridized with whole chromosome painting probes derived from domestic pig chromosomes 1-18 and X. For both peccary species, a total of 31 autosomal segments that are conserved between pig and peccary could be identified. The painting results confirm conclusions inferred from G-band analyses that the karyotypes of the collared peccary and the white-lipped peccary are largely different. The karyotypic heterogeneity of the Dicotylidae contrasts with the relative homogeneity among the karyotypes of the Suidae. For this difference between the Dicotylidae and the Suidae, a number of explanations are being postulated: 1) the extant peccaries are phylogenetically less closely related than is usually assumed; 2) the peccary genome is less stable than the genome of the pigs; and 3) special (e.g. biogeographical or biosocial) circumstances have facilitated the fixation of chromosome rearrangements in ancestral dicotylid populations.

CD47 is a integrin-associated protein originally discovered as a plasma membrane molecule that co-purified with the integrin ·vs3 from leukocytes and placenta (Brown et al., 1990). CD47 is an unusual member of the immunoglobulin (Ig) superfamily of membrane proteins, with a single IgV-like domain at its N-terminus, a highly hydrophobic stretch with five membrane-spanning segments and an alternatively spliced cytoplasmic C-terminus ranging in length from 3 to 36 amino acids (Schwartz and Baron, 1999). In human, CD47 is a receptor for thrombospondin family members, a ligand for the transmembrane signaling protein SIRP· and a component of a supramolecular complex containing specific integrins, heterotrimeric G proteins and cholesterol (Brown and Frazier, 2001). Mouse, rat, porcine and bovine CD47 molecules have been cloned and show about 70% overall amino acid identity with the human molecule suggesting that this gene might play a similar role in different species. Materials and methods

A porcine bacterial artificial chromosome (BAC) clone, containing the melanocortin 2 receptor gene (MC2R) was isolated. The complete coding sequence of the MC2R gene, contained in 1 exon, was determined. Polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) was performed on a 241-bp coding fragment. An AluI polymorphism, detecting a silent mutation, was found and typed on unrelated animals of five different pig breeds. The Meishan, Piétrain and Large White breeds differ significantly in allele frequencies from the Landrace and Czech Meat Pig breeds. The melanocortin 5 receptor gene (MC5R) was detected by PCR in the same BAC clone, as could be expected from the human and porcine mapping data. PCR-SSCP was performed on a 200-bp coding of MC5R, but no polymorphisms were detected. The BAC clone was mapped to Sscr6q27 by fluorescent in situ hybridization (FISH). A (CA)n microsatellite (SGU0002), isolated from the BAC, was localized on chromosome 6 by RH mapping near marker SW1473 and by linkage mapping on the MARC reference family at the same position as the marker SW2173 (97 cM). Allele frequencies, heterozygosity and polymorphism information contents (PIC) values were calculated for the five different pig breeds examined. The transcription of both genes in porcine liver, heart, kidney, fat, brain, pancreas, stomach, bladder, ovaries, lung, spleen, skin, adrenal gland and muscle tissues was examined by reverse transcriptase-polymerase chain reaction. Transcription was detected in skin and adrenal gland tissues for MC2R, while a positive signal was detected for MC5R in kidney, fat, pancreas, skin, adrenal gland and spleen tissues.

Previous comparative mapping suggested that at least five pig chromosomes (Sscr4, 6, 9, 10 and 14) share homology with human chromosome 1 (Hsap1). A significant quantitative trait loci (QTL) for fat deposition has been identified on Sscr4 that appears to be near the junction region between Sscr4 and Sscr9 relative to Hsap1. It is of interest to define the boundaries of conserved synteny between pig chromosomes and Hsap1 to use human map information to identify putative comparative positional candidates for this QTL. Eleven genes, including Janus kinase 1 (JAK1), Prostaglandin E receptor3 (PTGER3), urate oxidase (UOX), coagulation factor 3 (F3), vascular cell adhesion molecule 1 (VCAM1), ribosomal protein L5 (RPL5), POU domain, class 2, transcription factor 1 (POU2F1), coagulation factor 5 (F5), Prostaglandin endoperoxide synthase-2 (PTGS2), myosin binding protein H (MYBPH) and Antithrombin III (SERPINC1), were selected to refine the boundaries of the blocks of conserved synteny between Hsap1 and pig chromosomes. Pig sequence tagged sites (STSs) were developed and used to physically map these 11 genes using a somatic cell hybrid panel. Eight loci have been mapped by using fluorescent in situ hybridization (FISH) to improve map resolution. Heterologous FISH was used to refine the location of VCAM1 on human chromosomes. In addition, human yeast artificial chromosomes (YACs) were mapped by heterologous FISH on pig metaphases to refine the boundaries of the regions of homology between Sscr4 and Sscr9 on Hsap1. Results from this study suggest the precise break in conserved synteny on Hsap1 corresponding to the Sscr4/6 and Sscr4/9 transitions are most likely on the Hsap1p22 and Hsap1q24-25 regions, respectively. Further, our data predict that Hsap1q21-24 is a candidate region for the backfat QTL localized to Sscr4.

Excessive softness is a serious defect of dry cured hams which seems related to high activity of lysosomal cysteine proteinases, such as cathepsin B, in fresh pork muscles a few days after slaughtering. As it has been shown that cathepsin B activity has a moderate heritability in Italian Large White pigs we started a candidate gene approach to identify the gene(s) that affect(s) this parameter. Here, we studied two candidate genes: cathepsin B (CTSB) and cystatin B (CSTB). We amplified and sequenced porcine DNA fragments for these two genes that were used to identify polymorphisms by SSCP and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Four and two alleles were detected at the CTSB and CSTB loci, respectively. Sequencing of the CSTB alleles showed a missense mutation that changes a codon for aspartic acid into a codon for asparagine in exon 3 of the gene. Allele frequencies for the two loci differed among the pig breeds studied (Large White, Landrace, Duroc, Belgian Landrace, Hampshire, Piétrain, Meishan, Cinta Senese, Casertana, Calabrese and Nero di Sicilia). Linkage, somatic cell hybrid panel and radiation hybrid panel analyses assigned CTSB to porcine chromosome (Sscr) 14 and CSTB to Sscr 13. The markers identified at the CTSB and CSTB loci were used in association studies with several traits of economic importance including parameters that may indicate the suitability of pig meat to produce dry-cured hams. Significant associations were observed between CTSB and back-fat thickness and between CSTB and average daily gain. In this study, cathepsin B activity was not associated with the polymorphisms identified at the CTSB and CSTB loci.

We have isolated 14 differentially displayed and 10 further expressed sequence tags (ESTs) from Musculus biceps femoris of newborn healthy and splay leg piglets. By comparison with EMBL/GenBank data we could identify nine porcine homologues to human genes (TATA box binding protein associated factor B TAF1B; B-cell CLL/lymphoma 7B BCL7B; pyruvate dehydrogenase kinase, isoenzyme 4 PDK4; ribosomal protein S10 RPS10; SPARC-like 1 SPARCL1; epithelial protein lost in neoplasm beta EPLIN; N-myc downstream-regulated gene 2 NDRG2; pleiomorphic adenoma gene like 2 PLAGL and, BCL-2 associated transcription factor short form BTFS). Eight fragments correspond to uncharacterized ESTs and 7 ESTs had no significant match with database sequences. These data provide the first expression profiles in skeletal muscle of neonatal piglets and are a basis for candidate gene investigations for congenital splay leg in piglets. Eleven ESTs were physically mapped to porcine chromosomes 1, 3, 4, 5, 7, 8, 9 and 10 and contribute to the comparative map of humans and pigs.

In our studies of the genes constituting the porcine A0 blood group system, we have characterized a cDNA, encoding an alpha(1,3)N-acetylgalactosaminyltransferase, that putatively represents the blood group A transferase gene. The cDNA has a 1095-bp open reading frame and shares 76.9% nucleotide and 66.7% amino acid identity with the human ABO gene. Using a somatic cell hybrid panel, the cDNA was assigned to the q arm of pig chromosome 1, in the region of the erythrocyte antigen A locus (EAA), which represents the porcine blood group A transferase gene. The RNA corresponding to our cDNA was expressed in the small intestinal mucosae of pigs possessing EAA activity, whereas expression was absent in animals lacking this blood group antigen. The UDP-N-acetylgalactosamine (UDP-GalNAc) transferase activity of the gene product, expressed in Chinese hamster ovary (CHO) cells, was specific for the acceptor fucosyl-alpha(1,2)galactopyranoside; the enzyme did not use phenyl-beta-D-galactopyranoside (phenyl-beta-D-Gal) as an acceptor. Because the alpha(1,3)GalNAc transferase gene product requires an alpha(1,2)fucosylated acceptor for UDP-GalNAc transferase activity, the alpha(1,2)fucosyltransferase gene product is necessary for the functioning of the alpha(1,3)GalNAc transferase gene product. This mechanism underlies the epistatic effect of the porcine S locus on expression of the blood group A antigen.CDS: coding sequence; CHO: Chinese Hamster Ovary; EAA: erythrocyte antigen A; FCS: foetal calf serum; Fucalpha(1,2)Gal: fucosyl-alpha(1,2)galactopyranoside; Gal: galactopyranoside; GGTA1: Galalpha(1,3)Gal transferase; PCR: polymerase chain reaction; phenyl-beta-D-Gal: phenyl-beta-D-galactopyranoside; R: Galbeta1-4Glcbeta1-1Cer; UDP-GalNAc: uridine diphosphate N-acetylgalactosamine

Interleukin 18 (IL-18), a recently described cytokine, plays an important role in the cell-mediated immune response, in particular through its ability to induce the production of interferon (IFN)-gamma. We cloned pig IL-18 cDNA from the intestinal epithelial cell line IPI-2I using a reverse transcriptase-polymerase chain reaction method with primers derived from the human IL-18 sequence. The amino acid sequence deduced from pig IL-18 cDNA encodes a 192 amino-acid polypeptide that exhibits 92, 90, 81, and 71% similarity to IL-18 from horse, dog, human, and rodents (mouse and rat), respectively. Structural comparison of the IL-18 protein with IL-1alpha and IL-1beta showed that IL-18 shares several characteristics with the IL-1 cytokine family: the IL-1 signature-like sequence, a potential caspase-1 (ICE) cleavage site, and the presence of 12 predicted beta strands. Fluorescence in situ hybridization was used to localize the IL-18 gene on the short arm (p13) of pig chromosome 9. Analysis of IL-18 expression in different organs of piglets demonstrated that IL-18 mRNA is weakly expressed in the kidney and the lung. By contrast, we observed highly constitutive expression of IL-18 mRNA in the spleen, mesenteric lymph nodes, and the intestine, particularly in the small intestine, indicating a potential role for IL-18 as a first line of host defense in the intestinal mucosa.

Previous mapping between the human and pig genomes suggested extensive conservation of human chromosome 13 (HSA13) to pig chromosome 11 (SSC11). The objectives of this study were comparative gene mapping of pig homologs of HSA13 genes and examining gene order within this conserved synteny group by physical assignment of each locus. A detailed HSA13 to SSC11 comparison was chosen since the comparative gene map is not well developed for these chromosomes and a rearranged gene order within conserved synteny groups was observed from the comparison between HSA13 and bovine chromosome 12 (BTA12). Heterologous primers for PCR were designed and used to amplify pig homologous fragments. The pig fragments were sequenced to confirm the homology. Six pig STSs (FLT1, ESD, RB1, HTR2A, EDNRB, and F10) were physically mapped using a somatic cell hybrid panel to SSC11, and fluorescent in situ hybridization (FISH) mapping was also applied to improve map resolution and determine gene order. Results from this study increase the comparative information available on SSC11 and suggest a conserved gene order on SSC11 and HSA13, in contrast to human:bovine comparisons of this syntenic group.

We report the use of dual-colour chromosome painting to determine the exact nature of certain chromosome rearrangements observed in the pig (Sus scrofa domestica). The chromosomal abnormalities were detected by GTG- and RBG-banding techniques. The initially proposed interpretations were: (1) rcp(6;13)(p1.5;q4.1); (2) rcp(11;16)(p1.4;q1.4); (3) rcp(6;16)(p1.1;q1.1); (4) rcp(13;17)(q4.1;q1.1); (5) rcp(6;14)(q2.7;q2.1); (6) rcp(3;5)(p1.3;q2.3); (7) rcp(2; 14)(q1.3;q2.7); (8) rcp(15;17)(q1.3;q2.1). Hybridizations were carried out with biotin- and digoxigenin-labelled probes obtained by priming authorizing random mismatches polymerase chain reaction (PARM-PCR) amplification of porcine flow-sorted chromosomes. In some cases, i.e. (1), (4), (5), (6), (7) and (8), the fluorescence in situ hybridization (FISH) results allowed confirmation of the interpretations proposed with classical cytogenetic methods. Chromosome painting proved the reciprocity of the translocation in cases (1), (6) and (8), whereas modifications of the formula were proposed for case (2). Primed in situ DNA labelling (PRINS) experiments have also been carried out in case (3) using a primer specific for the centromeres of acrocentric chromosomes (first experiment) or a primer specific for the centromeres of a subset of meta- and submetacentric chromosomes including chromosome 6 (second experiment). It allowed us to demonstrate that the breakpoints occurred in the centromeric region of chromosome 16 and in the p. arm of chromosome 6, just above the centromere.