Analysis of the non-recombining Y chromosome defines polymorphisms in domestic pig breeds: ancestral bases identified by comparative sequencing.

Sequences from 20 amplicons representing nine different loci and 11369bp from the short arm of the pig Y chromosome were compared using pools of DNA from different European and Chinese breeds. A total of 33 polymorphic sites were identified, including five indels and 28 single nucleotide polymorphisms (SNPs). Three high frequency SNPs within the coding regions of SRY were further analysed across 889 males representing 25 European and 25 Asian breeds or Lines, plus a European Line of Meishan. Two haplotypes seen to be associated with 'European' or 'Chinese' origin in the initial SNP discovery phase were found to be the most common in their respective groups of breeds in a more detailed genotyping study. Two further SRY haplotypes are relatively rare. One was found exclusively within Tamworth, at low frequency in Retinto, and in three Chinese breeds (Huai, Sahwutou and Xiaomeishan). The other uncommon haplotype is found exclusively in Bamajiang, two further Chinese breeds (Hangjiang Black and Longling) and two European rare breeds (Mangalica and Linderödssvin), but appears based on comparison with other suids to represent an ancestral sequence.

Mapping markers linked to porcine salmonellosis susceptibility.

The goal of this study was to identify pig chromosomal regions associated with susceptibility to salmonellosis. Genomic DNA from pig reference populations with differences in susceptibility to Salmonella enterica serovar Choleraesuis as quantified by spleen and liver bacterial colonization at day 7 post-infection (dpi; Van Diemen et al. 2002) was used. These samples belonged to the offspring of a sire thought to be heterozygous for genes involved in susceptibility to salmonellosis. Amplified fragment length polymorphism (AFLP) markers were created and used to determine associations with spleen or bacterial counts at 7 dpi. To position linked markers, two mapping populations, the Roslin and Uppsala PiGMaP pedigrees were used to create an integrated map which included the AFLP markers associated with salmonellosis. Twenty-six AFLP markers located in 14 different chromosomal regions in the porcine genome were found to be significantly associated with susceptibility (Chi-square P < 0.05). More than one linked marker was found on chromosomes 1, 7, 13, 14 and 18. It is likely that these regions contain genes involved in Salmonella susceptibility. Regions on chromosomes 1, 7 and 14 were significantly associated with Salmonella counts in the liver and regions on chromosomes 11, 13 and 18 with counts in spleen. The identification of these chromosomal regions highlights specific areas to search for candidate genes that may be involved in innate or adaptive immunity. Further investigation into these chromosomal regions would be useful to improve our understanding of host responses to infection with this widespread pathogen.

Genetic diversity analysis using lowly polymorphic dominant markers: the example of AFLP in pigs.

DNA markers are commonly used for large-scale evaluation of genetic diversity in farm animals, as a component of the management of animal genetic resources. AFLP markers are useful for such studies as they can be generated relatively simply; however, challenges in analysis arise from their dominant scoring and the low level of polymorphism of some markers. This paper describes the results obtained with a set of AFLP markers in a study of 59 pig breeds. AFLP fingerprints were generated using four primer combinations (PC), yielding a total of 148 marker loci, and average harmonic mean of breed sample size was 37.3. The average proportion of monomorphic populations was 63% (range across loci: 3%-98%). The moment-based method of Hill and Weir (2004, Mol Ecol 13:895-908) was applied to estimate gene frequencies, gene diversity (F(ST)), and Reynolds genetic distances. A highly significant average F(ST) of 0.11 was estimated, together with highly significant PC effects on gene diversity. The variance of F(ST) across loci also significantly exceeded the variance expected under the hypothesis of AFLP neutrality, strongly suggesting the sensitivity of AFLP to selection or other forces. Moment estimates were compared to estimates derived from the square root estimation of gene frequency, as currently applied for dominant markers, and the biases incurred in the latter method were evaluated. The paper discusses the hypotheses underlying the moment estimations and various issues relating to the biallelic, dominant, and lowly polymorphic nature of this set of AFLP markers and to their use as compared to microsatellites for measuring genetic diversity.

Genetic diversity within and between European pig breeds using microsatellite markers.

An important prerequisite for a conservation programme is a comprehensive description of genetic diversity. The aim of this study was to use anonymous genetic markers to assess the between- and the within-population components of genetic diversity for European pig breeds at the scale of the whole continent using microsatellites. Fifty-eight European pig breeds and lines were analysed including local breeds, national varieties of international breeds and commercial lines. A sample of the Chinese Meishan breed was also included. Eleven additional breeds from a previous project were added for some analyses. Approximately 50 individuals per breed were genotyped for a maximum of 50 microsatellite loci. Substantial within-breed variability was observed, with the average expected heterozygosity and observed number of alleles per locus being 0.56 [range 0.43-0.68] and 4.5 respectively. Genotypic frequencies departed from Hardy-Weinberg expectations (P < 0.01) in 15 European populations, with an excess of homozygotes in 12 of them. The European breeds were on average genetically very distinct, with a Wright F(ST) index value of 0.21. The Neighbour-Joining tree drawn from the Reynolds distances among the breeds showed that the national varieties of major breeds and the commercial lines were mostly clustered around their breeds of reference (Duroc, Hampshire, Landrace, Large White and Piétrain). In contrast, local breeds, with the exception of the Iberian breeds, exhibited a star-like topology. The results are discussed in the light of various forces, which may have driven the recent evolution of European pig breeds. This study has consequences for the interpretation of biodiversity results and will be of importance for future conservation programmes.

Genetic diversity in European pigs utilizing amplified fragment length polymorphism markers.

The use of DNA markers to evaluate genetic diversity is an important component of the management of animal genetic resources. The Food and Agriculture Organisation of the United Nations (FAO) has published a list of recommended microsatellite markers for such studies; however, other markers are potential alternatives. This paper describes results obtained with a set of amplified fragment length polymorphism (AFLP) markers as part of a genetic diversity study of European pig breeds that also utilized microsatellite markers. Data from 148 AFLP markers genotyped across samples from 58 European and one Chinese breed were analysed. The results were compared with previous analyses of data from 50 microsatellite markers genotyped on the same animals. The AFLP markers had an average within-breed heterozygosity of 0.124 but there was wide variation, with individual markers being monomorphic in 3-98% of the populations. The biallelic and dominant nature of AFLP markers creates a challenge for their use in genetic diversity studies as each individual marker contains limited information and AFLPs only provide indirect estimates of the allelic frequencies that are needed to estimate genetic distances. Nonetheless, AFLP marker-based characterization of genetic distances was consistent with expectations based on breed and regional distributions and produced a similar pattern to that obtained with microsatellites. Thus, data from AFLP markers can be combined with microsatellite data for measuring genetic diversity.

A survey of pre-slaughter conditions, halothane gene frequency, and carcass and meat quality in five Spanish pig commercial abattoirs.

A total of 116 deliveries, comprising 15,695 commercial pigs delivered to five abattoirs, were surveyed during winter and summer. Information about on-farm fasting, transport duration and stocking density, and lairage time was collected. Cortisol, creatine phospho-kinase (CPK), and lactate, and DNA for halothane genotype were analysed in a subsample of pigs at exsanguination in every journey. Electrical conductivity (PQM) in semimembranosus muscle (SM) and carcass characteristics (Fat-o-Meater and skin damage) were measured in each carcass. pHu of SM was analysed in the laboratory in a subsample in every journey. Carcasses were identified as PSE or DFD based on PQM and pHu, respectively. The n gene frequency ranged among abattoirs from 54 to 8%. Mean lean content was 58.9% for nn, 57.3% for Nn, and 55.8% for NN pigs, though a difference of 2.5% lean was observed between two abattoirs with the same n gene frequency. A straight relationship of the incidence of serious PSE carcasses and n gene frequency was found. The overall incidence of serious PSE and DFD carcasses was 6.5 and 12.5%, respectively. A higher incidence of PSE carcasses was found in summer; in deliveries with <12 h on-farm fasting; with transport stocking densities >0.40 m(2)/100 kg pig; and in transports of <2 h duration. A higher incidence of DFD carcasses was found in winter; with transport stocking densities <0.40 m(2)/100 kg pig; transports of >2 h duration; and lairage times >9 h. Cortisol level in blood increased in winter and decreased after 12-18 h fasting time. A rise in the lactate concentration was observed in pigs transported in high stocking density (<0.40 m(2)/100 kg pig) and for a longer time (>2 h). All blood stress indices increase as increasing lairage time. Carcasses with more skin damage had higher levels of cortisol, CPK and lactate, and higher incidence of DFD meat, compared with non and low skin damage carcasses.

Detection of Listeria species and Listeria monocytogenes using polymerase chain reaction.

Five oligonucleotide sequences are described that were used as primers in the polymerase chain reaction (PCR) to amplify specific sequences from Listeria DNA. When all five primers were used in combination, three PCR products were possible; a Listeria specific product that occurs with DNA from any Listeria sp., a Listeria monocytogenes specific product that occurs only in the presence of DNA from this organism and a universal product that is found using DNA from any bacterial source. The occurrence of these PCR products was used as a diagnostic test on bacteria isolated from various food samples to detect Listeria sp. and L. monocytogenes.

Molecular cloning and characterization of the beta-amylase gene from Bacillus circulans.

A gene encoding the beta-amylase of Bacillus circulans was isolated from a lambda library and sequenced. The structural gene consists of a 1725 bp open reading frame encoding a polypeptide with a predicted molecular wt of 62830 Daltons. Two active forms of the enzyme were found when the gene was expressed in E. coli. The larger 60 kD form was approximately 3 kD larger than the mature beta-amylase secreted from B. circulans, suggesting that processing of this protein is different between the two species. The smaller 49 kD form is also present at a low level in B. circulans and may result from proteolytic cleavage. The enzyme has a temperature optimum of 50 degrees C. Two other genes, one encoding an alpha-amylase and one a pullulanase, were also isolated from the lambda library.

Interferon gamma mRNA "superinduction" in human lymphocytes.

Human peripheral blood mononuclear lymphocytes produce interferon gamma (IFN-gamma) in response to stimulation by mitogens. Previous studies on the kinetics of IFN-gamma mRNA production upon mitogen induction, showed that steady-state levels of mRNA increased to a maximum at 12-24 h post-induction after which they declined to levels not detectable by the assay used. We show here that in mitogen induced peripheral blood lymphocytes, inhibition of protein synthesis using three different inhibitors (cycloheximide, puromycin, pactamycin) resulted in an increase in the steady-state levels of IFN-gamma mRNA. The levels of mRNA in cells treated with inhibitor 16 h post-induction were up to 3-fold higher than in untreated cells. Superinduction was possible up to 40 h post-induction after which the steady-state levels of mRNA had declined to limits below detection; IFN-gamma mRNA was not superinduced by cycloheximide in the presence of actinomycin D.

Role of protein synthesis in induction of interferon-gamma by mitogens in human lymphocytes.

The effect of inhibition of protein synthesis on interferon gamma (IFN-gamma) mRNA induction has been examined in human peripheral blood leukocytes and growing T lymphoblasts. Inhibition of protein synthesis by cycloheximide or puromycin when lymphocytes were stimulated with mitogen alone had little effect on the steady-state levels of IFN-gamma mRNA induced. Activation of the IFN-gamma gene appears to occur independently of synthesis of protein factors. Synergistic induction of IFN-gamma by mitogen plus the phorbol ester mezerein was at least in part accounted for by increased levels of IFN-gamma mRNA in both fresh lymphocytes and growing lymphoblasts. Inhibition of protein synthesis abolished the synergistic effect on mRNA leading to levels similar to those observed in cells treated with mitogen alone. Synergistic induction is dependent upon synthesis of protein factors either within the cells or produced as soluble mediators; these factors are not simply lymphokines since addition of exogenous factors to the cultures did not reverse the block on synergy by the inhibition. These data suggest that protein factors though they may be important in exerting qualitative control on the level of production of IFN-gamma have no role in the initial activation of the gene.

Differences in the expression of the human interferon-gamma gene in fresh lymphocytes and cultured lymphoblasts.

Fresh human peripheral blood mononuclear lymphocytes and lymphoblasts that had been grown for a period in T-cell growth-factor containing medium were stimulated with staphylococcal enterotoxin A plus mezerein to produce interferon-gamma (IFN-gamma). Growing lymphoblasts produced peak levels of IFN-gamma much earlier after induction than fresh lymphocytes. Quantitation of the steady-state levels of IFN-gamma mRNA showed these to differ markedly between the two cell types over a period of time post-induction. In fresh lymphocytes the steady-state levels of IFN-gamma mRNA increased to a peak level over a period of 4 days while in growing lymphoblasts the peak level occurred after 8 hours. These differences in IFN-gamma mRNA production were shown to be not the result of gross alteration of RNA metabolism following blast transformation.

Expression of the human interferon-beta gene cloned in phage M13 mp7.

The single-stranded DNA phage, M13 mp7 was used in the construction of an expression vector containing the coding sequence for mature interferon-beta (IFN-beta). Two clones expressed a fused polypeptide showing the biological and physicochemical properties of IFN-beta, despite the fact that the N-terminal amino acid sequence had been changed; 10(6) I.U./l of culture were produced with a molecular weight of 20 000.

Translation of mRNA for Limulus polyphemus haemocyanin polypeptides in vitro: studies on subunit heterogeneity.

The haemocyanin of Limulus polyphemus is composed of a number (possibly 10-15) of polypeptides and is believed to be synthesised in cells called cyanoblasts. In vitro translation in the rabbit reticulocyte haemolysate system and in Xenopus oocytes, of mRNA isolated from cyanoblast-containing tissue, allowed the detection of several haemocyanin polypeptides amongst the products of translation. At least seven polypeptides with molecular weights in the range 68 000-71 000 were identified by an immunological method followed by electrophoretic characterisation on two-dimensional polyacrylamide gels. Comparison of the polypeptide patterns of authentic haemocyanin, reticulocyte lysate translation products and Xenopus oocyte translation products led to the conclusion that the polypeptides are unlikely to undergo significant post-translational modification or to possess cleavable signal sequences. It is proposed that release of haemocyanin into the haemolymph in vivo may involve bursting of the cyanoblasts.