A frameshift deletion in the GDF9 gene in Icelandic Loa prolific sheep.

Several genetic variants have been shown to affect the mean number of offspring in different sheep breeds. Here, we analyzed samples from Icelandic sheep with the aim of identifying the genetic cause of the Icelandic Loa phenotype using three previously identified prolificacy genes as candidates. We demonstrate that a 4-bp frameshift deletion positioned in the mature region of the GDF9 protein in the Loa animals is a likely causal mutation for the observed increase in prolificacy; however, sequencing showed that not all ewes with a high number of offspring carried the deletion, suggesting the presence of a second mutation segregating within this group of animals.

Authoritative subspecies diagnosis tool for European honey bees based on ancestry informative SNPs.

BACKGROUND: With numerous endemic subspecies representing four of its five evolutionary lineages, Europe holds a large fraction of Apis mellifera genetic diversity. This diversity and the natural distribution range have been altered by anthropogenic factors. The conservation of this natural heritage relies on the availability of accurate tools for subspecies diagnosis. Based on pool-sequence data from 2145 worker bees representing 22 populations sampled across Europe, we employed two highly discriminative approaches (PCA and FST) to select the most informative SNPs for ancestry inference. RESULTS: Using a supervised machine learning (ML) approach and a set of 3896 genotyped individuals, we could show that the 4094 selected single nucleotide polymorphisms (SNPs) provide an accurate prediction of ancestry inference in European honey bees. The best ML model was Linear Support Vector Classifier (Linear SVC) which correctly assigned most individuals to one of the 14 subspecies or different genetic origins with a mean accuracy of 96.2% ± 0.8 SD. A total of 3.8% of test individuals were misclassified, most probably due to limited differentiation between the subspecies caused by close geographical proximity, or human interference of genetic integrity of reference subspecies, or a combination thereof. CONCLUSIONS: The diagnostic tool presented here will contribute to a sustainable conservation and support breeding activities in order to preserve the genetic heritage of European honey bees.

Origin and diversification of the plasminogen activation system among chordates.

BACKGROUND: The plasminogen (PLG) activation system is composed by a series of serine proteases, inhibitors and several binding proteins, which together control the temporal and spatial generation of the active serine protease plasmin. As this proteolytic system plays a central role in human physiology and pathophysiology it has been extensively studied in mammals. The serine proteases of this system are believed to originate from an ancestral gene by gene duplications followed by domain gains and deletions. However, the identification of ancestral forms in primitive chordates supporting these theories remains elusive. In addition, evolutionary studies of the non-proteolytic members of this system are scarce. RESULTS: Our phylogenetic analyses place lamprey PLG at the root of the vertebrate PLG-group, while lamprey PLG-related growth factors represent the ancestral forms of the jawed-vertebrate orthologues. Furthermore, we find that the earliest putative orthologue of the PLG activator group is the hyaluronan binding protein 2 (HABP2) gene found in lampreys. The prime plasminogen activators (tissue- and urokinase-type plasminogen activator, tPA and uPA) first occur in cartilaginous fish and phylogenetic analyses confirm that all orthologues identified compose monophyletic groups to their mammalian counterparts. Cartilaginous fishes exhibit the most ancient vitronectin of all vertebrates, while plasminogen activator inhibitor 1 (PAI-1) appears for the first time in cartilaginous fishes and is conserved in the rest of jawed vertebrate clades. PAI-2 appears for the first time in the common ancestor of reptiles and mammals, and represents the latest appearing plasminogen activator inhibitor. Finally, we noted that the urokinase-type plasminogen activator receptor (uPAR)-and three-LU domain containing genes in general-occurred later in evolution and was first detectable after coelacanths. CONCLUSIONS: This study identifies several primitive orthologues of the mammalian plasminogen activation system. These ancestral forms provide clues to the origin and diversification of this enzyme system. Further, the discovery of several members-hitherto unknown in mammals-provide new perspectives on the evolution of this important enzyme system.

Analyses of pig genomes provide insight into porcine demography and evolution.

For 10,000 years pigs and humans have shared a close and complex relationship. From domestication to modern breeding practices, humans have shaped the genomes of domestic pigs. Here we present the assembly and analysis of the genome sequence of a female domestic Duroc pig (Sus scrofa) and a comparison with the genomes of wild and domestic pigs from Europe and Asia. Wild pigs emerged in South East Asia and subsequently spread across Eurasia. Our results reveal a deep phylogenetic split between European and Asian wild boars ∼1 million years ago, and a selective sweep analysis indicates selection on genes involved in RNA processing and regulation. Genes associated with immune response and olfaction exhibit fast evolution. Pigs have the largest repertoire of functional olfactory receptor genes, reflecting the importance of smell in this scavenging animal. The pig genome sequence provides an important resource for further improvements of this important livestock species, and our identification of many putative disease-causing variants extends the potential of the pig as a biomedical model.

A synteny-based draft genome sequence of the forage grass Lolium perenne.

Here we report the draft genome sequence of perennial ryegrass (Lolium perenne), an economically important forage and turf grass species that is widely cultivated in temperate regions worldwide. It is classified along with wheat, barley, oats and Brachypodium distachyon in the Pooideae sub-family of the grass family (Poaceae). Transcriptome data was used to identify 28,455 gene models, and we utilized macro-co-linearity between perennial ryegrass and barley, and synteny within the grass family, to establish a synteny-based linear gene order. The gametophytic self-incompatibility mechanism enables the pistil of a plant to reject self-pollen and therefore promote out-crossing. We have used the sequence assembly to characterize transcriptional changes in the stigma during pollination with both compatible and incompatible pollen. Characterization of the pollen transcriptome identified homologs to pollen allergens from a range of species, many of which were expressed to very high levels in mature pollen grains, and are potentially involved in the self-incompatibility mechanism. The genome sequence provides a valuable resource for future breeding efforts based on genomic prediction, and will accelerate the development of new varieties for more productive grasslands.

A-to-I RNA editing of the IGFBP7 transcript increases during aging in porcine brain tissues.

The IGFBP7 gene encodes insulin-like growth factor protein 7. IGFPB7 is involved in diverse biological functions including cell growth regulation, senescence and apoptosis, and also acts as a tumor suppressor in multiple cancers. The IGFBP7 mRNA is subject to A-to-I RNA editing mediated by adenosine deaminases acting on RNA 1 and 2 (ADAR1 and ADAR2). In the current study we have examined molecular characteristics of the porcine IGFBP7 gene, and determined the mRNA editing in different tissues. The A-to-I RNA editing of human IGFBP7 in positions Arg78 and Lys95 was shown to be conserved in the porcine homologue. In addition, a novel editing site was discovered in position Lys97 in the porcine IGFBP7 transcript. A differential editing was demonstrated at the three positions in the IGFBP7 transcript with very high degrees of editing in frontal cortex, cerebellum and lung. Interestingly, the degree of editing increased during aging in porcine frontal cortex and cerebellum. The IGFBP7 gene was mapped to pig chromosome 8. The porcine IGFBP7 gene was found to be ubiquitously expressed in examined organs and tissues. The methylation status of the IGFBP gene was examined in brain and liver by bisulfate sequencing and a high degree of methylation was found in the two tissues, 52% and 54%, respectively.

Ancient human genome sequence of an extinct Palaeo-Eskimo.

We report here the genome sequence of an ancient human. Obtained from approximately 4,000-year-old permafrost-preserved hair, the genome represents a male individual from the first known culture to settle in Greenland. Sequenced to an average depth of 20x, we recover 79% of the diploid genome, an amount close to the practical limit of current sequencing technologies. We identify 353,151 high-confidence single-nucleotide polymorphisms (SNPs), of which 6.8% have not been reported previously. We estimate raw read contamination to be no higher than 0.8%. We use functional SNP assessment to assign possible phenotypic characteristics of the individual that belonged to a culture whose location has yielded only trace human remains. We compare the high-confidence SNPs to those of contemporary populations to find the populations most closely related to the individual. This provides evidence for a migration from Siberia into the New World some 5,500 years ago, independent of that giving rise to the modern Native Americans and Inuit.

Deep sequencing of Danish Holstein dairy cattle for variant detection and insight into potential loss-of-function variants in protein coding genes.

BACKGROUND: Over the last few years, continuous development of high-throughput sequencing platforms and sequence analysis tools has facilitated reliable identification and characterization of genetic variants in many cattle breeds. Deep sequencing of entire genomes within a cattle breed that has not been thoroughly investigated would be imagined to discover functional variants that are underlying phenotypic differences. Here, we sequenced to a high coverage the Danish Holstein cattle breed to detect and characterize single nucleotide polymorphisms (SNPs), insertion/deletions (Indels), and loss-of-function (LoF) variants in protein-coding genes in order to provide a comprehensive resource for subsequent detection of causal variants for recessive traits. RESULTS: We sequenced four genetically unrelated Danish Holstein cows with a mean coverage of 27X using an Illumina Hiseq 2000. Multi-sample SNP calling identified 10,796,794 SNPs and 1,295,036 indels whereof 482,835 (4.5 %) SNPs and 231,359 (17.9 %) indels were novel. A comparison between sequencing-derived SNPs and genotyping from the BovineHD BeadChip revealed a concordance rate of 99.6-99.8 % for homozygous SNPs and 93.3-96.5 % for heterozygous SNPs. Annotation of the SNPs discovered 74,886 SNPs and 1937 indels affecting coding sequences with 2145 being LoF mutations. The frequency of LoF variants differed greatly across the genome, a hot spot with a strikingly high density was observed in a 6 Mb region on BTA18. LoF affected genes were enriched for functional categories related to olfactory reception and underrepresented for genes related to key cellular constituents and cellular and biological process regulation. Filtering using sequence derived genotype data for 288 Holstein animals from the 1000 bull genomes project removing variants containing homozygous individuals retained 345 of the LoF variants as putatively deleterious. A substantial number of the putative deleterious LoF variants had a minor allele frequency >0.05 in the 1000 bull genomes data set. CONCLUSIONS: Deep sequencing of Danish Holstein genomes enabled us to identify 12.1 million variants. An investigation into LoF variants discovered a set of variants predicted to disrupt protein-coding genes. This catalog of variants will be a resource for future studies to understand variation underlying important phenotypes, particularly recessively inherited lethal phenotypes.

Cloning and characterization of the porcine DBC1 gene encoding deleted in bladder cancer.

Deleted in bladder cancer 1 (DBC1) is a tumour suppressor which is involved in the regulation of cell growth and programmed cell death. In this study we report the cloning and characterization of porcine DBC1 cDNA. RT-PCR cloning produced a cDNA with an open reading frame of 2,283 bp encoding a polypeptide of 761 amino acids with a predicted molecular mass of 88.6 kDa and estimated isoelectric point of 9.1. The encoded pig DBC1 protein shows a very high amino acid similarity to human (99 %) and to mouse (98 %) DBC1. The porcine DBC1 gene was mapped to chromosome 1. The nucleotide sequence of the promoter displayed a high degree of conservation of elements responsible for neuron-specific expression. The porcine DBC1 gene was found to be highly expressed in brain tissues. The methylation status of the porcine DBC1 gene was examined in brain and liver by bisulfite sequencing. Methylation percentages of 53-61 were observed for the gene body whereas significantly lower values (1-4 %) were found in exon 1 and the promoter sequence of DBC1. The sequences of the porcine DBC1 cDNA and the DBC1 promoter and exon 1 sequence have been submitted to DDBJ/EMBL/GenBank under the accession numbers KF733442 and KJ396193, respectively.

Surfactant protein-D-encoding gene variant polymorphisms are linked to respiratory outcome in premature infants.

OBJECTIVE: Associations between the genetic variation within or downstream of the surfactant protein-D-encoding gene (SFTPD), which encodes the collectin surfactant protein-D (SP-D) and may lead to respiratory distress syndrome or bronchopulmonary dysplasia, recently were reported. Our aim was to investigate whether SFTPD variations affect serum SP-D levels in infants and pulmonary outcome in premature infants. STUDY DESIGN: Serum SP-D levels were measured in 211 mature and 202 premature infants, and 7 SFTPD single-nucleotide polymorphisms (SNPs) were genotyped. SNP analysis and haplotype analysis were used to associate genetic variation to SP-D, respiratory distress (RD), oxygen requirement, and respiratory support. RESULTS: The 5'-upstream SFTPD SNP rs1923534 and the 3 structural SNPs rs721917, rs2243639, and rs3088308 were associated with the SP-D level. The same SNPs were associated with RD, a requirement for supplemental oxygen, and a requirement for respiratory support. Haplotype analyses identified 3 haplotypes that included the minor alleles of rs1923534, rs721917, and rs3088308 that exhibited highly significant associations with decreased SP-D levels and decreased ORs for RD, oxygen supplementation, and respiratory support. CONCLUSION: These findings extend and validate previous observations of SFTPD association with the risk of respiratory outcomes and suggest SFTPD as an essential factor affecting pulmonary adaptation in premature infants.

The genome and transcriptome of perennial ryegrass mitochondria.

BACKGROUND: Perennial ryegrass (Lolium perenne L.) is one of the most important forage and turf grass species of temperate regions worldwide. Its mitochondrial genome is inherited maternally and contains genes that can influence traits of agricultural importance. Moreover, the DNA sequence of mitochondrial genomes has been established and compared for a large number of species in order to characterize evolutionary relationships. Therefore, it is crucial to understand the organization of the mitochondrial genome and how it varies between and within species. Here, we report the first de novo assembly and annotation of the complete mitochondrial genome from perennial ryegrass. RESULTS: Intact mitochondria from perennial ryegrass leaves were isolated and used for mtDNA extraction. The mitochondrial genome was sequenced to a 167-fold coverage using the Roche 454 GS-FLX Titanium platform, and assembled into a circular master molecule of 678,580 bp. A total of 34 proteins, 14 tRNAs and 3 rRNAs are encoded by the mitochondrial genome, giving a total gene space of 48,723 bp (7.2%). Moreover, we identified 149 open reading frames larger than 300 bp and covering 67,410 bp (9.93%), 250 SSRs, 29 tandem repeats, 5 pairs of large repeats, and 96 pairs of short inverted repeats. The genes encoding subunits of the respiratory complexes - nad1 to nad9, cob, cox1 to cox3 and atp1 to atp9 - all showed high expression levels both in absolute numbers and after normalization. CONCLUSIONS: The circular master molecule of the mitochondrial genome from perennial ryegrass presented here constitutes an important tool for future attempts to compare mitochondrial genomes within and between grass species. Our results also demonstrate that mitochondria of perennial ryegrass contain genes crucial for energy production that are well conserved in the mitochondrial genome of monocotyledonous species. The expression analysis gave us first insights into the transcriptome of these mitochondrial genes in perennial ryegrass.

Integration of mate pair sequences to improve shotgun assemblies of flow-sorted chromosome arms of hexaploid wheat.

BACKGROUND: The assembly of the bread wheat genome sequence is challenging due to allohexaploidy and extreme repeat content (>80%). Isolation of single chromosome arms by flow sorting can be used to overcome the polyploidy problem, but the repeat content cause extreme assembly fragmentation even at a single chromosome level. Long jump paired sequencing data (mate pairs) can help reduce assembly fragmentation by joining multiple contigs into single scaffolds. The aim of this work was to assess how mate pair data generated from multiple displacement amplified DNA of flow-sorted chromosomes affect assembly fragmentation of shotgun assemblies of the wheat chromosomes. RESULTS: Three mate pair (MP) libraries (2 Kb, 3 Kb, and 5 Kb) were sequenced to a total coverage of 89x and 64x for the short and long arm of chromosome 7B, respectively. Scaffolding using SSPACE improved the 7B assembly contiguity and decreased gene space fragmentation, but the degree of improvement was greatly affected by scaffolding stringency applied. At the lowest stringency the assembly N50 increased by ~7 fold, while at the highest stringency N50 was only increased by ~1.5 fold. Furthermore, a strong positive correlation between estimated scaffold reliability and scaffold assembly stringency was observed. A 7BS scaffold assembly with reduced MP coverage proved that assembly contiguity was affected only to a small degree down to ~50% of the original coverage. CONCLUSION: The effect of MP data integration into pair end shotgun assemblies of wheat chromosome was moderate; possibly due to poor contig assembly contiguity, the extreme repeat content of wheat, and the use of amplified chromosomal DNA for MP library construction.

Pairwise comparisons of ten porcine tissues identify differential transcriptional regulation at the gene, isoform, promoter and transcription start site level.

The transcriptome is the absolute set of transcripts in a tissue or cell at the time of sampling. In this study RNA-Seq is employed to enable the differential analysis of the transcriptome profile for ten porcine tissues in order to evaluate differences between the tissues at the gene and isoform expression level, together with an analysis of variation in transcription start sites, promoter usage, and splicing. Totally, 223 million RNA fragments were sequenced leading to the identification of 59,930 transcribed gene locations and 290,936 transcript variants using Cufflinks with similarity to approximately 13,899 annotated human genes. Pairwise analysis of tissues for differential expression at the gene level showed that the smallest differences were between tissues originating from the porcine brain. Interestingly, the relative level of differential expression at the isoform level did generally not vary between tissue contrasts. Furthermore, analysis of differential promoter usage between tissues, revealed a proportionally higher variation between cerebellum (CBE) versus frontal cortex and cerebellum versus hypothalamus (HYP) than in the remaining comparisons. In addition, the comparison of differential transcription start sites showed that the number of these sites is generally increased in comparisons including hypothalamus in contrast to other pairwise assessments. A comprehensive analysis of one of the tissue contrasts, i.e. cerebellum versus heart for differential variation at the gene, isoform, and transcription start site (TSS), and promoter level showed that several of the genes differed at all four levels. Interestingly, these genes were mainly annotated to the "electron transport chain" and neuronal differentiation, emphasizing that "tissue important" genes are regulated at several levels. Furthermore, our analysis shows that the "across tissue approach" has a promising potential when screening for possible explanations for variations, such as those observed at the gene expression levels.

Orthology Guided Assembly in highly heterozygous crops: creating a reference transcriptome to uncover genetic diversity in Lolium perenne.

Despite current advances in next-generation sequencing data analysis procedures, de novo assembly of a reference sequence required for SNP discovery and expression analysis is still a major challenge in genetically uncharacterized, highly heterozygous species. High levels of polymorphism inherent to outbreeding crop species hamper De Bruijn Graph-based de novo assembly algorithms, causing transcript fragmentation and the redundant assembly of allelic contigs. If multiple genotypes are sequenced to study genetic diversity, primary de novo assembly is best performed per genotype to limit the level of polymorphism and avoid transcript fragmentation. Here, we propose an Orthology Guided Assembly procedure that first uses sequence similarity (tBLASTn) to proteins of a model species to select allelic and fragmented contigs from all genotypes and then performs CAP3 clustering on a gene-by-gene basis. Thus, we simultaneously annotate putative orthologues for each protein of the model species, resolve allelic redundancy and fragmentation and create a de novo transcript sequence representing the consensus of all alleles present in the sequenced genotypes. We demonstrate the procedure using RNA-seq data from 14 genotypes of Lolium perenne to generate a reference transcriptome for gene discovery and translational research, to reveal the transcriptome-wide distribution and density of SNPs in an outbreeding crop and to illustrate the effect of polymorphisms on the assembly procedure. The results presented here illustrate that constructing a non-redundant reference sequence is essential for comparative genomics, orthology-based annotation and candidate gene selection but also for read mapping and subsequent polymorphism discovery and/or read count-based gene expression analysis.

Genome-wide association scan and phased haplotype construction for quantitative trait loci affecting boar taint in three pig breeds.

BACKGROUND: Boar taint is the undesirable smell and taste of pork meat derived from some entire male pigs. The main causes of boar taint are the two compounds androstenone and skatole (3-methyl-indole). The steroid androstenone is a sex pheromone produced in the testis of the boars. Skatole is produced from tryptophan by bacteria in the intestine of the pigs. In many countries pigs are castrated as piglets to avoid boar taint, however, this is undesirable for animal welfare reasons. Genetic variations affecting the level of boar taint have previously been demonstrated in many breeds. In the study presented in this paper, markers and haplotypes, which can be applied to DNA-based selection schemes in order to reduce or eliminate the boar taint problem, are identified. RESULTS: Approximately 30,000 SNPs segregating in 923 boars from three Danish breeds; Duroc, Landrace, and Yorkshire, were used to conduct genome wide association studies of boar taint compounds. At 46 suggestive quantitative trait loci (QTL), 25 haplotypes and three single markers with effects were identified. Furthermore, 40% of the haplotypes mapped to previously identified regions. Haplotypes were also analysed for effects of slaughter weight and meat content. The most promising haplotype was identified on Sus scrofa chromosome 1. The gain in fixed effect of having this haplotype on level of androstenone in Landrace was identified to be high (1.279 µg/g). In addition, this haplotype explained 16.8% of the phenotypic variation within the trait. The haplotype was identified around the gene CYB5A which is known to have an indirect impact on the amount of androstenone. In addition to CYB5A, the genes SRD5A2, LOC100518755, and CYP21A2 are candidate genes for other haplotypes affecting androstenone, whereas, candidate genes for the indolic compounds were identified to be SULT1A1 and CYP2E1. CONCLUSIONS: Despite the small sample size, a total of 25 haplotypes and three single markers were identified including genomic regions not previously reported. The haplotypes that were analysed showed large effects on trait level. However, little overlap of QTL between breeds was observed.

A transcriptome map of perennial ryegrass (Lolium perenne L.).

BACKGROUND: Single nucleotide polymorphisms (SNPs) are increasingly becoming the DNA marker system of choice due to their prevalence in the genome and their ability to be used in highly multiplexed genotyping assays. Although needed in high numbers for genome-wide marker profiles and genomics-assisted breeding, a surprisingly low number of validated SNPs are currently available for perennial ryegrass. RESULTS: A perennial ryegrass unigene set representing 9,399 genes was used as a reference for the assembly of 802,156 high quality reads generated by 454 transcriptome sequencing and for in silico SNP discovery. Out of more than 15,433 SNPs in 1,778 unigenes fulfilling highly stringent assembly and detection parameters, a total of 768 SNP markers were selected for GoldenGate genotyping in 184 individuals of the perennial ryegrass mapping population VrnA, a population being previously evaluated for important agronomic traits. A total of 592 (77%) of the SNPs tested were successfully called with a cluster separation above 0.9. Of these, 509 (86%) genic SNP markers segregated in the VrnA mapping population, out of which 495 were assigned to map positions. The genetic linkage map presented here comprises a total of 838 DNA markers (767 gene-derived markers) and spans 750 centi Mogan (cM) with an average marker interval distance of less than 0.9 cM. Moreover, it locates 732 expressed genes involved in a broad range of molecular functions of different biological processes in the perennial ryegrass genome. CONCLUSIONS: Here, we present an efficient approach of using next generation sequencing (NGS) data for SNP discovery, and the successful design of a 768-plex Illumina GoldenGate genotyping assay in a complex genome. The ryegrass SNPs along with the corresponding transcribed sequences represent a milestone in the establishment of genetic and genomics resources available for this species and constitute a further step towards molecular breeding strategies. Moreover, the high density genetic linkage map predominantly based on gene-associated DNA markers provides an important tool for the assignment of candidate genes to quantitative trait loci (QTL), functional genomics and the integration of genetic and physical maps in perennial ryegrass, one of the most important temperate grassland species.

The genetic regulation of the terminating phase of liver regeneration.

BACKGROUND: After partial hepatectomy (PHx), the liver regeneration process terminates when the normal liver-mass/body-weight ratio of 2.5% has been re-established. To investigate the genetic regulation of the terminating phase of liver regeneration, we performed a 60% PHx in a porcine model. Liver biopsies were taken at the time of resection, after three weeks and upon termination the sixth week. Gene expression profiles were obtained using porcine oligonucleotide microarrays. Our study reveals the interactions between genes regulating the cell cycle, apoptosis and angiogenesis, and the role of Transforming Growth Factor-ß (TGF-ß) signalling towards the end of liver regeneration. RESULTS: Microarray analysis revealed a dominance of genes regulating apoptosis towards the end of regeneration. Caspase Recruitment Domain-Containing Protein 11 (CARD11) was up-regulated six weeks after PHx, suggesting the involvement of the caspase system at this time. Zinc Finger Protein (ZNF490) gene, with a potential negative effect on cell cycle progression, was only up-regulated at three and six weeks after PHx indicating a central role at this time. TGF-ß regulation was not found to be significantly affected in the terminating phase of liver regeneration. Vasohibin 2 (VASH2) was down-regulated towards the end of regeneration, and may indicate a role in preventing a continued vascularization process. CONCLUSIONS: CARD11, ZNF490 and VASH2 are differentially expressed in the termination phase of liver regeneration. The lack of TGF-ß up-regulation suggests that signalling by TGF-ß is not required for termination of liver regeneration.

Gene expression profiling of porcine skeletal muscle in the early recovery phase following acute physical activity.

Acute physical activity elicits changes in gene expression in skeletal muscles to promote metabolic changes and to repair exercise-induced muscle injuries. In the present time-course study, pigs were submitted to an acute bout of treadmill running until near exhaustion to determine the impact of unaccustomed exercise on global transcriptional profiles in porcine skeletal muscles. Using a combined microarray and candidate gene approach, we identified a suite of genes that are differentially expressed in muscles during postexercise recovery. Several members of the heat shock protein family and proteins associated with proteolytic events, such as the muscle-specific E3 ubiquitin ligase atrogin-1, were significantly upregulated, suggesting that protein breakdown, prevention of protein aggregation and stabilization of unfolded proteins are important processes for restoration of cellular homeostasis. We also detected an upregulation of genes that are associated with muscle cell proliferation and differentiation, including MUSTN1, ASB5 and CSRP3, possibly reflecting activation, differentiation and fusion of satellite cells to facilitate repair of muscle damage. In addition, exercise increased expression of the orphan nuclear hormone receptor NR4A3, which regulates metabolic functions associated with lipid, carbohydrate and energy homeostasis. Finally, we observed an unanticipated induction of the long non-coding RNA transcript NEAT1, which has been implicated in RNA processing and nuclear retention of adenosine-to-inosine edited mRNAs in the ribonucleoprotein bodies called paraspeckles. These findings expand the complexity of pathways affected by acute contractile activity of skeletal muscle, contributing to a better understanding of the molecular processes that occur in muscle tissue in the recovery phase.

Spatio-temporal regulation of ADAR editing during development in porcine neural tissues.

Editing by ADAR enzymes is essential for mammalian life. Still, knowledge of the spatio-temporal editing patterns in mammals is limited. By use of 454 amplicon sequencing we examined the editing status of 12 regionally extracted mRNAs from porcine developing brain encompassing a total of 64 putative ADAR editing sites. In total 24 brain tissues, dissected from up to five regions from embryonic gestation day 23, 42, 60, 80, 100 and 115, were examined for editing.   Generally, editing increased during embryonic development concomitantly with an increase in ADAR2 mRNA level. Notably, the Gria2 (GluR-B) Q/R site, reported to be ~100% edited in previous studies, is only 54% edited at embryonic day 23. Transcripts with multiple editing sites in close proximity to each other exhibit coupled editing and an extraordinary incidence of long-range coupling of editing events more than 32 kb apart is observed for the kainate glutamate receptor 2 transcript, Grik2. Our study reveals complex spatio-temporal ADAR editing patterns of coordinated editing events that may play important roles in the development of the mammalian brain.

Characterization of the porcine FBX07 gene: the first step towards generation of a pig model for Parkinsonian pyramidal syndrome.

Parkinsonian pyramidal syndrome, also named pallido-pyramidal syndrome (PKPS), is the combination of early-onset progressive Parkinsonism with pyramidal tract signs. FBXO7, an F-box protein, is a component of modular E3 ubiquitin protein ligases called SCFs (SKP1, cullin, F-box proteins), which functions in phosphorylation-dependent ubiquitination. FBXO7 mutations cause autosomal recessive, early-onset PKPS. Here we report the molecular cloning and characterization of two isoforms of FBXO7 cDNA from pigs. The encoded FBXO7 protein displays a very high homology to human FBXO7 with an amino acid identity of 90%. Phylogenetic analysis demonstrated that porcine FBXO7 is closely related to other mammalian FBXO7 proteins. Furthermore, the genomic structure of the porcine FBXO7 gene was determined. The intron-exon structure is similar to that of the human FBXO7 gene. The promoter sequence for the porcine FBXO7 gene was also identified. A recognition site for miR-301a was found in the 3'UTR region of porcine FBXO7. Investigating the genetic variation in the porcine FBXO7 gene revealed a missense A/G SNP in exon 5. The A/G SNP results in a substitution of an asparagine to a serine residue (N269S). Using a radiation hybrid map the FBXO7 gene was mapped to pig chromosome 5. Real-time quantitative RT-PCR analysis revealed that FBXO7 mRNA is differentially expressed in many tissues and organs, and that FBXO7 transcript can be detected early in embryo development.