Molecular Characterization and SNP Development for the Porcine Il6 and Il10 Genes

Different cytokines are secreted in response to specific microbial molecules referred to as pathogen associated molecular patterns (PAMPs). Interleukin 6 (IL6) and interleukin 10 (IL10), both secreted by macrophages and lymphocytes, play a central role in the immunological response. In this work we obtained the genomic structure and complete DNA sequence of the porcine IL6 and IL10 genes and identified polymorphisms in the genomic sequences of these genes on a panel of ten different pig breeds. Comparative intra- and interbreed sequence analysis revealed a total of eight polymorphisms in the porcine IL6 gene and 21 in the porcine IL10 gene, which include single nucleotide polymorphisms (SNPs) and insertion deletion polymorphisms (indels). Additionally, the chromosomal localization of the IL10 gene was determined by FISH and RH mapping.     

Molecular characterization,tissue expression profile and SNP analysis of the porcine NR1H4 gene

Nuclear Receptor subfamily 1, group H, member 4 (NR1H4) is a receptor for bile acids and has an important role in regulating energy metabolism in liver, muscle and adipose tissues in humans and animals. In this study, we cloned the full coding region of NR1H4 gene from porcine Longissimus dorsi by Rapid amplification of cDNA end (RACE). Results indicated that the open reading frame of NR1H4 covered 1461 bp encoding 486 amino acid residues and the deduced amino acid sequence was 91–94 % identical to that of Homo sapiens, Bos taurus, Macaca mulatta, Gorilla gorilla, and Ovis aries. Bioinformatic analysis indicated that NR1H4 contained 31 phosphorylation sites with 14 serine, 6 threonine and 11 tyrosine. One single nucleotide polymorphism (SNP) was detected by PCR–RFLP in 3′ untranslated region of exon 9 (NR1H4) and the allele frequency analysis showed that A allele frequency was low among 396 pigs from five breeds. The NR1H4 mRNA expression pattern showed that NR1H4 gene was expressed highly in live and Longissimus dorsi. This work provided an important experimental basis for further research on mechanism of lipid metabolism and fat deposition in pigs.     

Isolation and characterization of marsupial IL5 genes

The genomic nucleotide sequence and chromosomal position of the interleukin 5 (IL5) gene has been described for the model marsupial Macropus eugenii (tammar wallaby). A 272 base pair genomic IL5 polymerase chain reaction (PCR) product spanning exon 3, intron 3, and exon 4 was generated using stripe-faced dunnart (Sminthopsis macroura) DNA. This PCR product was used to isolate a genomic lambda clone containing the complete IL5 gene from a tammar wallaby EMBL3 lambda library. Sequencing revealed that the tammar wallaby IL5 gene consists of four exons separated by three introns. Comparison of the marsupial coding sequence with coding sequences from eutherian species revealed 61 to 69% identity at the nucleotide level and 48 to 63% identity at the amino acid (aa) level. A polymorphic complex compound microsatellite was identified within intron 2 of the tammar wallaby IL5 gene. This microsatellite was also found in other marsupials including the swamp wallaby, tree kangaroo, stripe-faced dunnart, South American opossum, brushtail possum, and koala. Fluorescence in situ hybridization using DNA from the IL5 clone on tammar wallaby chromosomes indicated that the IL5 gene is located on Chromosome 1.     

Association of IL-10 receptor 2 (IL10RB) SNP with systemic sclerosis

Interleukin-10 (IL-10) signaling has been suggested to play a role in systemic sclerosis (SSc). IL10RB codes for IL-10 receptor 2 (IL-10R2), a component shared in receptor complexes for IL-10, IL-22, IL-26 and interferon (IFN)-λ. In this study, we examined association of IL10RB polymorphism with susceptibility to SSc. Genotype A/A at rs2834167 (47K/K) was significantly increased in diffuse cutaneous SSc (dcSSc) (41.3% in dcSSc, 20.9% in controls, P = 0.0018, odds ratio = 2.67). A SNP in the 5′ flanking region of IL10RB, rs999788, also showed association with dcSSc; however, this association was shown to be secondarily caused by linkage disequilibrium with rs2834167. Significant association was not observed in limited cutaneous SSc (lcSSc). Presence of anti-topoisomerase I antibody was also associated with rs2834167A/A genotype (P = 0.0019). Serum IL-10 level was significantly associated with the number of rs2834167A allele (P = 0.007). These findings suggested that signaling through IL-10R2 may play a causative role in dcSSc.     

Molecular characterization, expression and mapping of porcine LMP2 and MECL-1 genes.

Low molecular mass polypeptides 2 (LMP2) and Multicatalytic endopeptidase complex subunit (MECL-1) are two of three catalytic beta-type subunits of the 20s proteasome and upon interferon gamma-induction. LMP2 is critical for the production of major histocompatibility complex (MHC) class I ligand and T-lymphocytes. The LMP2 gene is located in the MHC region, but MECL-1 is located outside the MHC region. They are involved in the antigen presentation and are important candidate genes for an initial exploration of relationships between the antigen processing genes and disease resistance. In this report, the porcine LMP2 and MECL-1 cDNA were cloned and A 5099 bp LMP2 genomic DNA structure was identified, then two single nucleotide polymorphisms were detected in the exon2 and exon5 of LMP2 gene in 367 individuals. The LMP2 and MECL-1 genes putative protein included 219,274 amino acids, respectively. Alignment and phylogenetic of predicted porcine LMP2 and MECL-1 amino acid sequence with their homologies were analyzed. Tissues expression of LMP2 and MECL-1 mRNA were observed by real time quantitative PCR (Q-PCR) method, the results revealed MECL-1 expressed widely in all tissues, but LMP2 was not detected in muscle. The porcine MECL-1 gene was mapped to chromosome 6, closely linked to microsatellite SW1108 (LOD = 4.09, 84cR) by radiation hybrid panel.     

Single nucleotide polymorphism (SNP) discovery in porcine expressed genes

High-throughput genotyping of swine populations is a potentially efficient method for establishing animal lineage and identification of loci important to animal health and efficient pork production. Markers were developed based upon single nucleotide polymorphisms (SNPs), which are abundant and amenable to automated genotyping platforms. The focus of this research was SNP discovery in expressed porcine genes providing markers to develop the porcine/human comparative map. Locus specific amplification (LSA) and comparative sequencing were used to generate PCR products and allelic information from parents of a swine reference family. Discovery of 1650 SNPs in 403 amplicons and strategies for optimizing LSA-based SNP discovery using alternative methods of PCR primer design, data analysis, and germplasm selection that are applicable to other populations and species are described. These data were the first large-scale assessment of frequency and distribution of porcine SNPs.     

Association of polymorphisms of cytokine genes (IL1B, IL1RN, TNFA, LTA, IL6, IL8, and IL10) with chronic obstructive pulmonary disease

To assess the role that polymorphisms of cytokine genes play in genetic predisposition to chronic obstructive pulmonary disease (COPD), the allele and genotype distributions of IL1B, IL1RN, TNFA, LTA, IL6, IL8, and IL10 were studied in COPD patients (N = 319) and healthy individuals (N = 403), residents of Ufa, Bashkortostan. Genotype IL1RN*2/IL1RN*2 of IL1RN was identified as a risk factor for COPD, its frequency being 9.80% in the COPD patients and 4.67% in the healthy subjects (x ² = 5.45, df = 1, P = 0.02, OR = 2.21). Genotype GG of the LTA polymorphism A252G was significantly more common in the COPD patients than in the controls (7.84% vs. 3.72%; x ² = 5.00, df = 1, P = 0.026). In patients with COPD stage IV, the frequency of this genotype was twice as high as in those with COPD stages II and III (11.18% vs. 4.79%; x ² = 3.08, df = 1, P = 0.08). Genotype GG of the TNFA polymorphism G(?308)A in combination with genotype AA of the LTA polymorphism A252G was significantly less frequent in the COPD patients than in the healthy subjects (38.55% vs. 46.93%; x ² = 8.82, df = 1, P = 0.0039). Genotype GG of the IL6 polymorphism G(?174)C was more frequent in the patients with COPD stage IV (43.75% vs. 31.54% in the patients with COPD stages II and III, x ² = 4.15, P = 0.042). No significant differences were found between the groups of COPD patients and healthy subjects concerning the genotype frequencies of the polymorphisms T(?511)C and T3953C of IL1B, G(?308)A of TNFA, G(?174)C of IL6, A(?251)C of IL8, and C(?627)A of IL10.     

Molecular and enzymatic characterization of the porcine endogenous retrovirus protease

The protease of the porcine endogenous retrovirus (PERV) subtypes A/B and C was recombinantly expressed in Escherichia coli as proteolytically active enzyme and characterized. The PERV Gag precursor was also recombinantly produced and used as the substrate in an in vitro enzyme assay in parallel with synthetic nonapeptide substrates designed according to cleavage site sequences identified in the PERV Gag precursor. The proteases of all PERV subtypes consist of 127 amino acid residues with an M(r) of 14,000 as revealed by determining the protease N and C termini. The PERV proteases have a high specificity for PERV substrates and do not cleave human immunodeficiency virus (HIV)-specific substrates, nor are they inhibited by specific HIV protease inhibitors. Among the known retroviral proteases, the PERV proteases resemble most closely the protease of the murine leukemia retrovirus.     

Molecular characterization of porcine MMP19 and MMP23B genes and its association with immune traits

MMP19 and MMP23B belong to the Matrix metalloproteases (MMPs) family, which are zinc-binding endopeptidases that are capable of degrading various components of the extracellular matrix. They are thought to play important roles in embryonic development, reproduction and tissue remodeling, as well as in cell proliferation, differentiation, migration, angiogenesis, apoptosis and host defense. However, they are poorly understood in pigs. Here, we obtained the full length coding region sequence and genomic sequence of the porcine MMP19 and MMP23B genes and analyzed their genomic structures. The deduced amino acid sequence shares similar precursor protein domains with human and mouse MMP19 and MMP23B protein, respectively. Using IMpRH panel, MMP19 was mapped to SSC5p12-q11 (closely linked to microsatellite DK) and MMP23B was mapped to SSC8q11-q12 (linked to microsatellite Sw2521). Quantitative real-time PCR showed that MMP19 was abundantly expressed in the liver, while MMP23B was strongly expressed in the ovarian and heart. Furthermore, both genes were all expressed increasingly in prenatal skeletal muscle during development. Three SNPs were detected by sequencing and PCR-RFLP methods, and association analysis indicated that C203T at exon 5 of MMP19 has a significant association with the blood parameters WBC (G/L) and IgG2 (mg/mL) (P<0.05), SNP C131T at exon 3 of MMP23B is significantly associated with the blood parameters HGB (g/L) and MCH (P<0.05), and A150G in exon 4 has no significant association with the economic traits in pigs.     

Molecular characteristics of the porcine DLK1 and MEG3 genes

Imprinted genes play important roles in embryo survival and postnatal growth regulation. The DLK1 and MEG3 (previously GTL2) genes are linked and reciprocally imprinted in several mammals, but their imprinting status is still unknown in pigs. In this study, we report polymorphisms, imprinting status and QTL analyses of the porcine DLK1 and MEG3 genes. Muscle and adipose DNA and RNA samples from 30-day-old animals generated with reciprocal crosses between the Korean native pig (KNP) and Yorkshire breeds were used to analyse DLK1 and MEG3 variation and expression. The samples exhibited paternal expression of DLK1 and maternal expression of MEG3 in pigs. These results indicated that the imprinting status of the DLK1 and MEG3 genes is conserved across mammalian species. By linkage analyses, we assigned the DLK1 and MEG3 genes to the telomeric region of SSC7. By QTL analyses, we confirmed a significant polar overdominance (POD) effect in DLK1 , which was previously detected for several growth traits in pigs. However, no significant POD effect was found with the MEG3 locus.     

Molecular identification and characterization of the Arabidopsis AtADF1, AtADF5 and AtADF6 genes

Actin depolymerizing factor (ADF) is a key regulator of the organization of the actin cytoskeleton during various cellular activities. We found that ADF genes in Arabidopsis form a large family consisting of at least nine members, four of which were cloned and sequenced in this study. Comparison of genomic and cDNA sequences showed that the AtADF1, AtADF5, and AtADF6 genes all contain two introns at conserved positions. Analysis of transgenic Arabidopsis plants carrying promoter-GUS fusion constructs revealed that AtADF1 and AtADF6 are expressed in the vascular tissues of all organs, whereas expression of AtADF5 is restricted to the root tip meristem. GFP-AtADF1, GFP-AtADF5, and GFP-AtADF6 fusion proteins were found to bind to actin filaments in vivo, and to reorganize the actin cytoskeleton when transiently expressed in plant cells.     

Molecular characterization of the porcine MHC class I region

A porcine cosmid library was screened with a human MHC class I cDNA. Four positive clones were isolated and mapped with different restriction endonucleases. Altogether nine SLA class I genes were identified and their positions located within restriction maps. Sizes of class I homologous DNA sequences varied between 3600 and 5800bp. The distances between these regions ranged from 11900 to 22200bp.     

Molecular characterization and expression patterns of the actinin-associated LIM protein (ALP) subfamily genes in porcine skeletal muscle

The actinin-associated LIM protein (ALP) subfamily has important functions in cell signal transduction, cell proliferation, and integration of cytoskeletal architecture. To detect their functions in pig skeletal muscle, we cloned and characterized the pig ALP subfamily genes, drew their genomic structure maps, and detected their tissue expression patterns. We identified a new spliced variant of PDLIM3 in pig skeletal muscle and named it as PDLIM3-4, which was only expressed in the heart and skeletal muscle. Our results showed that PDLIM3-4 was expressed in adult pig skeletal muscle with the highest expression level, and both PDLIM3-4 isoform and PDLIM4 had different expression profiles during the prenatal and postnatal stages of skeletal muscle development among the three pig breeds. These studies provide useful information for further research on the functions of pig ALP subfamily genes in skeletal muscle development.     

Molecular characterization and association analysis of porcine CA3

Carbonic anhydrase 3 (CA3) is a member of the carbonic anhydrase family, which plays an important role in various cell processes. In this paper, molecular characterization revealed that CA3 genomic DNA consists of seven exons and six introns, spans about 10.5 kb and maps to porcine chromosome 4q11-->q14. Results of expression profiles showed that the expression levels of CA3 increased in skeletal muscles from prenatal 33- to 65-day-old Chinese Tongcheng pigs. These levels subsequently decreased to a steady state in prenatal 90-day-old, postnatal 2-day-old, postnatal 28-day-old, and pregnant 65-day-old pigs. The expression patterns of Chinese Tongcheng pig embryos were different from that of Landrace pig embryos. CA3 was expressed at higher levels in skeletal muscle and liver than in kidney, lung, stomach, intestine, and brain, but was not detected in heart and spleen. Statistical analysis showed the CA3 gene polymorphism was different between Chinese indigenous and introduced commercial western pig breeds, and was associated with intramuscular fat content and percentage of ham of pigs.     

Molecular characterization of the neuronatin gene in the porcine placenta

Imprinted genes play important roles in placental and embryonic development. Neuronatin (NNAT), first identified as an imprinted gene in human and mouse brains, played important roles in neuronal differentiation in the brain and in glucose-mediated insulin secretion in pancreatic β cells. In the pig, NNAT was reported to be imprinted in eleven tissues. Our previous microarray hybridization study showed that NNAT was differentially expressed in Yorkshire and Meishan pig placentas, but the imprinting status and function of NNAT in the placenta have not been investigated. We demonstrated for the first time that NNAT was monoallelically expressed in the placenta. Immunochemistry analysis showed that NNAT was located in the uterine luminal and glandular epithelium in placentas. We also confirmed the differential expression of NNAT in Meishan and Yorkshire pig placentas by qPCR. Using IPA software and the published literature, we created a model network of the possible relationships between NNAT and glucose transporter genes. A dual luciferase reporter assay demonstrated that the crucial promoter region of NNAT contained a CANNTG sequence in the +210 to +215 positions, which corresponded to the E-box. Our findings demonstrated important roles of NNAT in placenta function.