Molecular characterization,chromosomal location,alternative splicing and polymorphism of porcine <Emphasis Type="Italic">GFAT1</Emphasis> gene

Glutamine: fructose-6-phosphate amidotransferase (GFAT) is the rate-limiting enzyme of the hexosamine synthesis pathway, which plays important roles in insulin resistance and glucose toxicity. GFAT1 is one of the two isoenzymes of GFAT. In the present study, we cloned cDNA sequence of the porcine GFAT1 gene and identified a GFAT1 splice variant (designed GFAT1-L) that contains a 54 bp insertion within the coding region. Nested RT–PCR revealed that GFAT1 was ubiquitously expressed in all tested tissues, but GFAT1-L was only expressed in skeletal muscle and heart, not in liver, spleen, lung, kidney, small intestine, stomach and fat tissue, suggested that GFAT1-L was selectively expressed in striate muscle in pig. Using both the somatic cell hybrid panel and radiation hybrid panel, the GFAT1 gene was mapped to porcine chromosome 3q21-q27, in which several significant QTLs for carcass traits were found. Among the SNPs we found in porcine GFAT1 gene, only the g. 101A>G polymorphism which located in intron 8 was polymorphic in two pig populations we investigated in the study. Association analyses revealed that the g. 101A>G polymorphism has a significant effect on lean meat percentage (P < 0.05), corrected backfat thickness (P < 0.05) and backfat at the rump (P < 0.05).     

Molecular cloning,expression patterns and subcellular localization of porcine <Emphasis Type="Italic">TMCO1</Emphasis> gene

The product of transmembrane and coiled-coil domains 1 (TMCO1) gene is a member of DUF841 superfamily of several eukaryotic proteins with unknown function. The partial DNA sequence of porcine TMCO1 was first cloned with a pig 567 bp ORF encoding 188 amino acids. By tissues expression analysis, the TMCO1 was found highly expressed in the liver, kidney and heart. The porcine TMCO1 protein was subsequently demonstrated to localize in the mitochondrion by confocal fluorescence microscopy. This data provides an important basis for conducing further studies on the functions and regulatory mechanisms underlying the role of TMCO1 gene.     

Cloning and ontogenetic expression of the uncoupling protein 1 gene <Emphasis Type="Italic">UCP1</Emphasis> in sheep

The uncoupling protein 1 (UCP1) is an indicator of brown adipocytes and is involved in the control of body temperature and regulation of energy balance. It abundantly expresses in newborns and has important functions in adults. However, little information was known on UCP1 gene expression in young and adolescent sheep. In this study, we cloned and identified the full-length DNA and cDNA sequences of the ovine UCP1 gene, which were 6659 bp and 1621 bp, respectively, and predicted the location of the gene on chromosome 17. Forty-eight animals with an equal number of males and females each for both Guangling Large Tail sheep (GLT) and Small Tail sheep Han (STH) sheep were used to study the ontogenetic expression of UCP1 mRNA in eight adipose tissues by quantitative real-time polymerase chain reaction (PCR). The results showed that the mRNA was expressed in all tissues studied and at all stages from 2 to 12 months of age. Nevertheless, the mRNA in perirenal fat was expressed significantly higher than that in other tissues and lower in superficial fat than in deep deposits. The highest expression was observed in animals at 2 months of age and then decreased gradually with age. Global expression in GLT was significantly higher than that in STH. Interactions between tissue and breed and age also influenced the mRNA expression significantly. In addition, the mRNA expression was associated with the single nucleotide polymorphism (SNP) haplotypes detected in the cDNA of the gene.     

Partial molecular characterization,expression pattern,polymorphism and association analysis of porcine <Emphasis Type="Italic">SKP2</Emphasis> gene

As a component of E3 ubiquitin protein ligases called SCFs, SKP2 protein belongs to a member of FBLs protein which is the biggest eukaryotic subfamily of F-BOX proteins with 12 members. In this study, we cloned and sequenced partial cDNA, intron 1 and intron 6 of porcine SKP2 gene. The partial cDNA is 1,402 bp long and has an open reading frame of 1,272 bp which encodes 424 putative amino acids. The deduced protein comprises a conserved F-BOX domain at position from the 90th to 140th amino acid. The phylogenetic tree indicated that porcine SKP2 has the closest genetic relationship with bovine SKP2 than other selected animal species. Quantitative RT-PCR analysis displayed that the tissue expression level of porcine SKP2 fluctuated remarkably in a large range, and it expressed in thymus with the highest level and in longissimus dorsi muscle with the lowest level. Two SNPs were identified, meanwhile, further polymorphism analysis with Cfr42I showed that AA genotype was in dominance absolutely among four kinds of unrelated Chinese indigenous miniature and one introduced Landrace pig breeds. In addition, association analysis with immune traits and blood parameters revealed that the SNP Cfr42I in intron 1 was significantly associated with red cell distribution width of neonate piglets at 0 day (P = 0.027).     

Identification and characterization of interferon regulatory factor-1 from orange-spotted grouper (<Emphasis Type="Italic">Epinephelus coioides</Emphasis>)

Interferon-regulatory factor 1 (IRF-1) is the first member of IRF family, which is involved in many biological processes such as immune response, antiviral defense, cell growth regulation, and apoptosis. In this study, an IRF-1 gene, EcIRF-1, was isolated and characterized from orange-spotted grouper (Epinephelus coioides). The full-length cDNA of EcIRF-1 is 1,730 bp, including an open reading frame of 906 bp, a 5′-terminal untranslated region (5′-UTR) of 153 bp, and a 3′-UTR of 671 bp. The EcIRF-1 gene consists of 10 exons and 9 introns, spanning over approximate 4.3 kb of genomic sequence. The 5′-UTR sequence contains an exon and an intron, and the 3′-UTR sequence is included in the last exon. Expression analysis by real-time PCR reveals that the EcIRF-1 gene is ubiquitously expressed in various healthy fish tissues, whereas its expression is upregulated in vivo in response to polyinosinic–polycytidylic acid or lipopolysaccharide stimulation. Subcellular localization analysis shows the EcIRF-1 is an intranuclearly localized and immobile protein in the cultured fish cells. Data presented in this paper provide an important base to further understand EcIRF-1 gene function and its regulation associated with interferon immune system in orange-spotted grouper.     

Characterization of the porcine differentially expressed <Emphasis Type="Italic">PDK4</Emphasis> gene and association with meat quality

To investigate the differential expression of genes in the skeletal muscle between Yorkshire and Chinese indigenous breed Meishan pigs, suppression subtractive hybridization was carried out and many genes were proved to be expressed significantly different in the two breeds. One gene highly expressed in Meishan but lowly expressed in Yorkshire specific library, shared strong homology with human pyruvate dehydrogenase kinase 4 (PDK4). Using semi-quantity and quantity PCR, We confirmed its differential expression between the two breeds. Temporal and spatial expression analysis indicated that porcine PDK4 gene is highly expressed in skeletal muscle and the highest in neonatal pigs. Complete cDNA cloning and sequence analysis revealed that porcine PDK4 gene contains an open reading frame of 1,221 bp. The deduced amino acid sequence showed conservation in evolution. A G/A mutation in intron 9 was identified and association analysis showed that it was significantly associated with intramuscular fat, muscle water content.     

Two common nonsynonymous paraoxonase 1 (<Emphasis Type="Italic">PON1</Emphasis>) gene polymorphisms and brain astrocytoma and meningioma

Background  

Human serum paraoxonase 1 (PON1) plays a major role in the metabolism of several organophosphorus compounds. The enzyme is encoded by the polymorphic gene PON1, located on chromosome 7q21.3. Aiming to identify genetic variations related to the risk of developing brain tumors, we investigated the putative association between common nonsynonymous PON1 polymorphisms and the risk of developing astrocytoma and meningioma.     

Molecular characterization,chromosomal localization and association analysis with back-fat thickness of porcine <Emphasis Type="Italic">LPIN2</Emphasis> and <Emphasis Type="Italic">LPIN3</Emphasis>

The products of mammalian LPIN2 and LPIN3 are phosphatidate phosphatase type 1 enzymes, which play an important role in the de novo biosynthesis of triacylglycerol, phosphatidylcholine and phosphatidylethanolamine. In this study, we obtained a 2,985-bp cDNA sequence of porcine LPIN2, which contains a 2,676-bp open reading frame flanked by an 11-bp 5′UTR and a 298-bp 3′UTR, and a 2,843-bp cDNA sequence of porcine LPIN3, which contains a 111-bp 5′UTR, a 2,580-bp open reading frame and a 152-bp 3′UTR. RT-PCR analysis showed that both LPIN2 and LPIN3 mRNA were ubiquitously expressed with a very high level in liver. By using the somatic cell hybrid panel (SCHP) and the radiation hybrid (IMpRH) panel, porcine LPIN2 and LPIN3 were assigned to 6q24-(1/2)q31 and 17(1/2)q21-q23, respectively. One T2193C single nucleotide polymorphism in LPIN2 was identified and was detected by Hin6I PCR-RFLP. Association analysis showed that different genotypes of LPIN2 were associated with back-fat thickness between the 6th and 7th ribs (P < 0.01).     

Identification and characteristics of a novel gene, <Emphasis Type="Italic">EJO1</Emphasis>, in the Chinese mitten crab (<Emphasis Type="Italic">Eriocheir japonica sinensis</Emphasis>) ovary

EJO1, a novel gene presumably involved in the ovary development of the Chinese mitten crab (Eriocheir japonica sinensis), was identified and characterized by suppression subtractive hybridization and cDNA macroarray analysis. EJO1 expression was 2.6-fold higher at stage III than at stage II during the ovary development of the mitten crab. EJO1 is 876 bp in length containing a 759 bp open reading frame which encodes a 252-amino-acid protein. Homology analysis showed that no sequence significantly matching EJO1 was found in SwissProt, so it was deduced as a novel gene (GenBank accession number: AY185917). The EJO1 protein is probably a secretion protein with a signal peptide of 17 amino acids. The pI/Mw deduced from the amino acid sequence was 6.18/28.18 kDa. Expression profile showed that EJO1 mRNA is highly expressed in the heart, intestine, and ovary of the crab, while there is little or no expression in muscle and hepatopancreas. The differential expression of EJO1 at the different developmental stages of the ovary was further confirmed by Northern blot analysis. In conclusion, EJO1 is a novel gene differentially expressed in the ovary of the Chinese mitten crab, which may play an important role in the ovary development.     

Molecular characterization and association analysis of porcine adipose triglyceride lipase (<Emphasis Type="Italic">PNPLA2</Emphasis>) gene

The adipose triglyceride lipase (PNPLA2, also known as ATGL) is a novel triacylglycerol (TG) lipase which specifically removes the first fatty acid from the triglyceride molecule generating free fatty acid and diglyceride (DG) in mammalian cells. Here we describe the molecular characterization of the porcine ATGL gene. The full-length cDNA sequence contains a 1,461 bp open reading frame encoding a protein of 486 amino acids with a calculated molecular mass of 53.2 kDa and an isoelectric point of 7.90. The porcine ATGL protein shares high identity with other mammalian ATGL. The ATGL gene contains 9 coding exons, spans approximately 6 kb. The porcine ATGL mRNA was expressed predominantly in backfat, mildly in muscle, small intestine and heart, and almost absent in liver, spleen, lung, stomach, kidney and ovary. Statistical analysis showed the ATGL gene polymorphism (G/A³⁹²) was different between Chinese indigenous and introduced commercial western pig breeds, and was highly associated with almost all the fat deposition and carcass traits, including subcutaneous fat thickness, viscera adipose tissue, lean percentage, loin eye traits and even rib numbers.     

Porcine insulin receptor substrate 4 (<Emphasis Type="Italic">IRS4</Emphasis>) gene: cloning,polymorphism and association study

Using PCR and inverse PCR techniques we obtained a 4,498 bp nucleotide sequence FN424076 encompassing the complete coding sequence of the porcine insulin receptor substrate 4 (IRS4) gene and its proximal promoter. The 1,269 amino acid porcine protein deduced from the nucleotide sequence shares 92% identity with the human IRS4 and possesses the same domains and the same number of tyrosine phosphorylation motifs as the human protein. We detected substitution FN424076:g.96C<G in the promoter region that segregates in Meishan and a synonymous substitution FN424076:g.1829T<C in the coding sequence with allele C present only in Meishan. Linkage mapping placed the IRS4 gene at position 82 cM on the current USDA–USMARC linkage map of porcine chromosome X. Association analyses were performed on 555 animals of 12th–15th generation of the Meishan × Large White cross and showed that both SNPs were highly significantly associated with backfat depth (P = 0.0005) and that the SNP FN424076:g1829T<C was also associated with loin depth (P = 0.017). The Meishan alleles increased back fat depth and decreased loin depth. IRS4 can be considered a positional candidate gene for at least some of the QTL located at the centromeric region of porcine chromosome X.     

Cloning,characterization and expression of the extracellular lipase gene from <Emphasis Type="Italic">Aureobasidium </Emphasis><Emphasis Type="Italic">pullulans</Emphasis> HN2-3 isolated from sea saltern

The extracellular lipase structural gene was isolated from cDNA of Aureobasidium pullulans HN2-3 by using SMART^TM RACE cDNA amplification kit. The gene had an open reading frame of 1245 bp long encoding a lipase. The coding region of the gene was interrupted by only one intron (55 bp). It encodes 414 amino acid residues of a protein with a putative signal peptide of 26 amino acids. The protein sequence deduced from the extracellular lipase structural gene contained the lipase consensus sequence (G-X-S-X-G) and three conserved putative N-glycosylation sites. According to the phylogenetic tree of the lipases, the lipase from A. pullulans was closely related to that from Aspergillus fumigatus (XP_750543) and Neosartorya fischeri (XP_001257768) and the identities were 50% and 52%, respectively. The mature peptide encoding cDNA was subcloned into pET-24a (+) expression vector. The recombinant plasmid was expressed in Escherichia coli BL21(DE3). The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass of about 47 kDa was found. Enzyme activity assay verified the recombinant protein as a lipase. A maximum activity of 0.96 U/mg was obtained from cellular extract of E. coli BL21(DE3) harboring pET-24a(+)LIP1. Optimal pH and temperature of the crude recombinant lipase were 8.0 and 35 °C, respectively and the crude recombinant lipase had the highest hydrolytic activity towards peanut oil.     

Cloning,characterization and expression of two new polyphenol oxidase cDNAs from <Emphasis Type="Italic">Agaricus bisporus</Emphasis>

Two new polyphenol oxidase (PPO) cDNAs (PPO3 and PPO4 cDNAs, accession numbers GQ354801 and GQ354802, respectively) were obtained by RACE-PCR from Agaricus bisporus. PPO3 cDNA was 1844 bp in length with an open reading frame of 1731 bp, while PPO4 cDNA was 2042 bp with an open reading frame of 1836 bp. PPO3 and PPO4 cDNAs, with 52% identity at the nucleic acid level, encoded a 576-amino acid protein of 66.3 kDa and 611-amino acid protein of 68.3 kDa, respectively. Mature forms of PPO3 and PPO4 were characterized after removing the specific C-terminal region and expressed in Escherichia coli BL21 (DE3) RIPL using pGEX-4T-1 vector. The expressed proteins were probed by the anti-A. bisporus PPO antibody but without PPO activity. This indicated that the recombinant mature PPO3 and mature PPO4 could not form an active center in prokaryotic expression system.