Molecular cloning,chromosomal location and expression pattern of porcine CIDEa and CIDEc

Cell death-inducing DFF45-like effectors a and c (CIDEa and CIDEc) are two members of the novel CIDE family of apoptosis-inducing factors. Except as pro-apoptotic proteins, it has been reported that CIDEa and CIDEc could be involved in lipid or fat metabolism. Here we first reported the cDNA cloning, chromosome mapping and expression analysis of CIDEa and CIDEc in pigs. Sequence analysis showed that porcine CIDEa contains an open reading frame of 660 bp, which encodes 219 amino acids, and CIDEc contains a coding region of 717 bp that encodes 238 amino acids. The deduced amino acid sequence of porcine CIDEa and CIDEc shows high similarities to their corresponding human and mouse homologues. Radiation hybrid mapping demonstrated that porcine CIDEa and CIDEc are located at chromosome 6q21-26 and 13q31 respectively, syntenic with the loci of their corresponding homologues on human chromosomes. Tissue distribution analysis indicated that porcine CIDEa and CIDEc mRNAs are co-expressed in various tissues. Both of them were highly expressed in white adipose tissue, and CIDEc mRNA was also expressed at relatively high level in porcine small intestine, lymph and brain. Furthermore, CIDEa and CIDEc mRNA level in white adipose tissues and liver were significantly higher in obese pigs than in their lean counterparts. Our studies provide basic molecular information useful for the further investigation on the function of the two genes, which will be helpful in better understanding of the roles of CIDEs in lipid metabolism.     

Cloning, Expression Pattern, Chromosomal Localization, and Evolution Analysis of Porcine Gnaq, Gna11, and Gna14

The Gq class is one subfamily of the G protein alpha subunits multigene family. It comprises four genes: Gnaq, Gna11, Gna14, and Gna15. In mice and humans, the alpha subunit is an essential component of G protein interaction with receptors and effectors. We report here the cloning and characterization of porcine Gnaq, Gna11, and Gna14. We have cloned the full-length coding sequences of porcine Gnaq, Gna11, and Gna14 (1,080, 1,080, and 1,068 bp, respectively) and then mapped them chromosomally to regions 1q21-27, 2q21-24, and 1q21-27 by radiation hybrid mapping. Tissue distribution analysis indicated that Gnaq and Gna11 were coexpressed in liver, heart, muscle, spleen, adipose tissue, brain, and uterus, but Gna14 mRNA was detected only in kidney and lung. The phylogenetic trees reveal that porcine Gnaq, Gna11, and Gna14 are evolutionarily closer to their human homologs. This is the first report of molecular cloning and characterization of porcine Gnaq, Gna11, and Gna14, which will be helpful for further understanding of the physiological role of Gq class genes in pigs.     

A novel porcine gene,alpha-1-antichymotrypsin 2 (SERPINA3-2): sequence,genomic organization,polymorphism and mapping

A novel porcine gene, alpha-1-antichymotrypsin 2 (SERPINA3-2), a member of the serpin superfamily, was isolated from a porcine genomic library and sequenced. The genomic organization of the approximately 9.0 kb gene was determined on the basis of the porcine liver cDNA of SERPINA3-1 and SERPINA3-2, and comprises five exons and four introns. The coding sequence of SERPINA3-2 shares 86% identity with the paralogue, SERPINA3-1. Porcine SERPINA3-2 was found to be an orthologue of human SERPINA3 (71% identity of the coding sequences) and both genes have a similar genomic organization. Polymorphisms were found in intron 4 of the porcine gene using polymerase chain reaction-restriction fragment length polymorphism. The gene was mapped by linkage analysis and radiation hybrid mapping to the distal end of chromosome 7q, to the gene cluster of the protease inhibitors including PI1 (SERPINA1), PI2, PI3, PI4 (apparently paralogues of SERPINA3), and PO1A and PO1B. SERPINA3-2 is the first porcine serpin gene whose genomic organization has been determined.     

Cloning,chromosome mapping and expression pattern of porcine PLIN and M6PRBP1 genes

The PAT proteins, named after the three PLIN/ADRP/TIP47 (PAT) proteins, PLIN for perilipin, ADRP for adipose differentiation-related protein and TIP47 for tail-interacting protein of 47 kDa, now officially named M6PRBP1 for mannose-6-phosphate receptor binding protein 1, is a set of intracellular lipid droplet binding proteins. They are localized in the outer membrane monolayer enveloping lipid droplets and are involved in the metabolism of intracellular lipid. This work describes the cloning and sequencing of porcine PLIN and M6PRBP1 cDNAs, the chromosome mapping of these two genes, as well as the expression pattern of porcine PAT genes. Sequence analysis shows that the porcine PLIN cDNA contains an open reading frame of 1551 bp encoding 516 amino acids and that the porcine M6PRBP1 cDNA contains a coding region of 1320 bp encoding 439 amino acids. Comparison of PLIN and M6PRBP1 amino-acid sequences among various species reveals that porcine and bovine proteins are the most conserved. Porcine PLIN and M6PRBP1 genes have been mapped to pig chromosomes 7 and 2, respectively, by radiation hybrid analysis using the IMpRH panel. Expression analyses in pig showed a high expression of PLIN mRNA in adipose tissue, M6PRBP1 mRNA in small intestine, kidney and spleen and ADRP mRNA in adipose tissue, lung and spleen.     

Molecular cloning of porcine alveolar macrophage-derived neutrophil chemotactic factors I and II; identification of porcine IL-8 and another intercrine-alpha protein.

Alveolar macrophages (AM) mediate lung inflammation by producing lipid and peptide molecules that attract neutrophils (PMN) to the lung. Recently we described two porcine proteins called alveolar macrophage-derived chemotactic factors, AMCF-I and -II, that are potent, efficacious, and specific PMN chemoattractants both in vitro and in vivo. We report here the cloning of the full-length cDNAs which code for each protein. Porcine AM were stimulated for 4 h in vitro with Escherichia coli endotoxin (LPS), and a cDNA library was created from poly(A)(+)-selected mRNA. Specific oligonucleotide probes for AMCF-I and AMCF-II were amplified from the porcine AM cDNA library by the polymerase chain reaction using degenerate oligonucleotide primer pairs derived from the N-terminal amino acid sequences of the proteins. These probes were used to isolate 2 full-length cDNAs of 1466 (AMCF-I) and 1515 (AMCF-II) base pairs. Both cDNAs code for proteins with four cysteine residues containing the C-X-C sequence characteristic of the intercrine-alpha family of neutrophil chemoattractants. AMCF-I shares 74% identity with human IL-8 and 84% identity with rabbit IL-8, and likely represents the porcine homologue of IL-8. By contrast, AMCF-II has no obvious human homologue. AMCF-II shares 53% identity with human neutrophil activating peptide 2. Its shared identity with the GRO-related proteins is as high as 61% (rat CINC/GRO), and its shared identity with the 78 amino acid epithelial cell-derived neutrophil activator (ENA-78) is 67%. AMCF-II may represent a new member of the intercrine-alpha family of neutrophil chemoattractants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization and chromosome assignment of the porcine AHCY gene for S-adenosylhomocysteine hydrolase

The gene for S-adenosylhomocysteine hydrolase (AHCY) is involved in the regulation of cellular methylation reactions. Here we report the cloning, sequencing, and chromosomal assignment of the porcine AHCY gene. The gene consists of 10 exons spanning approximately 19 kb of genomic DNA. It encodes a protein of 432 amino acids that shows about 96% identity to the orthologous S-adenosylhomocysteine hydrolases from human, rat, and mouse. The porcine AHCY gene is located very close to the agouti signaling protein gene (ASIP) on SSC17q21. The chromosomal localization was subsequently confirmed by RH mapping and the genetic mapping of an intragenic microsatellite that maps to 57 cM on the linkage map of SSC17.     

Structure and Polymorphism Analysis of Transforming Growth Factor Beta Receptor 1 (TGFBR1) in Pigs

Many quantitative trait loci (QTL) for growth and reproductive traits have been detected on the porcine chromosome region 1qter (SSC1qter), making it one of the most important genomic regions for pig breeding. SSC1q corresponds to human chromosome 9, on which lies transforming growth factor beta receptor 1 (TGFBR1). We cloned the porcine TGFBR1 cDNA and gene (as a candidate for QTL) and analyzed the gene structure and polymorphism. Porcine TGFBR1 consists of 9 exons and 8 introns. Intron 2 is alternatively spliced at the acceptor site, resulting in two kinds of mRNA, with putative open reading frames of 1500 and 1512 bp in length. The shorter one encodes 499 amino acid residues. The amino acid sequence has 96.2 and 97.2% sequence similarity to those of human and bovine TGFBR1, respectively. The sequence similarity between porcine and murine TGFBR1 is 95.6%. We detected three single-nucleotide substitutions in exons 1, 2, and 7. Those in exons 1 and 7 are nonsynonymous substitutions resulting in Pro8Ser and Ile413Val substitutions, respectively.     

一个新的猪肌肉组织EST的分离、定位与表达

利用mRNA差异显示技术,从猪骨骼肌组织中分离到一个新的表达序我标签（expressed sequence tag ,EST)ESThp9-1(其GenBan登录号：BI596262）,其序列长196bp,经BLAST程序与GenBank中存在的序列比对后,发现与猪的所有序列无同源性,但与大鼠的U3A核内小RNA基因及小鼠的U3B．4核内小RNA基因同源性分别为87％（93个碱基）和85％（96个碱基）。半定量RT－PCR表明,此EST在猪的多数组织中均有表达。通过体细胞杂种板（somatic cell hybrid panel,SCHP)及辐射杂种板（radiation hybrid panel,RH)分析,EST hp9-1被定位于猪的12号染色体长臂,与微卫星标记S0090连锁。根据同源性比对和物理定痊结果,推测EST hp9-1为猪的U3基因家族中一员。     

Human BTR1, a new bicarbonate transporter superfamily member and human AE4 from kidney

We report the cloning, characterization, and chromosomal localization of two novel human members of the bicarbonate transporter superfamily, BTR1 (Bicarbonate Transporter Related protein-1) and AE4 (Anion Exchange protein 4). BTR1 is a novel mammalian protein. The BTR1 gene maps to chromosome 20p12 and encodes a 100 kDa protein predominantly expressed in the kidney, salivary glands, testis, thyroid glands, and trachea. The AE4 gene maps to chromosome 5q23-31 and encodes a 104 kDa protein expressed mainly in the kidney. Human AE4 shares 84% identity with the recently reported rabbit AE4, a sodium independent, Cl(-)/HCO(-)(3) exchanger located on the apical membrane of beta-intercalated kidney cells.     

ANGPTL4 regulate glutamine metabolism and fatty acid oxidation in nonsmall cell lung cancer cells

Angiopoietin‐like protein (ANGPTL) 4 is a key factor in the regulation of lipid and glucose metabolism in metabolic diseases. ANGPTL4 is highly expressed in various cancers, but the regulation of energy metabolism in tumours remains to be determined. This study explored the role of ANGPTL4 in aerobic glycolysis, glutamine consumption and fatty acid oxidation in nonsmall cell lung cancer (NSCLC) cells. Two NSCLC cell lines (A549 and H1299) were used to investigate the role of ANGPTL4 in energy metabolism by tracer techniques and with Seahorse XF technology in ANGPTLs4 knockdown cells. RNA microarrays and specific inhibitors were used to identify targets in ANGPTLs4‐overexpressing cells. The results showed that knockdown of ANGPTLs4 could inhibit energy metabolism and proliferation in NSCLC. ANGPTLs4 had no significant effect on glycolysis but affected glutamine consumption and fatty acid oxidation. Knockdown of ANGPTLs4 also significantly inhibited tumour metastasis and energy metabolism in mice and had a weak effect on glycolysis. RNA microarray analysis showed that ANGPTLs4 significantly affected glutaminase (GLS) and carnitine palmitoyl transferase 1 (CPT1). ANGPTLs4‐overexpressing cells were exposed to a glutamine deprivation environment, and cell proliferation and energy metabolism were significantly decreased but still differed from normal NSCLC cells. Treatment of ANGPTLs4‐overexpressing cells with GLS and CPT1 inhibitors simultaneously prevented the regulatory effects on cell proliferation and energy metabolism. ANGPTLs4 could promote glutamine consumption and fatty acid oxidation but not glycolysis or accelerate energy metabolism in NSCLC.