Characterization and Physical Mapping of the Porcine CDS1 and CDS2 Genes

CDP-diacylglycerol synthase (CDS) catalyzes the conversion of phosphatidic acid to CDP-diacylglycerol, an important precursor for the synthesis of phosphatidylinositol, phosphatidylglycerol, and cardiolipin. We amplified and sequenced 2,053 bp of the pig CDS1 mRNA. The structure of the pig CDS1 gene was determined, being very similar to that of the human, rat, and mouse genes with respect size and organization of the 13 exons. In addition, we identified three polymorphic positions in exons 10 and 11. One of them, the A/C₁₀₀₆, was genotyped in samples belonging to Iberian, Landrace, Large White, Pietrain, and Meishan pig breeds. Expression of this gene was also analyzed by real-time polymerase chain reaction (PCR) in different tissues showing a high CDS1 expression in testis. Moreover, a 1240-bp fragment of the pig CDS2 mRNA was amplified and sequenced. Finally, the CDS1 and CDS2 genes were physically mapped to porcine chromosomes 8 and 17, respectively, by using the INRA, University of Minnesota porcine Radiation Hybrid panel (IMpRH).     

Sustained phospholipase C stimulation of H9c2 cardiomyoblasts by vasopressin induces an increase in CDP-diacylglycerol synthase 1 (CDS1) through protein kinase C and cFos

Chronic stimulation (24 h) with vasopressin leads to hypertrophy in H9c2 cardiomyoblasts and this is accompanied by continuous activation of phospholipase C. Consequently, vasopressin stimulation leads to a depletion of phosphatidylinositol levels. The substrate for phospholipase C is phosphatidylinositol (4, 5) bisphosphate (PIP₂) and resynthesis of phosphatidylinositol and its subsequent phosphorylation maintains the supply of PIP₂. The resynthesis of PI requires the conversion of phosphatidic acid to CDP-diacylglycerol catalysed by CDP-diacylglycerol synthase (CDS) enzymes. To examine whether the resynthesis of PI is regulated by vasopressin stimulation, we focussed on the CDS enzymes. Three CDS enzymes are present in mammalian cells: CDS1 and CDS2 are integral membrane proteins localised at the endoplasmic reticulum and TAMM41 is a peripheral protein localised in the mitochondria. Vasopressin selectively stimulates an increase CDS1 mRNA that is dependent on protein kinase C, and can be inhibited by the AP-1 inhibitor, T-5224. Vasopressin also stimulates an increase in cFos protein which is inhibited by a protein kinase C inhibitor. We conclude that vasopressin stimulates CDS1 mRNA through phospholipase C, protein kinase C and cFos and provides a potential mechanism for maintenance of phosphatidylinositol levels during long-term phospholipase C signalling.     

牛卵巢PRSS35基因CDS序列分析

为测定牛卵巢丝氨酸蛋白酶35 (PRSS35)的CDS序列并进行生物信息学分析。试验根据NCBI上已公布牛PRSS35基因的mRNA序列设计特异性引物,使用RT-PCR技术扩增牛卵泡中PRSS35的CDS序列。结果显示,牛PRSS35基因CDS区序列全长为1 239 bp,共编码412个氨基酸,PRSS35与其他10个物种的同源序列相似性较高,且该蛋白具有一个长度为20个氨基酸的信号肽,具有11个O-糖基化位点和2个N-糖基化位点,以及3个磷酸化位点,并发现有一个典型的Tryp_Spc结构域,即胰蛋白酶样丝氨酸蛋白酶结构域。为进一步研究该基因及其编码蛋白在卵泡发育过程中所起的作用提供了一定的理论依据。     

Construction of a bacterial artificial chromosome library for the Rongchang pig breed and its use for the identification of genes involved in intramuscular fat deposition

In a search for genes affecting intramuscular fat deposition, we constructed a bacterial artificial chromosome (BAC) library for the whole genome of Rongchang pig, a domestic Chinese swine breed. The library consisted of approximately 192,000 clones, with an averaged insert size of 116 kb. Frequency of non-insert clone of the BAC library was no higher than 1.8%, based on estimation of 220 BAC clones randomly selected. We estimated the coverage of the library to be more than seven porcine genome equivalents. Subsequent screening of the BAC library with a three-step PCR procedure resulted in identification of seven candidate genes that were potentially involved in intramuscular fat deposition. The number of positive BAC clones ranged from 2 to 4 for each of the seven genes. One positive clone, containing the lipin1 gene, was fully sequenced by shotgun method to generate 118,041 bp porcine genomic sequences. The BAC clone contained complete DNA sequence of porcine lipin1 gene including all the exons and introns. Our results indicate that this BAC library is a useful tool for gene identification and help to serve as an important resource for future porcine genomic study.     

猪PID1基因CDS区的克隆及其mRNA表达与肌内脂肪沉积关系

为了探索PID1(Phosphotyrosine interaction domain containing1)基因的表达与脂肪沉积的关系,文章利用兼并引物进行RT-PCR从猪脂肪和肌肉组织中克隆PID1基因CDS(Coding region)区全序列,并采用荧光定量PCR方法对大白猪、鲁莱黑猪、莱芜猪3个猪品种的肝脏、脂肪和肌肉组织PID1基因mRNA表达进行了相对定量分析。结果表明:经克隆、测序,得到了猪PID1基因654bp全编码区序列,通过Blast比对,与人、大鼠、牛有93.88%、66.94%、88.07%的同源性。PID1基因在同一个品种猪中mRNA表达水平总体表现为:肝脏脂肪肌肉。在不同品种3种组织中PID1基因mRNA表达水平总体表现为:莱芜猪鲁莱黑猪大白猪,其中肝脏中差异显著(P0.05),但是在脂肪和肌肉组织中莱芜猪与鲁莱黑猪差异不显著(P0.05)。对于高肌内脂肪(LWH)、中等肌内脂肪(LWI)和低肌内脂肪(LWL)沉积的3组莱芜猪,PID1基因在肝脏组织中的表达水平是LWH显著高于LWL(P0.05),在肌肉组织中则是LWH显著高于LWI和LWL(P0.05)。PID1基因在莱芜猪品种内3个组织中mRNA表达量与IMF含量相关均不显著,而在品种间3个组织中mRNA表达量与IMF含量呈显著正相关(P0.05)。结果提示:PID1的表达可能与脂肪沉积性状存在一定的关系。     

Cloning and expression of the porcine myogenin gene

Abstract

Myogenin is a member of the myoD family of myogenic regulatory factors which direct the initiation of myogenesis during skeletal muscle development. Myogenin is responsible for the differentiation of proliferating myoblasts into myotubes and the absence of this factor results in severe muscular deficiency. Using PCR primers designed from mammalian myogenin sequences, a 215 bp PCR product was amplified from porcine genomic DNA and found to be 89.7% homologous to mouse myogenin. A porcine cosmid library was screened with the myogenin PCR probe and a 6.5 kb portion of a cosmid containing the pig myogenin gene was sequenced. The sequence includes 5´ flanking region, the entire protein coding region composed of three exons and two introns and 3´ flanking region. The protein coding region was highly conserved (≥92.5%) with mouse and human myogenin coding regions. Transfection of pig myogenin into C3H10T1/2 cells, a multipotential fibroblast cell line, resulted in 28% myogenic conversion assayed by cell fusion to form multinucleated cells and synthesis of sarcomeric myosin. These results indicate that the cloned myogenin gene was capable of initiating myogenesis in a fibroblast cell line.     

Improving the comparative map of SSC2p-q13 by sample sequencing of BAC clones

To improve the comparative map for pig chromosome 2 and increase the gene density on this chromosome, a porcine bacterial artificial chromosome (BAC) library was screened with 17 microsatellite markers and 18 genes previously assigned to pig chromosome 2. Fifty-one BAC clones located in the region of a maternally imprinted quantitative trait locus for backfat thickness (BFT) were identified. From these BACs 372 kb were sample sequenced. The average read length of a subclone was 442 basepair (bp). Contig assembly analysis showed that every bp was sequenced 1.28 times. Subsequently, sequences were compared with sequences in the nucleotide databases to identify homology with other mammalian sequences. Sequence identity was observed with sequences derived from 35 BACs. The average percentage identity with human sequences was 87.6%, with an average length of 143 bp. In total, sample sequencing of all BACs resulted in sequence identity with 29 human genes, 13 human expressed sequence tags (ESTs), 17 human genomic clones, one rat gene, one porcine gene and nine porcine ESTs. Eighteen genes located on human chromosome 11 and 19, and seven genes from other human locations, one rat gene and one porcine gene were assigned to pig chromosome 2 for the first time. The new genes were added to the radiation hybrid map at the same position as the locus from which the BAC that was sequenced was derived. In total 57 genes were placed on the radiation hybrid map of SSC2p-q13.     

Cloning and characterization of porcine insulin gene

The complete porcine preproinsulin cDNA and 1022 bp of its 5'-flanking region have been cloned by PCR-based technology and characterized. The porcine insulin gene has the same structure of three exons and two introns as that found in all insulin genes sequenced to date. Northern blot analysis of isolated adult porcine islets demonstrated an increase in steady-state insulin mRNA levels in response to high concentrations of glucose. Highly conserved cis-acting elements were found in the 5'-flanking region of the porcine insulin gene including multiple E and A elements as well as a cAMP responsive element (CRE). Tissue-specific activity of the proximal promoter was confirmed by transient transfection of the promoter/reporter gene constructs. This information now makes it possible for regulation and expression of the porcine insulin gene to be analyzed.     

Genetic and physical mapping, expression analysis and partial sequence of porcine PER1

The porcine PER1 gene was mapped to chromosome 12q1.4-->q1.5 using fluorescence in situ hybridisation. A polymorphic microsatellite marker (S0601) was isolated from a BAC clone shown to contain the PER1 gene. Linkage analysis assigned S0601 distal to ALOX12 on SSC12, providing further evidence for the conservation of synteny between HSA17 and SSC12. RT-PCR analysis demonstrated the expression of PER1 in all 11 tissues tested, consistent with the data from other mammalian species. Part of the PER1 gene was sequenced, homologous to exons 2-14 of the human gene and encoding the N-terminus of porcine PER1. The predicted amino acid sequence of the partial pig PER1 protein shares over 96% identity with its human orthologue.     

cDNA cloning,sequence identification and tissue expression distribution of three novel porcine genes: UCHL3, RIT1 and CCND3

The complete CDS sequences of three porcine genes: UCHL3, RIT1 and CCND3 were amplified using RT-PCR based on the sequence information of the mouse or other mammals and referenced highly homologous pig ESTs. Sequence analysis of these three genes revealed that the porcine UCHL3 gene encodes a protein of 230 amino acids and has high homology with the ubiquitin carboxyl-terminal hydrolase isozyme L3 (UCHL3) of four species-bovine (97%), human (96%), mouse (95%) and rat (94%). The porcine RIT1 gene encodes a protein of 219 amino acids and has high homology with the GTP-binding protein Rit1 (RIT1) of two species-human (97%), mouse (97%). The porcine CCND3 gene encodes a protein of 292 amino acids and has high homology with the G1/S-specific cyclin-D3 (CCND3) of four species-bovine (98%), human (97%), mouse (93%) and rat (92%). The phylogenetic tree analysis revealed that the swine UCHL3 has a closer genetic relationship with the UCHL3 of bovine, and the swine RIT1 has closer genetic relationships with the RIT1 of human, but the swine CCND3 has a closer genetic relationship with the CCND3 of bovine. The RT-PCR gene expression analysis indicated that the swine UCHL3, RIT1 and CCND3 genes were differentially expressed in tissues including small intestine, large intestine, liver, muscle, fat, lung, spleen and kidney. Our experiment established the primary foundation for further research on these three swine genes.     

Length polymorphism of PCR-amplified genomic fragments of the Pregnancy-Associated Glycoprotein (PAG) gene family in the pig and some other domestic and wild mammals

Porcine pregnancy-associated glycoprotein genes (pPAG) are known as a multigene family, in which five members have been cloned and sequences of their cDNAs identified. Porcine PAG1 and pPAG3 genes, belonging to the pPAG1-like subfamily, both encode enzymatically inactive precursors. In contrast, cDNAs of pPAG2, pPAG4 and pPAG6 represent the pPAG2-like gene subfamily, encoding enzymatically active precursors. The objective of this study was to investigate the polymorphism of both pPAG-like gene subfamilies in the pig in comparison to other domestic species, including cattle, sheep and goat (Artiodactyla), their wild relatives (red deer and wild pig) and horse (Perissodactyla). This is the first paper indicating the polymorphism of the pPAG gene family, examined by lengths of amplified genomic fragments (PCR). Obtained PCR products were analysed in relation to five characterised cDNAs of pPAGs (pPAG1-like and/or pPAG2-like subfamilies) and according to one recognised structural exon-intron organisation of the pPAG2 gene, among at least eight pPAG2-like genes expected in the porcine genome. The highest polymorphism frequency of both pPAG1- and pPAG2-like gene subfamilies was found in the second region, exons 5 and 6 (with intron E). The length of PCR-amplified genomic fragments was approximately: 1043, 700, 600 and 193 bp. A high polymorphism frequency was found in the 3'-terminal fragment, corresponding to exons 7-9 (with introns G and H), more frequent the pPAG2-like gene subfamily. The length of PCR-amplified genomic fragments was approximately: 733, 650 and 356 bp. In contrast, PAG polymorphism was not detected in another region, encompassing exons 2-4 (with introns B and C). The length of PCR-amplified genomic fragments was approximately 279 bp in all examined genomes. In conclusion, amplification of various regions of the PAG gene family presents a relatively inexpensive PCR method of animal pre-selection with different genotypes. Such a pre-selection of animals is helpful for further gene number inquiry of the PAG gene family in each animal, then in related generations. The obtained results provide a useful background for a genetic marker preparation (by Southern analysis of the PAG family) that will presumably enable an economical early selection of young animals for effective reproduction.     

Molecular characteristics and expression profiles of glycerol-3-phosphate dehydrogenase 1 (GPD1) gene in pig

The cytosolic activity of glycerol-3-phosphate dehydrogenase 1 (GPD1, EC 1.1.1.8) plays an important role in the synthesis of triacylglycerol and in the transport of reducing equivalents from the cytosol to mitochondria. Here we report the full-length genomic sequence of porcine GPD1 gene including promoter region. Porcine GPD1 gene contains eight exons and seven introns. Using the ImpRH, the GPD1 gene was mapped on chromosome 5. Sub-cellular localization of the pig GPD1 was localized in cytoplasm by GFP reporter gene. The full-length CDS of porcine GPD1 gene comprises 1050 nucleotides and it encodes 349 amino acids. Using the CDS sequences of 17 species, we built the phylogeny tree of GPD1 gene. We investigated the expression level of the gene in 13 different tissues and time course from birth to postnatal day 28 in longissinus doris muscle (LD) and in cerebrum. The result shows that porcine GPD1 gene is expressed in almost all tissues we tested but its levels of expression varies widely over 2 orders of magnitude. LD and the cerebrum have similar expression pattern that is at a low level at birth and increasing with aging to the highest level at postnatal day 8 in LD and postnatal day 14 in cerebrum. But weaning decreased the expression level of the GPD1 gene. This may partially explains the effects of weaning on energy metabolism.     

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.