Molecular cloning and expression analyses of a novel swine gene-ARF4

The mRNA differential display technique was performed to investigate the differences of gene expression in the longissimus muscle tissues from Meishan and Large White pigs. One novel gene that was differentially expressed was identified through semi-quantitative RT-PCR and the cDNA complete sequence was then obtained using the rapid amplification of cDNA ends (RACE) method. The nucleotide sequence of the gene is not homologous to any of the known porcine genes. The sequence prediction analysis revealed that the open reading frame of this gene encodes a protein of 180 amino acids that contains the putative conserved domain of ADP-ribosylation factor (ARF) which has high homology with the ADP-ribosylation factor 4 (ARF4) of six species-bovine (98%), human and orangutan (96%), African clawed frog (96%), mouse and rat (98%)-so that it can be defined as swine ADP-ribosylation factor 4 (ARF4). This novel porcine gene was finally assigned to GeneID:595108. The phylogenetic tree analysis revealed that the swine ARF4 has a closer genetic relationship with the rat and mouse ARF4 than with those of human and African clawed frog. The tissue expression analysis indicated that the swine ARF4 gene is over expressed in muscle, fat, heart, spleen, liver, and ovary and moderately expressed in lung and kidney but weakly expressed in small intestine. Our experiment is the first to establish the primary foundation for further research on the swine ARF4 gene.     

Molecular characterization and expression profile of a novel porcine gene differentially expressed in the muscle and backfat tissues from Chinese Meishan and Russian Large White pigs

The mRNA differential display technique was performed to investigate the differences of gene expression in the longissimus dorsi muscle and backfat tissues from Chinese Meishan and Russian Large White pigs. One novel gene that was differentially expressed was identified through semi-quantitative RT-PCR and the cDNA complete sequence was then obtained using the rapid amplification of cDNA ends (RACE) method. The cDNA sequence of this gene is not homologous to any of the known porcine genes. The sequence prediction analysis revealed that the open reading frame of this gene encodes a protein of 402 amino acids that contains the putative conserved transposase DDE domain and further Blast analysis revealed that this protein has 100% homology with the Tn10 transposase from Oryza sativa, Serratia marcescens, and Salmonella, and therefore, this gene can be defined as the swine Tn10 transposase gene. This novel porcine gene was finally assigned to Gene ID: 100049649. The RT-PCR analysis of the tissue expression profile was carried out using the tissue cDNAs of one Meishan pig as the templates, and the result indicated that this novel swine gene is moderately expressed in fat, and weakly expressed in small intestine, liver, kidney, and spleen but almost not expressed in heart, ovary, muscle, and lung. Our experiment established the primary foundation for further research into the biological significance of swine Tn10 transposase gene.     

Molecular characterization and expression profile of a novel porcine gene differentially expressed in the muscle and backfat tissues from Chinese Meishan and Russian Large White pigs

The mRNA differential display technique was performed to investigate the differences of gene expression in the longissimus dorsi muscle and backfat tissues from Chinese Meishan and Russian Large White pigs. One novel gene that was differentially expressed was identified through semiquantitative RT-PCR, and the cDNA complete sequence was then obtained using the rapid amplification of the cDNA ends (RACE) method. The cDNA sequence of this gene is not homologous to any of the known porcine genes. The sequence prediction analysis revealed that the open reading frame of this gene encodes a protein of 402 amino acids that contains the putative conserved transposase DDE domain, and further Blast analysis revealed that this protein has 100% homology with the Tn10 transposase from Oryza sativa, Serratia marcescens, and Salmonella, and, therefore, this gene can be defined as the swine Tn10 transposase gene. This novel porcine gene was finally assigned to Gene ID: 100049649. The RT-PCR analysis of the tissue expression profile was carried out using the tissue cDNAs of one Meishan pig as the templates, and the result indicated that this novel swine gene is moderately expressed in fat and weakly expressed in small intestine, liver, kidney, and spleen but almost not expressed in heart, ovary, muscle, and lung. Our experiment established the primary foundation for further research into the biological significance of swine Tn10 transposase gene.     

A novel porcine gene,MAPKAPK3, is differentially expressed in the pituitary gland from mini-type Diannan small-ear pigs and large-type Diannan small-ear pigs

The mRNA differential display technique was performed to investigate the differences of gene expression in the pituitary gland from mini-type Diannan small-ear pigs and large-type Diannan small-ear pigs. One novel gene differentially expressed was identified through semi-quantitative RT-PCR and its cDNA complete sequence was then obtained using the rapid amplification of cDNA ends (RACE) method. Nucleotide sequence of the gene is not homologous to any of the known porcine genes. The sequence analysis revealed that the open reading frame of this gene encodes a protein of 384 amino acids has high homology with the mitogen-activated protein kinase-activated protein kinase 3 (MAPKAPK3) of eight species-cattle (96%), horse (96%), rhesus monkey (93%), rabbit (77%), human (98%), chimpanzee (98%), mouse (94%) and rat (91%)-so that it can be defined as swine MAPKAPK3 gene. This gene was finally assigned GENE ID: 100233192. Computer assisted analysis revealed that the genomic DNA of open reading frame of the swine MAPKAPK3 gene consists of ten exons and nine introns. The phylogenetic tree analysis revealed that the swine MAPKAPK3 gene has a closer genetic relationship with the MAPKAPK3 of cattle. The tissue expression analysis indicated that the MAPKAPK3 mRNA expression levels of large-type Diannan small-ear pigs are ubiquitously increased in various tissues compared to the mini-type Diannan small-ear pigs. Our experiment suggested that the swine MAPKAPK3 gene might play an important role in the growth and development differences between mini-type Diannan small-ear pigs and large-type Diannan small-ear pigs.     

A novel porcine gene, POT1, differentially expressed in the longissimus muscle tissues from Wujin and Large White pigs

The mRNA differential display technique was performed to investigate the differences of gene expression in the longissimus muscle tissues from Wujin and Large White pigs. One novel gene differentially expressed was identified through quantitative real time PCR and the cDNA complete sequence was then obtained using the rapid amplification of cDNA ends (RACE) method. The nucleotide sequence of the gene is not homologous to any of the known porcine genes. The sequence prediction analysis revealed that the open reading frame of this gene encodes a protein of 507 amino acids that shares high homology with the protection of telomeres 1 isoform 4 (POT1) of human (86%)-so that this gene can be defined as swine POT1 gene. This gene is structured in 12 exons and 11 introns as revealed by computer-assisted analysis. The tissue expression analysis indicated that the swine POT1 gene is differentially expressed in tissues including muscle, heart, liver, fat, kidney, lung, pancreas and spleen. Our experiment is the first to establish the primary foundation for further research on the swine POT1 gene.     

A porcine gene, PBK, differentially expressed in the longissimus muscle from Meishan and Large White pig

An investigation of differences in gene expression in the longissimus muscle of Meishan and Large White pigs was undertaken, using the mRNA display technique. A fragment of one differentially expressed gene was isolated and sequenced, whereupon the complete cDNA sequence was then obtained by using the rapid amplification of cDNA ends (RACE). The nucleotide sequence of the gene is not related to any known porcine gene. Sequence analysis revealed that the open reading frame of this gene encodes a protein with 322 amino acids, thus displaying high sequence identity with the PDZ binding kinase (PBK) of eleven other animal species - dog, horse, cattle, human, chimpanzee, crab-eating macaque, rhesus monkey, rat, mouse, gray short-tailed opossum and platypus, so it can be defined as the porcine PBK gene. This gene was finally assigned GeneID:100141310. Phylogenetic tree analysis revealed that the swine PBK gene has a closer genetic relationship with the PBK gene of platypus. Gene expression analysis of eight tissues of a Meishan x Large White cross showed that the porcine PBK gene is differentially expressed in various tissues. Our experiment established the primary foundation for further research on this gene.     

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.     

Differential display reveals a novel pig gene,PRPF3, which is differentially expressed in Large White versus Wujin skeletal muscle tissues

The mRNA differential display technique was performed to investigate gene expression differences in the longissimus dorsi muscle from Wujin and Large White pigs. A fragment of one differentially expressed gene was isolated and sequenced. A complete cDNA sequence of the gene was obtained using the rapid amplification of cDNA ends method. The open reading frame of this gene encodes a protein of 683 amino acids, which is homologous with the PRP3 pre-mRNA processing factor 3 (PRPF3) of five species: bovine (99%), human (99%), sumatran orangutan (99%), mouse (99%) and chicken (94%). This newly identified gene was respectively defined as the swine PRPF3 gene. This gene is structured in 15 exons and 14 introns as revealed by computer-assisted analysis. The phylogenetic tree analysis revealed that the swine PRPF3 gene has a closer genetic relationship with the PRPF3 gene of sumatran orangutan.     

Molecular Cloning, Mapping, and Expression Analysis of the EIF4A2 Gene in Pig

A full-length cDNA clone encoding the eukaryotic translation initiation factor 4A, isoform 2 (EIF4A2), was cloned from the fetal skeletal cDNA library from the pig (Sus scrofa). EIF4A2 is a highly conserved gene for one of the protein-synthesis initiation factors involved in the binding of mRNA to the ribosome. Based on this cDNA sequence, the deduced protein of 407 amino acids contains the characteristic motifs shared by the DEAD-box supergene family. The genomic nucleotide sequence of this gene was determined and a single nucleotide polymorphism located in the 5′ untranslated region was genotyped. The porcine EIF4A2 was expressed in all tissues examined but in variable amounts. The EIF4A2 expression level in muscle was upregulated through embryonic and neonatal development until adult, suggesting that porcine EIF4A2 was possibly involved in translation regulation of other muscle-related genes in muscle formation and development. In addition, we mapped porcine EIF4A2 to q4.1 of SSC13, in agreement with comparative mapping data.     

Identification of a differentially expressed gene PPP1CB between porcine Longissimus dorsi of Meishan and Large WhitexMeishan hybrids

To study the molecular basis of heterosis, suppression subtractive hybridization was used to investigate the differences in gene expression between porcine Longissimus dorsi of F1 hybrids Large WhitexMeishan and their female parents Meishan. From two specific subtractive cDNA libraries, the clones selected by reverse Northern high-density blot screening were chosen to clone full-length cDNA by rapid amplification of cDNA ends. An expression-upregulated gene for Meishan skeletal muscle, designated protein phosphatase 1, catalytic subunit, beta isoform (PPP1CB), was identified. Porcine PPP1CB contains an open reading frame encoding 327 amino acid residues with 13 and 1763 nucleotides in the 5' and 3' untranslated regions, respectively. A DNA fragment of 721 nucleotides was amplified and a mutation that creates/disrupts a restriction site for endonuclease RsaI was found. The derived amino acid sequence of PPP1CB has high homology with the PPP1CB of three species, Mus musculus (99%), human (99%) and mouse (100%). The tissue expression analysis indicated that the swine PPP1CB gene is generally expressed in most tissues. The possible role of PPP1CB and its relation to porcine heterosis are discussed.