Somatic Cell Hybrid Panel Research Articles

Molecular characterization, chromosome mapping, and expression profile of porcine CDC16.

Cell division cycle16 (CDC16) is a core component among the eight protein subunits of the anaphase-promoting complex (APC). APC is a cyclin degradation system that governs the exit of cells from mitosis. Not much information is available for CDC16 in pig. In this study, a 2284-bp cDNA of porcine CDC16 was obtained by rapid amplification of cDNA ends (RACE). Porcine CDC16 was assigned to SSC11 q11-17, and was determined to be significantly linked with SW1452 by using somatic cell hybrid panel and radiation hybrid panel. One novel A/G SNP anchored in intron 7 of the gene was genotyped by restriction enzyme polymerase chain reaction (PCR)-restriction fragment length polymorphism-Csp6I. In five pig breeds, Shaziling, Taoyuan, Duroc, Landrace, and Yorkshire, the A allele frequency was dominant. Quantitative PCR revealed that porcine CDC16 was expressed in ten selected tissues of 25-day-old Shaziling and Yorkshire piglets, and that the mRNA expression of CDC16 in longissimus dorsi muscle of Shaziling was higher than that of Yorkshire. Expression levels of CDC16 were highest in longissimus dorsi muscle followed by that in pancreas. CDC16 protein was detected in longissimus dorsi muscle of 25-day-old Shaziling and Yorkshire piglets by immunohistochemistry with abundant protein expression index (P > 0.05). This study provides an insight into the role of porcine CDC16 in the formation of meat.

Molecular characterization, expression analysis and association study with immune traits of porcine PSMB6 gene

PSMB6 subunit belongs to the 20S proteasomal subunit family, which plays an importance role in the antigen presentation by MHC class I molecular. The full-length genomic DNA sequence was obtained by PCR and the full-length CDS of porcine PSMB6 was isolated by the reverse transcriptase-polymerase chain reaction (RT-PCR). The deduced protein of 239 amino acids showed 86% identity to the corresponding human sequence. RT-PCR revealed that porcine PSMB6 gene was expressed in seven tissues studied (heart, liver, spleen, lung, kidney, skeletal muscle and fat) and SYBR Green real-time RT-PCR analysis showed PSMB6 gene was expressed highest in spleen. A single nucleotide substitution resulting in the amino acid change (19Pro-19His) was detected within exon 1 and other two single nucleotide polymorphisms were detected in intron 1. The Genetic variations were identified in eight pig breeds showed diverse results. Furthermore, we found that the single nucleotide polymorphisms (SNP, G/T300) was significantly associated with delayed type hypersensitivity (DTH, P < 0.05), immunoglobin G (IgG1, P < 0.05), white blood cell count (WBC, P < 0.05). Using the pig/rodent somatic cell hybrid panel (SCHP), we mapped the porcine PSMB6 gene to SSC12, in agreement with comparative mapping data.

Cloning, chromosomal localization, expression profile and association analysis of the porcine WNT10B gene with backfat thickness

The Wingless-type MMTV integration site (Wnt) family encodes secreted glycoproteins that are ligands for the frizzled family of seven-transmembrane receptors and the low density lipoprotein receptor-related protein family of co-receptors. The WNT10B gene inhibits differentiation of preadipocytes invitro and impairs adipose development invivo. In the present study, a 1,615-bp cDNA sequence of the porcine WNT10B gene was obtained by RT-PCR. The porcine WNT10B gene was assigned to 5p11-p15 by using the somatic cell hybrid panel (SCHP) and the radiation hybrid (IMpRH) panel. One SNP in the 3'-untranslated region (3'-UTR) was found and association analysis suggested that the SNP was associated with backfat thickness. Semi-quantitative RT-PCR showed that the porcine WNT10B gene was expressed in all tissues examined in 35d and adult pigs and the mRNA expression of WNT10B in fat tissue of Tongcheng pigs was dramatically higher than that in Large White pigs.

Molecular Characterization, Chromosomal Localizations, Expression Profile, and Association Analysis of the Porcine PECI Gene with Carcass Traits

The full-length cDNA of the porcine peroxisomal Δ 3 ,Δ 2 -enoyl-CoA isomerase (PECI) gene encodes a monofunctional peroxisomal Δ 3 ,Δ 2 -enoyl-CoA isomerase. Cloning and sequencing of the porcine PECI cDNA revealed the presence of an 1185-base pair open reading frame predicted to encode a 394-amino acid protein by the 5' rapid amplification of cDNA ends (5'-RACE) and EST sequences. The porcine PECI gene was expressed in seven tissues (heart, liver, spleen, lung, kidney, skeletal muscle, fat) which was revealed by reverse transcriptase-polymerase chain reaction (RT-PCR). The porcine PECI was mapped to SSC71/2 p11-13 using the somatic cell hybrid panel (SCHP) and the radiation hybrid panel (RH) (LOD score 12.84). The data showed that PECI was closely linked to marker S0383. A C/T single nucleotide polymorphism in PECI exon 10 (3'-UTR) was detected as a PvuII PCR-RFLP. Association analysis in our experimental pig population showed that different genotypes of PECI gene were significantly associated with the Average Backfat thickness (ABF) (p<0.05) and Buttock backfat thickness (p<0.01).

Molecular characterization, chromosomal location, alternative splicing and polymorphism of porcine GFAT1 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 characterization, chromosomal localization and association analysis with back-fat thickness of porcine LPIN2 and LPIN3

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).

Molecular Characterization and Expression Pattern of the Porcine STARS, a Striated Muscle-Specific Expressed Gene

STARS (striated muscle activator of Rho signaling) promotes the nuclear localization of MRTFs and mediates SRF transcription, which provides a potential muscle-specific mechanism for linking changes in the actin cytoskeleton structure with muscle gene expression. In this study, the full-length cDNA of the porcine STARS was cloned. The open reading frame of this gene contains 1,155 bp and encodes a protein of 384 amino acids, which is 79, 73, and 77% identical with human, mouse, and rat STARS genes, respectively. RT-PCR revealed that STARS is specifically expressed in heart and skeletal muscles. STARS is also distinctly different in different muscle developmental stages. The result indicates that its expression increased gradually from 33 dpc (days postcoitum) to postnatal muscles, and peaked 28 days postnatal. The porcine STARS was mapped to SSC4p13 using the somatic cell hybrid panel and the radiation hybrid panel IMpRH (LOD score 11.98). The data show that STARS is closely linked to marker SW871. A T/G single nucleotide polymorphism in the coding sequence, detected as Bsh1236I PCR-RFLP, displays allele frequency differences in six pig breeds.

Glioma-expressed antigen 2 (GLEA2): a novel protein that can elicit immune responses in glioblastoma patients and some controls.

Glioma constitutes the most frequent brain tumour in man with glioblastoma as the most prevalent and malignant type. The average survival time of less than 16 months underlines the need for improvements in diagnosis and therapy. Here, we report the identification of a novel antigen termed glioma-expressed antigen 2 (GLEA2) causing a frequent immune response in glioma patients. Screening of 450 000 clones from a glioblastoma lambda zap expression library with autologous patient serum revealed a group of five serum-positive clones sharing a high sequence homology. Further sequence analysis showed a sequence homology to a hepatocellular carcinoma associated antigen 58 (HCA58). We localized the novel HCA homologous gene termed glioma-expressed antigen 2 (GLEA2) on chromosome 20 by somatic cell hybrid panel mapping. Using allogenic sera from 39 glioblastoma patients, we found an immune response against GLEA2 in 17 patients (43%). In addition, screening with allogenic sera from other glioma patients revealed GLEA2 directed antibodies in two out of five pilocytic astrocytomas and in one out of two astrocytomas. Unrelated tumour sera revealed no immune response and sera from healthy persons showed an immune response in two out of 14 cases (14%). Northern blot hybridization and RT-PCR showed ubiquitous GLEA2 gene expression in glioma and normal tissues. The novel HCA homologous gene, GLEA2, appears to induce a frequent immune response in glioma. In the light of the lack of useful glioma markers, it appears reasonable to consider GLEA2 as a potential future diagnostic marker.

Investigation of Lpin1 as a candidate gene for fat deposition in pigs

Lpin1 deficiency prevents normal adipose tissue development and remarkably reduces adipose tissue mass, while overexpression of the Lpin1 gene in either skeletal muscle or adipose tissue promotes adiposity in mice. However, little is known about the porcine Lpin1 gene. In the present study, a 5,559-bp cDNA sequence of the porcine Lpin1 gene was obtained by RT-PCR and 3'RACE. The sequence consisted of a 111-bp 5'UTR, a 2,685-bp open reading frame encoding a protein of 894 amino acids and a 2,763-bp 3'UTR. Semi-quantitative RT-PCR analysis revealed that Lpin1 had a high level of expression in the liver, spleen, skeletal muscle and fat, a low level of expression in the heart, lung and kidney. The porcine Lpin1 gene was assigned to 3q21-27 by using the somatic cell hybrid panel (SCHP) and the radiation hybrid (IMpRH) panel. One C93T single nucleotide polymorphism (SNP) was identified and genotyped using the TaqI PCR-RFLP method. Association analysis between the genotypes and fat deposition traits suggested that different genotypes of the Lpin1 gene were associated with percentage of leaf fat and intramuscular fat.

Chromosomal Assignments of Eight Expressed Sequence Tags for Unknown Genes Showing Early Embryonic Death-Associated Changes of Expression Patterns in the Fetal Placenta of the Cow Carrying Somatic Nuclear-Derived Cloned Embryo

Eight expressed sequence tags for unknown novel genes showing early embryonic death-associated changes of expression patterns in the fetal placenta of the cow carrying somatic nuclear-derived cloned embryo were assigned to bovine chromosomes using deoxyribonucleic acids (DNAs) of bovine/murine somatic cell hybrid panel.

Assignment of solute carrier family 2 (facilitated glucose transporter), members &amp;lt;i&amp;gt;SLC2A2&amp;lt;/i&amp;gt;, &amp;lt;i&amp;gt;SLC2A3&amp;lt;/i&amp;gt;, &amp;lt;i&amp;gt;SLC2A5&amp;lt;/i&amp;gt;, &amp;lt;i&amp;gt;SLC2A8&amp;lt;/i&amp;gt; and &amp;lt;i&amp;gt;SLC2A12&amp;lt;/i&amp;gt; to porcine chromosomes by somatic cell and radiation hybrid panel mapping (Brief report)

Abstract. The transport of glucose plays an important role in cellular glucose homeostasis and metabolism [1]. Due to the hydrophilic character of glucose, the transport of glucose in and out of cells requires specific carrier proteins. The mammalian facilitative glucose transport family, which contains the energy-independent transporters (gene symbol SLC2A, protein symbol GLUT), catalyzes the entry of glucose into mammalian cells by facilitative diffusion down a concentration gradient. Thirteen members of mammalian GLUT family have been now characterized [1]. In swine, the chromosomal locations for the five genes (SLC2A2, SLC2A3, SLC2A5, SLC2A8 and SLC2A12) have not yet been determined. In this study, as the first step to better understand of the roles of these GLUTs in pigs which could subsequently be beneficial for pig production, we report the mapping of the five genes using both porcine somatic cell hybrid panel (INRA-SCHP) and radiation hybrid panel (IMpRH).

Mapping of porcine BTG1, BTG4, TOB2, USP2 and PEG3 using somatic cell and radiation hybrid panels

Mapping of porcine <i>BTG1, BTG4, TOB2, USP2</i> and <i>PEG3</i> using somatic cell and radiation hybrid panels

The first linkage map of the American mink (Mustela vison)

Described herein, the first microsatellite linkage map for the American mink consists of 85 microsatellite markers resolved into 17 linkage groups. The map was constructed using 92 F(1) progeny from five sire families created by crossing mink with different colour types. The linkage groups ranged from 0 to 137 cM. These linkage groups were assigned to 12 of the 14 mink autosomes using a somatic cell hybrid panel. The total map covered 690 sex-averaged Kosambi units with an average marker spacing of 8 cM. This map will facilitate further genetic mapping of monogenic characters and QTL.

Mapping of porcine ANKRD1, ANKRD2, ANKRD23, VGLL2 and VGLL4 using somatic cell and radiation hybrid panels

Mapping of porcine <i>ANKRD1</i>, <i>ANKRD2</i>, <i>ANKRD23</i>, <i>VGLL2</i> and <i>VGLL4</i> using somatic cell and radiation hybrid panels

The Porcine FoxO1, FoxO3a and FoxO4 Genes: Cloning, Mapping, Expression and Association Analysis with Meat Production Traits

PoxO1, FoxO3a and FoxO4 belong to the FoxO gene family, which play important roles in the PI3K/PKB pathway. In this study, we cloned the porcine FoxOl, FoxO3a and FoxO4 sequences and assigned them to SSCllpll-15, SSC1p13 and SSC xq13 using somatic cell hybrid panel (SCHP) and radiation hybrid panel (IMpRH). RT-PCR results showed that these three genes are expressed in multiple tissues. Sequencing of PCR products from different breeds identified a synonymous T/C polymorphism in exon 2 of FoxO3a. This FoxO3a single nucleotide polymorphism (SNP) can be detected by AvaII restriction enzyme. The allele frequencies of this SNP were investigated in Dahuabai, Meishan, Tongcheng, Yushan, Large White, and Duroc pigs. Association of the genotypes with growth and carcass traits showed that different genotypes of FoxO3a were associated with carcass length and backfat thickness between 6th and 7th ribs (BTR) and drip loss (p<0.05).

Characterization of the Full-length cDNA, Chromosomal Localization, and Polymorphism of the Porcine RPLP0 Gene

RPLP0 gene encodes the acidic ribosomal phosphoprotein large P0 subunit, which is a component of the 60S subunit. The full-length cDNA sequence of porcine RPLP0 was obtained from skeletal muscle of fetal pig cDNA library and deposited in GenBank. The nucleotide sequence and the predicted protein sequence shared high sequence identity with other mammalian homologues. A C/A single nucleotide substitution in exon 5 was detected as Csp6?polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) shows allele frequency diversity among Tongcheng, Xiaomeishan, Yushan, Large White, Landrace, and Duroc breeds. Analyses of somatic cell hybrid panel (SCHP) and radiation hybrid (IMpRH) panel showed that the RPLP0 gene was mapped to SSC 14q22-q24 and was closely linked to locus SW1321 (25 cR, LOD = 14.54).

Somatic Cell Hybrid Mapping of Expressed Sequence Tags For Genes Showing Early Embryonic Death-Associated Changes of Expression Patterns in the Fetal Placenta of the Cow Carrying Somatic Nuclear-Derived Cloned Embryo

We previously detected 368 expressed sequence tags showing early embryonic death-associated changes of expression patterns in the fetal placenta of the cow carrying somatic nuclear-derived cloned embryo. In the present study 7 (presumed expressed sequence tags for HYPC, SPTBN1 and TNNC2, and four expressed sequence tags for unknown novel genes) out of the 368 expressed sequence tags were mapped to bovine chromosomes by analyzing deoxyribonucleic acids of bovine/murine somatic cell hybrid panel with polymerase chain reaction using primers specific for those bovine genes.

Imprinting analyses of the porcine GATM and PEG10 genes in placentas on days 75 and 90 of gestation

Imprinted genes are expressed monoallelically depending on their parental origin, and escape the Mendel's laws of heredity. They play important roles in the mammalian development, growth, and behavior. Placenta is a key tissue for the normal development and growth of fetus. It is also used to illuminate the evolution of genomic imprinting. In this study, we cloned the porcine GATM and PEG10 genes. Somatic cell hybrid panel (SCHP) and porcine radiation hybrid (IMpRH) panel were employed to locate GATM and PEG10 genes to SSC1q12-21 and SSC9p13-21, respectively. By sequencing PCR products, we detected several cSNPs in the two genes. The BseLI (GATM) and TaqI (PEG10) polymorphisms were used to investigate the allele frequencies in different pig breeds and the imprinting status in porcine placentas on days 75 and 90 of gestation. The results showed that for the GATM BseLI polymorphism, the Yorkshire and Duroc pigs had higher allele frequencies at the G allele, whereas the local pigs had higher allele frequencies at the A allele. Expression and sequencing analyses showed that both alleles were expressed for the GATM gene, indicating the GATM was not imprinted in the porcine placentas on days 75 and 90 of gestation. The allele frequencies of TaqI polymorphism for PEG10 gene were significantly different in native Chinese Erhualian breed comparing to Yorkshire. PEG10 was monoallelically expressed, showing the PEG10 gene may be imprinted in porcine placentas on days 75 and 90 of gestation.

Genomic organization, localization and polymorphism of porcine PSMB10, a gene encoding the third beta‐type proteasome subunit of 26S proteasome complex

The proteasome subunit, beta type 10 (PSMB10) gene regulated by interferon-gamma is a core part of the 26S proteasome complex, which is an important protein degrading system. Isolation and characterization of swine PSMB10 revealed a conserved structure with other mammalian PSMB10 genes. An A/G nucleotide polymorphism in PSMB10 intron 2 and a C/T single nucleotide polymorphism in exon 5 were detected by polymerase chain reaction restriction fragment length polymorphism. The allele frequencies were significantly different among Tongcheng, Landrace, Large White and Duroc. The porcine PSMB10 was mapped by somatic cell hybrid panel and radiation hybrid mapping on SSC6p14-p15, which is in good agreement with human-pig comparative maps.

Porcine OGN and ASPN: mapping, polymorphisms and use for quantitative trait loci identification for growth and carcass traits in a Meishan × Piétrain intercross

The porcine orthologues of human chromosome HSA9q22.31 genes osteoglycin (OGN) and asporin (ASPN) were mapped to porcine chromosome SSC3 using linkage analysis and a somatic cell hybrid panel. This mapping was refined to SSC3q11 using fluorescence in situ hybridization. These results confirm the existence of a small conserved synteny group between SSC3 and HSA9. Polymorphisms were revealed in both genes, including a pentanucleotide microsatellite (SCZ003) in OGN and two single nucleotide polymorphisms (AM181682.1:g.780G>T and AM181682.1:g.825T>C) in ASPN. The two genes were included in a set of markers for quantitative trait loci (QTL) mapping on SSC3 in the Hohenheim Meishan x Piétrain F2 family. Major QTL for growth and carcass traits were centred in the ASPN-SW902 region.