Identification and characterization of a differentially expressed protein (CAPZB) in skeletal muscle between Meishan and Large White pigs

Actin capping protein beta (CAPZB) protein was identified with considerable differences in the longissimus dorsi muscle between Large White and Meishan pigs using proteomics approach. However, in pigs, the information on CAPZB is very limited. In this study, we cloned and characterized the porcine actin capping protein beta (CAPZB) gene. In addition, we present two novel porcine CAPZB splice variants CAPZB1 and CAPZB2. CAPZB1 was expressed in all twenty tissues. However, CAPZB2 was predominantly expressed in the skeletal muscle and heart. In addition, the two isoforms had different expression profiles during the skeletal muscle development and between breeds. Moreover, the SNP T394G was identified in the coding region of the CAPZB gene, which was significantly associated with the carcass traits including the LFW, CFW, SFT and LEA. Data presented in our study suggests that the CAPZB gene may be a candidate gene of meat production trait and provides useful information for further studies on its roles in porcine skeletal muscle.     

Enzyme patterns in trisomy 19 of the mouse

Activity patterns of cytosolic and mitochondrial enzymes of carbohydrate and amino acid metabolism have been measured in murine trisomy 19. In spite of marked hypoplasia, no significant alterations of the patterns (per gram of organ weight) were observed, with the exception of glutamate oxaloacetate transaminase (GOT-1), and phosphoglycerate mutase (PGAM). Clear-cut gene dosage effects in liver, brain, heart, skeletal muscle, and erythrocytes of fetal and newborn mice, confirm the assignment of GOT-1 to chromosome 19. Data obtained for PGAM demonstrate that one of the two different subunits leading to organ-specific isozyme patterns of the dimer enzyme protein is coded on chromosome 19 (gene Pgam-1). Dosage effects are fully expressed in liver, brain, and erythrocytes (AA-type isozyme), but not in skeletal muscle (BB-type isozyme). Dosage effects on the hybrid AA-AB-BB-isozyme pattern in the course of development of the heart muscle, were demonstrated by means of quantitative activity measurement after electrophoretic separation. The comparison of enzyme patterns of eusomic and trisomic erythrocytes, produced after injection of fetal stem cells into irradiated adult carriers (transplantation chimaeras), revealed enzyme activity ratios that were similar to those produced by erythrocytes of adult euploid and trisomic mice. This is in agreement with the chromosome assignments and dosage effects mentioned above.     

Molecular characterization, expression patterns, and polymorphism of a differentially expressed porcine gene (PYGM) isolated by suppression subtractive hybridization and two-dimensional gel electrophoresis analysis

Suppression subtractive hybridization was performed to detect the differences in gene expression of porcine longissimus dorsi muscles between Large White and Chinese Meishan pigs. An upregulated gene in Large White that shared high homology with human muscle glycogen phosphorylase (PYGM) was identified. The porcine PYGM gene contains an open reading frame encoding 842 amino acid residues with 26 and 283 nucleotides in the 5' and 3' untranslated regions, respectively. Tissue distribution analysis indicated that porcine PYGM mRNAs are highly expressed in all tissues. Expression pattern of PYGM was similar in the two breeds. Both breeds had the highest expression levels when 120 days old (p<0.01), and PYGM was upregulated during skeletal muscle development. A similar expression pattern of PYGM in protein level was also observed by differential proteome analysis of skeletal muscle development using two-dimensional gel electrophoresis and mass spectroscopy. The mRNA abundance of PYGM in Large White was higher than Meishan at all four stages (p<0.05). Moreover, a G/T mutation in exon 8 was identified and association analysis with meat quality traits showed that it was significantly associated with lean meat percentage (p<0.05). Our data may provide further insight into the molecular mechanisms responsible for breed-specific differences in porcine growth and meat quality.     

Porcine skeletal muscle differentially expressed gene ATP5B: molecular characterization, expression patterns, and association analysis with meat quality traits

The 2-DE/MS-based proteomics approach was used to investigate the differences of porcine skeletal muscle, and ATP5B was identified as one differential expression protein. In the present study, ATP5B gene was further cloned by RT-PCR, the sequence was analyzed using the bioinformatics method, and the mRNA expression was detected by qRT-PCR. Sequence analysis showed that the porcine ATP5B gene contains an ORF encoding 528-amino-acid residues with 49 and 166 nucleotides in the 5′ and 3′ UTRs, respectively. The mRNA of ATP5B was widely expressed in all 14 tissues tested, but especially highly expressed in parorchis and fat. The expression pattern of ATP5B was similar in Large White and Meishan breeds, showing that the expression was upregulated by 3 days after birth and downregulated during postnatal development of skeletal muscle. Comparing the two breeds, the mRNA abundance of ATP5B in Large White was more highly expressed than in Meishan at all developmental stages (P < 0.05). Moreover, a synonymous mutation, G75A in exon 8, was identified and association analysis with the traits of meat quality showed that it was significantly associated with the RLF, FMP, IFR, IMF, and IMW (P < 0.05). These results suggested that ATP5B probably plays a key role in porcine skeletal muscle development and may provide further insight into the molecular mechanisms responsible for breed-specific differences in meat quality.     

Effects of thyroid hormone on mRNAs of phosphoglycerate mutase subunits in rat muscle during development

BACKGROUND: We previously showed that triiodothyronine (T3) stimulates muscle phosphoglycerate mutase (PGAM) activity and isozyme transition in rat skeletal and cardiac muscles. METHODS: The effects of T3 on PGAM types B and M subunit expression in rat muscle during development are reported. RESULTS: T3 administration during the first 21 days of rat life more than doubles type M PGAM mRNA levels, but produces minor effects on type B PGAM mRNA levels. The antihormone propylthiouracil (PTU) slightly decreases both type B and M mRNA levels, but this decrease is not statistically significant. CONCLUSION: Thyroid hormone influences PGAM mRNA isozyme levels differently and increases type M mRNA.     

Molecular characterization,transcriptional regulation and association analysis with carcass traits of porcine TCAP gene

TCAP (also known as titin-cap or telethonin) is one of the titin interacting Z-disk proteins involved in the regulation and development of normal sarcomeric structure. In this study, we cloned the cDNA and promoter sequences of porcine TCAP gene, which contained a 504 bp full-length coding region. Quantitative real-time PCR (qRT-PCR) analyses showed that porcine TCAP was highly expressed in the skeletal muscle, heart, and kidney. During postnatal muscle development, TCAP expression was down-regulated from 30 days to 120 days in Large White and Meishan pigs. One single nucleotide polymorphism c.334G>A in exon 2 of the TCAP gene was identified and detected by allele-specific primer-polymerase chain reaction (ASP-PCR). Association analysis revealed that the polymorphism had significant associations (P < 0.05 and P < 0.01) with some carcass traits. Analysis of the porcine TCAP promoter in different cell lines demonstrated that it is a muscle-specific promoter. In addition, we found that the porcine TCAP promoter can be activated by MyoD, MyoG and MEF2 in myotubes, which indicated that TCAP may play a role in the regulation of porcine skeletal muscle development. These findings provide useful information for the further investigation of the function of TCAP in porcine skeletal muscle.     

Molecular characterization and expression patterns of serine/arginine-rich specific kinase 3 (<Emphasis Type="Italic">SPRK3</Emphasis>) in porcine skeletal muscle

SRPK3 is a protein kinase belonging to serine/arginine protein kinases (SRPK) family, which phosphorylates serine/arginine repeat-containing proteins, and is controlled by a muscle-specific enhancer directly regulated by MEF2. In this study, a full-length cDNA of the porcine SRPK3 gene encoding a 566 amino acid protein was isolated. It contains 14 exons over approximately 4.3 kb. The deduced amino acid sequence of porcine SRPK3 contains a bipartite kinase domain, and shows high similarities to their corresponding human and cattle homologues. Tissue distribution analysis indicated that porcine SRPK3 mRNAs are highly expressed in heart and skeletal muscle especially in uterus and parorchis, but at low level in brain, stomach, small intestine, and ovary. Expression pattern of SRPK3 was similar in Large White and Chinese Meishan breeds. Both the two breeds had the highest expression levels at fetal 65 days (P < 0.01), and decreased while the age increased until 60 days old, then increased at 120 days (P < 0.01) and decreased at 180 days (P < 0.05). However, at fetal 65 days, the mRNA abundance of SRPK3 in Large White was 12.5-fold higher than in Meishan pigs (P < 0.01), whereas at 180 days, the abundance in Meishan was 3.4-fold higher than in Large White pigs (P < 0.01). These results suggest that the SRPK3 gene might be an important gene of skeletal muscle development and also provides basic molecular information useful for further studies on its roles in porcine skeletal muscle.     

Molecular characterization of the BTG2 and BTG3 genes in fetal muscle development of pigs

BTG2 and BTG3 are two members of the B-cell translocation gene family with anti-proliferative properties. BTG1 gene in this gene family has been reported to play a key role in muscle growth. In this study, we identified and characterized the porcine BTG2 and BTG3 genes, mapped the two genes to porcine chromosomes, and analyzed their expression differences in the longissimus dorci muscle of 33 dpc (day postconception), 65 dpc and 90 dpc in the lean Landrace and fatty Chinese Tongcheng pig breeds. Expression changes in differentiated C2C12 cells were also investigated with myogenin as internal control. The results showed that the porcine BTG2 and BTG3 genes were mapped on SSC9q21-25 and SSC13q47, respectively. BTG2 gene expressed at high levels in skeletal muscle and heart in both Tongcheng and Landrace pigs whereas BTG3 gene expressed at lower levels in skeletal muscle and heart than in other tissues. Furthermore, BTG3 expressed at higher levels in skeletal muscle of Tonghceng compared with Landrace pig. The expression of BTG2 and BTG3 was significantly different in skeletal muscle among different developmental stages and between the two breeds. Expression analysis in murine myoblast cells showed that both genes were induced in differentiated C2C12 cells, suggesting a role of them in myogenic differentiation. Our study indicated that BTG2 and BTG3, especially BTG3 gene, may be important genes for skeletal muscle growth.     

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.     

Molecular characterization,expression patterns and subcellular localization of Myotrophin (MTPN) gene in porcine skeletal muscle

Myotrophin (MTPN) is an effective growth factor in promoting skeletal muscle growth in vitro and vivo and has been purified from porcine skeletal muscle. However, in pigs, the information on MTPN gene is very limited. In this study, we cloned cDNA sequences and analyzed the genomic structure of porcine MTPN gene. The deduced amino acid sequence of porcine MTPN contains two the ankyrin repeat domains. RT-PCR analysis revealed that porcine MTPN gene was widely expressed in many tissues, a high expression level was observed in the spleen, liver and uterus, and transient transfection indicated that porcine MTPN proteins was located in cytoplasms within Pig Kidney Epithelial cells (PK15). Quantitative real-time PCR (qRT-PCR) analyses showed that MTPN expression peaked at embryonic 65 day post conception (dpc). During postnatal muscle development, MTPN expression was down-regulated from the 3 day to the 180 day in Yorkshire pigs. This result suggests that the MTPN gene may be important gene for skeletal muscle growth and provides useful information for further studies on its roles in porcine skeletal muscle.     

Molecular characterization of differentially expressed TXNIP gene and its association with porcine carcass traits

Thioredoxin interacting protein (TXNIP), which plays a regulatory role in lipid metabolism and immune regulation, is down-regulated expressed in F₁ hybrids Landrace?×?Yorkshire skeletal muscle. Here we described the molecular characterization of porcine TXNIP gene. The full-length cDNA contains a coding sequence of 1,176?bp nucleotides with untranslated regions of 263?bp at 5′-end and 441?bp at 3′-end, respectively. The predicted molecular mass and isoelectric point of porcine TXNIP is 43.81?kDa and 7.385, respectively. The deduced 391 amino acids exhibit high identity with other mammalian TXNIP. The TXNIP gene contains eight coding exons and seven non coding introns, spans approximately 3,348?bp. The expression of porcine TXNIP mRNA is almost absent in Landrace?×?Yorkshire and lower level in 6-month-old pigs during skeletal muscle development. Other stages and breeds were high level expressed. Statistical analysis showed the TXNIP gene polymorphism (c.575-4T>C) was different between F₁ hybrids and their parents, was highly associated with dressing percentage (DP) and thorax–waist fat thickness (TFT) in the Yorkshire?×?Meishan F₂ population. The possible role of TXNIP was discussed.     

Cloning of a differentially expressed tropomyosin isoform from cultured rabbit aortic smooth muscle cells

The four known tropomyosin genes have highly conserved DNA and amino acid sequences, and at least 18 isoforms are generated by alternative RNA splicing in muscle and non-muscle cells. No rabbit tropomyosin nucleotide sequences are known, although protein sequences for alpha- and beta-tropomyosin expressed by rabbit skeletal muscle have been described. Subtractive hybridisation was used to select for genes differentially expressed in rabbit aortic smooth muscle cells (SMC), during the change in cell phenotype in primary culture that is characterised by a loss of cytoskeletal filaments and contractile proteins. This led to the cloning of a tropomyosin gene predominantly expressed in rabbit SMC during this change. The full-length cDNA clone, designated "rabbit TM-beta", contains an open reading frame of 284 amino acids, 5' untranslated region (UTR) of 117 base pairs and 3' UTR of 79 base pairs. It is closely related to the beta-gene isoforms in other species, with the highest homology in DNA and protein sequences to the human fibroblast isoform TM-1 (91.7% identity in 1035 bp and 93.3% identity in the entire 284 amino acid sequence of the protein). It differs from rabbit skeletal muscle beta-tropomyosin (81.7% homology at the protein level) mainly in two regions at amino acids 189-213 and 258-283 suggesting alternative splicing of exons 6a for 6b and 9d for 9a. Since this TM-beta gene was the only gene strongly enough expressed in SMC changing phenotype to be observed by the subtractive hybridisation screen, it likely plays a significant role in this process.