Protein-protein interaction of FHL3 with FHL2 and visualization of their interaction by green fluorescent proteins (GFP) two-fusion fluorescence resonance energy transfer (FRET)

LIM domain proteins are found to be important regulators in cell growth, cell fate determination, cell differentiation and remodeling of the cell cytoskeleton. Human Four-and-a-half LIM-only protein 3 (FHL3) is a type of LIM-only protein that contains four tandemly repeated LIM motifs with an N-terminal single zinc finger (half LIM motif). FHL3 expresses predominantly in human skeletal muscle. In this report, FHL3 was shown to be a novel interacting partner of FHL2 using the yeast two-hybrid assay. Furthermore, site-directed mutagenesis of FHL3 indicated that the LIM2 of FHL3 is the essential LIM domain for interaction with FHL2. Green fluorescent protein (GFP) was used to tag FHL3 in order to study its distribution during myogenesis. Our result shows that FHL3 was localized in the focal adhesions and nucleus of the cells. FHL3 mainly stayed in the focal adhesion during myogenesis. Moreover, using site-directed mutagenesis, the LIM1 of FHL3 was identified as an essential LIM domain for its subcellular localization. Mutants of GFP have given rise to a novel technique, two-fusion fluorescence resonance energy transfer (FRET), in the determination of protein-protein interaction at particular subcellular locations of eukaryotic cells. To determine whether FHL2 and FHL3 can interact with one another and to locate the site of this interaction in a single intact mammalian cell, we fused FHL2 and FHL3 to different mutants of GFP and studied their interactions using FRET. BFP/GFP fusion constructs were cotransfected into muscle myoblast C2C12 to verify the colocalization and subcellular localization of FRET. We found that FHL2 and FHL3 were colocalized in the mitochondria of the C2C12 cells and FRET was observed by using an epi-fluorescent microscope equipped with an FRET specific filter set.     

Genetic Variation and Association of Insulin-Like Growth Factor Binding Protein-3 with Performance in Swine

Genetic variation of insulin-like growth factor binding protein-3 was analyzed in 17 pig breeds (14 native Chinese and 3 European). Using PCR-single strand conformation polymorphism, we found a polymorphism in intron 2, and this SNP was the combined mutation of G897T-G903A-C911T. The Chinese breeds carried a higher TAT/TAT genotype frequency (over 50%), except for Bamei (22%), Yujiang Black (0.0%), and Erhualian (10.0%); the European breeds had a higher GGC/GGC genotype frequency (Large White 1.67%, Landrace 13.89%, Duroc 0.0%). The allelic frequency of TAT in Chinese breeds was over 50%, except for Yujiang Black (12.5%); the allelic frequency of GGC was over 50% in all European breeds. The effect of genotype on 43 performance traits was investigated in one population (Lantang × Landrace). Pigs with the TAT/TAT genotype had higher B-point and C-point back-fat thickness than pigs with the GGC/GGC genotype. The TAT/TAT pigs also scored higher in meat color than the GGC/GGC pigs. These results implied that IGFBP-3 may affect meat quality and carcass traits.     

The LIM-only protein DRAL/FHL2 binds to the cytoplasmic domain of several alpha and beta integrin chains and is recruited to adhesion complexes

LIM proteins contain one or more double zinc finger structures (LIM domains) mediating specific contacts between proteins that participate in the formation of multiprotein complexes. We report that the LIM-only protein DRAL/FHL2, with four and a half LIM domains, can associate with alpha(3A), alpha(3B), alpha(7A), and several beta integrin subunits as shown in yeast two-hybrid assays as well as after overexpression in human cells. The amino acid sequence immediately following the conserved membrane-proximal region in the integrin alpha subunits or the C-terminal region with the conserved NXXY motif of the integrin beta subunits are critical for binding DRAL/FHL2. Furthermore, the DRAL/FHL2 associates with itself and with other molecules that bind to the cytoplasmic domain of integrin alpha subunits. Deletion analysis of DRAL/FHL2 revealed that particular LIM domains or LIM domain combinations bind the different proteins. These results, together with the fact that full-length DRAL/FHL2 is found in cell adhesion complexes, suggest that it is an adaptor/docking protein involved in integrin signaling pathways.     

LIM-only protein FHL3 interacts with CDC25B2 phosphatase

LIM domain proteins are important regulators of the growth, determination, and differentiation of cells. In this report, FHL3 (human four-and-a-half LIM-only protein 3) is shown to interact with human phosphatase CDC25B, a cell cycle regulator involved in the control of G2/M. We found that this interaction was specific to the CDC25B2 isoform. Deletion and point mutation studies indicated that the second LIM domain of FHL3 was essential for this interaction. FRET experiments in C2C12 cells showed that, although both proteins were colocated in the cytoplasm and the nucleus, they interacted only in the nucleus. Finally, we showed that FHL3 binding impaired neither CDC25B2 phosphatase activity nor its localization. Further work is now needed to elucidate the consequences of this interaction on myoblast fate decision and cycle control.     

Porcine growth differentiation factor 9 gene polymorphisms and their associations with litter size

Growth differentiation factor 9 (GDF9) is expressed in oocytes and is thought to be required for ovarian folliculogenesis.Given this function,GDF9 may be considered as a candidate gene controlling pig ovulate rate.In this study,the complete coding sequence was cloned (encoding a 444 amino acid),intron sequence and partial 5'-UTR of pig GDF9.RT-PCR results showed that GDF9 mRNA is expressed in a wide range of tissues of the ruttish Erhualian pig.The expression levels of GDF9 mRNA in pituitary,ovary,uterus and oviduct are higher in the Erhualian pigs than those in Duroc pigs,especially in pituitary with a significant difference (P＜0.05).Comparative sequencing revealed 12 polymorphisms,including 8 single nucleotide polymorphisms (SNPS) and one 314 bp indel in noncoding regions,and the other 3 SNPS in coding regions.Four polymorphisms,G359C,C1801T,T1806C and 314 bp indel,were developed as markers for further use in population variation and association studies.The G359C polymorphism segregates only in Chinese native pigs,Erhualian and Dahuabai,on the contrary,314 bp indel segregates only in Duroc and Landrace.C1801T and T1806C sites seem to be completely linked and segregate in Erhualian,Dahuabai and Landrace.In a word,GDF9 may be not associated with pig litter size in extensive populations as per the studies of allele distributions of the four polymorphisms and pilot association in four breeds.     

New SNP of the porcine Perilipin 2 (<Emphasis Type="Italic">PLIN2</Emphasis>) gene,association with carcass traits and expression analysis in skeletal muscle

PLIN2 (perilipin 2) is a cytosolic protein that promotes the formation and stabilization of the intracellular lipid droplets, organelles involved in the storage of lipid depots. Porcine PLIN2 gene represents a biological and positional candidate for fat deposition, a polygenic trait that affects carcass and meat quality. The aim of the present study was to screen PLIN2 gene for polymorphisms, to evaluate the association with carcass quality traits, and to investigate the gene expression in skeletal muscle. Six new single nucleotide polymorphisms (SNP) were detected by sequencing 32 samples from five pig breeds (Italian Large White, Italian Duroc, Italian Landrace, Belgian Landrace, Pietrain). Two SNP localized in introns, two in the 3′-untranslated region (UTR), and two missense SNP were found in exons. A 3′-UTR mutation (GU461317:g.98G>A), genotyped in 290 Italian Duroc pigs by High Resolution Melting, resulted significantly associated (P < 0.01) with average daily gain, feed conversion ratio, lean cuts and hams weight estimated breeding values. PLIN2 gene expression analysis in skeletal muscle of Italian Large White and Italian Duroc pigs divergent for backfat thickness and visible intermuscular fat showed a trend of higher expression level in pigs with higher intermuscular fat. These results suggest that PLIN2 can be a marker for carcass quality in pigs. Further investigation at both gene and protein level could elucidate its role on fat deposition.     

猪背最长肌中肌肉生长和脂肪沉积相关基因的差异表达和主成分分析

骨骼肌细胞和脂肪细胞在分化生长速度上相对竞争的平衡点是猪肉质和胴体性状的决定因素.利用Oligo功能分类芯片检测了瘦肉型的长白猪和脂肪型的太湖猪在初生、1、2、3、4和5月龄间背最长肌中肌肉生长和脂肪沉积相关基因的动态表达变化.差异表达分析结果显示,在初生至5月龄的品种间分别有15、16、11、13、18和20个基因的表达差异倍数大于2倍.品种内的方差分析表明,长白猪分别有18和22个基因,太湖猪分别有3和7个基因在月龄间的表达差异达极显著(Ｐ<0.01)和显著水平(Ｐ<0.05).主成分分析结果显示,先降后升是两品种内最具代表性的基因表达模式,且长白猪和太湖猪分别有7和6个基因的表达模式明显偏离其他基因,提示其可能受到了重要的调控. 此外,5个差异表达基因的荧光定量RT-PCR验证结果均与芯片结果呈正相关趋势.以上结果筛选出了对于猪肉质和胴体性状可能具有重要影响,值得深入研究的一些候选基因,为深入研究生长发育过程中参与肌纤维生长和脂肪酸合成关键基因的表达变化规律和互作调控机制提供了基础数据.     

Association of corticotropin-releasing hormone gene variation with performance and meat quality traits in commercial pig lines

The porcine corticotropin-releasing hormone(CRH) gene is a functional-positional candidate for quantitative tract loci on porcine chromosome 4 with major effects on growth and carcass composition. In addition, the central role of CRH in the neuroendocrine response to stress implicates the CRH gene as a functional candidate for meat quality. Association of a single nucleotide polymorphism (SNP) in the promoter region of the porcine CRH gene (g.233C > T) with several growth, carcass and meat quality traits was examined using more than 2000 individuals from four commercial lines: German Landrace (LR), Pietrain (Pi), German Large White x German Landrace (F1) and the German commercial fattening pig cross of Pietrain x F1 (PiF1). Significant association of the CRH SNP was found with feed conversion ratio in the PiF1 line, with carcass length in the LR line and with lean content in the F1, LR and Pi lines. Moreover, significant association with meat colour was found in the Pi and LR lines; however, the effects were in opposite directions. The presented results indicate that sequence variation in the porcine CRH gene has no major effect on growth and carcass composition in commercial pig lines, although it may significantly contribute to variation in meat quality. The g.233C>T SNP may be in incomplete linkage disequilibrium with causal mutations and/or exhibit effects in the context of DNA variation at other interacting loci.     

Chromosomal mapping,tissue distribution and cDNA sequence of Four-and-a-half LIM domain protein 1 (FHL1)

We have isolated and sequenced a human heart cDNA clone encoding a novel LIM-only protein. This full-length cDNA clone has a predicted open reading frame (ORF) encoding 280 amino acids. The ORF of this cDNA codes for a LIM-only protein that possesses four repeats of LIM domain and an extra zinc finger and this putative protein is named four-and-a-half LIM domain protein 1 (FHL1). FHL1 is unique when compared with other LIM-only proteins because it possesses an odd number of zinc fingers. When the FHL1 cDNA probe was used to hybridize with poly-(A) RNA of various human tissues, a very strong signal was detected in skeletal muscle, a moderate one in the heart; only weak signals were associated with the placenta, ovary, prostate, testis, small intestine, colon and spleen, and virtually no signal could be detected in brain, lung, liver, kidney, pancreas, thymus and peripheral blood leukocytes. The FHL1 gene was located to human chromosome at Xq27.2 by somatic cell hybrid mapping, fluorescent in situ hybridization (FISH) and radiation hybrid mapping.     

FHL家族与肿瘤的关系

FHL（four and half LIM domains）是LIM-only蛋白家族的重要成员。FHL家族包括FHLl-FHL5共5个成员,它们具有组织特异性。研究发现,FHL参与转录调节、信号转导、凋亡等,是细胞生长、分化的重要调节因子。已证实FHLl、FHL2与不同组织来源肿瘤的发生和发展有关。FHL表达水平是某些肿瘤预后的相关因素。研究FHL与肿瘤的关系,有利于阐明肿瘤的病理生理机制。     

Translocation of a human focal adhesion LIM-only protein, FHL2, during myofibrillogenesis and identification of LIM2 as the principal determinants of FHL2 focal adhesion localization

LIM domain proteins are found to be important regulators in cell growth, cell fate determination, cell differentiation, and remodeling of the cell cytoskeleton. Human Four-and-a-half LIM-only protein 2 (FHL2) is expressed predominantly in human heart and is only slightly expressed in skeletal muscle. Since FHL2 is an abundant protein in human heart, it may play an important role in the regulation of cell differentiation and myofibrillogenesis of heart at defined subcellular compartment. Therefore, we hypothesized that FHL2 act as a multi-functional protein by the specific arrangement of the LIM domains of FHL2 and that one of the LIM domains of FHL2 can function as an anchor and localizes it into a specific subcellular compartment in a cell type specific manner to regulate myofibrillogenesis. From our results, we observed that FHL2 is localized at the focal adhesions of the C2C12, H9C2 myoblast as well as a nonmyogenic cell line, HepG2 cells. Colocalization of vinculin-CFP and FHL2-GFP at focal adhesions was also observed in cell lines. Site-directed mutagenesis, in turn, suggested that the second LIM domain-LIM2 is essential for its specific localization to focal adhesions. Moreover, FHL2 was observed along with F-actin and focal adhesion of C2C12 and H9C2 myotubes. Finally, we believe that FHL2 moves from focal adhesions and then stays at the Z-discs of terminally differentiated heart muscle.     

Malic enzyme 1 genotype is associated with backfat thickness and meat quality traits in pigs

Malic enzyme 1 (ME1) is a part of the tricarboxylate shuttle that provides NADPH and acetyl-CoA required in fatty acid biosynthesis. The pig ME1 locus maps on the proximal end of chromosome 1, where a quantitative trait loci (QTL) affecting fat deposition has been previously described. We amplified fragments of 1457 and 1459 bp that corresponded to the complete coding region and the 3'-untranslated region (UTR), respectively, of the pig ME1 gene. The sequences of these two fragments in pigs from three breeds (Landrace, Large White and Piétrain) contained five single nucleotide polymorphisms (SNP) in the 3'-UTR: C1706T, G1762T, A1807C, C1857A and T1880A. Three haplotypes were found in two generations of a selected Landrace population: H1 (C1706 G1762 A1807 C1857 A1880), H2 (C1706 G1762 A1807 C1857 T1880) and H3 (T1706 T1762 C1807 A1857 T1880). Using Bayesian association analyses, significant associations (highest posterior density at 95%) between ME1 genotype and backfat (BF) thickness at 171 days and muscular pH were found in a Landrace population.     

山猪群体钙调蛋白酶抑制蛋白基因遗传变异研究

目的：研究猪钙调蛋白酶抑制蛋白（CAST）基因在山猪群体的遗传变异情况,为山猪肉质研究奠定基础。方法：应用PCR-SSCP技术和测序方法检测山猪及其杂种猪CAST基因的遗传多态性,并与其他品种猪相应序列进行比较。结果：用pCT1引物在山猪及其杂种群体中检测到2个多态位点（A,B）,用pCT2引物检测到3个多态位点（C,D,E）;序列分析表明,C和E位点共同拥有6处变异。结论：通过与其他猪品种比较,发现山猪的CASTMsp基因型分布与梅山猪的基因型分布完全一样,而与国外品种猪的基因型分布差异明显。     

An X-linked myopathy with postural muscle atrophy and generalized hypertrophy, termed XMPMA, is caused by mutations in FHL1

We have identified a large multigenerational Austrian family displaying a novel form of X-linked recessive myopathy. Affected individuals develop an adult-onset scapulo-axio-peroneal myopathy with bent-spine syndrome characterized by specific atrophy of postural muscles along with pseudoathleticism or hypertrophy and cardiac involvement. Known X-linked myopathies were excluded by simple-tandem-repeat polymorphism (STRP) and single-nucleotide polymorphism (SNP) analysis, direct gene sequencing, and immunohistochemical analysis. STRP analysis revealed significant linkage at Xq25-q27.1. Haplotype analysis based on SNP microarray data from selected family members confirmed this linkage region on the distal arm of the X chromosome, thereby narrowing down the critical interval to 12 Mb. Sequencing of functional candidate genes led to the identification of a missense mutation within the four and a half LIM domain 1 gene (FHL1), which putatively disrupts the fourth LIM domain of the protein. Mutation screening of FHL1 in a myopathy family from the UK exhibiting an almost identical phenotype revealed a 3 bp insertion mutation within the second LIM domain. FHL1 on Xq26.3 is highly expressed in skeletal and cardiac muscles. Western-blot analysis of muscle biopsies showed a marked decrease in protein expression of FHL1 in patients, in concordance with the genetic data. In summary, we have to our knowledge characterized a new disorder, X-linked myopathy with postural muscle atrophy (XMPMA), and identified FHL1 as the causative gene. This is the first FHL protein to be identified in conjunction with a human genetic disorder and further supports the role of FHL proteins in the development and maintenance of muscle tissue. Mutation screening of FHL1 should be considered for patients with uncharacterized myopathies and cardiomyopathies.     

Molecular Cloning of Porcine SUN5 Gene and Association between a SNP with Litter Size Trait

SUN domain-containing protein 5 (SUN5) is an important reproduction related gene. In this study, we cloned the full-length coding sequence of porcine SUN5 gene through RT-PCR. Sequence analysis of this gene revealed that the pig SUN5 gene encodes a protein of 383?amino acids that has high homology with the SUN5 protein of eight species: wild Bactrian camel (95%), alpaca (95%), Yangtze River dolphin (94%), sperm whale (94%), sheep (93%), black flying fox (93%), goat (92%), and horse (91%). This gene is structured into 13 exons and 12 introns as revealed by computer-assisted analysis. The prediction of transmembrane helices showed that pig SUN5 protein might be a transmembrane protein. PCR-Taq I-RFLP was established to detect the GU475008:c.138 G>A substitution of porcine SUN5 gene coding sequence and eight pig breeds displayed obvious genotype and allele frequency differences at this mutation locus. Association of this SNP with litter size traits was assessed in Large White (n?=?200) and Landrace (n?=?200) pig populations, and the results demonstrated that this polymorphic locus was significantly associated with the litter size of all parities in Large White and Landrace sows (P?相似文献