Myostatin-2 gene structure and polymorphism of the promoter and first intron in the marine fish Sparus aurata: evidence for DNA duplications and/or translocations

Background

Myostatin (MSTN) is a member of the transforming growth factor-ß superfamily that functions as a negative regulator of skeletal muscle development and growth in mammals. Fish express at least two genes for MSTN: MSTN-1 and MSTN-2. To date, MSTN-2 promoters have been cloned only from salmonids and zebrafish.

Results

Here we described the cloning and sequence analysis of MSTN-2 gene and its 5' flanking region in the marine fish Sparus aurata (saMSTN-2). We demonstrate the existence of three alleles of the promoter and three alleles of the first intron. Sequence comparison of the promoter region in the three alleles revealed that although the sequences of the first 1050 bp upstream of the translation start site are almost identical in the three alleles, a substantial sequence divergence is seen further upstream. Careful sequence analysis of the region upstream of the first 1050 bp in the three alleles identified several elements that appear to be repeated in some or all sequences, at different positions. This suggests that the promoter region of saMSTN-2 has been subjected to various chromosomal rearrangements during the course of evolution, reflecting either insertion or deletion events. Screening of several genomic DNA collections indicated differences in allele frequency, with allele 'b' being the most abundant, followed by allele 'c', whereas allele 'a' is relatively rare. Sequence analysis of saMSTN-2 gene also revealed polymorphism in the first intron, identifying three alleles. The length difference in alleles '1R' and '2R' of the first intron is due to the presence of one or two copies of a repeated block of approximately 150 bp, located at the 5' end of the first intron. The third allele, '4R', has an additional insertion of 323 bp located 116 bp upstream of the 3' end of the first intron. Analysis of several DNA collections showed that the '2R' allele is the most common, followed by the '4R' allele, whereas the '1R' allele is relatively rare. Progeny analysis of a full-sib family showed a Mendelian mode of inheritance of the two genetic loci. No clear association was found between the two genetic markers and growth rate.

Conclusion

These results show for the first time a substantial degree of polymorphism in both the promoter and first intron of MSTN-2 gene in a perciform fish species which points to chromosomal rearrangements that took place during evolution.     

Myostatin (MSTN) gene duplications in Atlantic salmon (Salmo salar): evidence for different selective pressure on teleost MSTN-1 and -2

Whereas the negative muscle regulator myostatin (MSTN) in mammals is almost exclusively expressed in the muscle by a single encoding gene, teleost fish possess at least two MSTN genes which are differentially expressed in both muscular and non-muscular tissues. Duplicated MSTN-1 genes have previously been identified in the tetraploid salmonid genome. From Atlantic salmon we succeeded in isolating the paralogous genes of MSTN-2, which shared about 70% identity with MSTN-1a and -1b. The salmon MSTN-2a cDNA encoded a predicted protein of 363 residues and included the conserved C-terminal bioactive domain. MSTN-2a seemed to be primarily expressed in the brain, and a functional role of teleost MSTN-2 in the neurogenesis similar to the inhibitory action of the closely related GDF-11 in the mammalian brain was proposed. In contrast, a frame-shift mutation in exon 1 of salmon MSTN-2b would lead to the synthesis of a putatively non-functional truncated protein. The absence of processed MSTN-2b mRNA in the examined tissues indicated that this gene has become a non-functional pseudogene. The differential, but partially overlapping, expression patterns of salmon MSTN-2a, -1a and -1b in muscular and non-muscular tissues are probably due to the different arrangement of the potential cis-acting regulatory elements identified in their putative promoter regions. Single and paired E-boxes in the MSTN-1b promoter were shown to bind both homo-and hetero-dimers of the myogenic regulatory factor MyoD and E47 in vitro of importance for initiating the myogenic program. Analyses of nucleotide substitution patterns indicated that the teleost MSTNs essentially have evolved under purifying selection, but a subset of amino acid sites under positive selective pressure were identified within the MSTN1 branch. The results may reflect the evolutionary forces related to adoption of the different functional roles proposed for the teleost MSTN isoforms. The phylogenetic analysis of multiple vertebrate MSTNs suggested at least two separate gene duplication events in the fish lineage. Linkage analysis of polymorphic microsatellites within intron 2 of salmon MSTN-1a and -1b mapped the two genes to different linkage groups in agreement with the tetraploid origin of the duplicated salmonid MSTN-1 and MSTN-2 genes.     

Single nucleotide polymorphisms in the upstream regulatory region alter the expression of myostatin

The expression of the gene encoding myostatin (MSTN), the product of which is a negative regulator of skeletal muscle growth and development in mammals, is regulated by many cis-regulatory elements, including enhancer box (E-box) motifs. While E-box motif mutants of MSTN exhibit altered expression of myostatin in many animal models, the phenotypes of these mutations in chicken are not investigated. In this study, we cloned and sequenced the full encoded DNA sequence of MSTN gene and its upstream promoter region in Wenshang Luhua chicken breed. After analysis of the sequence, 13 E-box motifs were identified in the MSTN promoter region, which were denoted by E1 to E13 according to their positions in the region. Although many single nucleotide polymorphisms (SNPs) were revealed in the MSTN promoter region, only two SNPs were in the E-boxes, i.e., the first nucleotide of the E3 and the fifth nucleotide of E4. The effects of these two polymorphisms on the expression of MSTN gene were explored both with MSTN-GFP reporter constructs in vitro and real-time PCR in vivo. The results suggested that the E-boxes in the chicken MSTN promoter region are involved in the regulation of myostatin expression and the polymorphisms in E3 and E4 altered the expression of myostatin.     

Phylogenetic analysis of the myostatin gene sub-family and the differential expression of a novel member in zebrafish

The myostatin (MSTN)-null phenotype in mammals is characterized by extreme gains in skeletal muscle mass or "double muscling" as the cytokine negatively regulates skeletal muscle growth. Recent attempts, however, to reproduce a comparable phenotype in zebrafish have failed. Several aspects of MSTN biology in the fishes differ significantly from those in mammals and at least two distinct paralogs have been identified in some species, which possibly suggests functional divergence between the different vertebrate classes or between fish paralogs. We therefore conducted a phylogenetic analysis of the entire MSTN gene sub-family. Maximum likelihood, Bayesian inference, and bootstrap analyses indicated a monophyletic distribution of all MSTN genes with two distinct fish clades: MSTN-1 and -2. These analyses further indicated that all Salmonid genes described are actually MSTN-1 orthologs and that additional MSTN-2 paralogs may be present in most, if not all, teleosts. An additional zebrafish homolog was identified by BLAST searches of the zebrafish Hierarchical Tets Generation System database and was subsequently cloned. Comparative sequence analysis of both genes (zebrafish MSTN (zfMSTN)-1 and -2) revealed many differences, primarily within the latency-associated peptide regions, but also within the bioactive domains. The 2-kb promoter region of zfMSTN-2 contained many putative cis regulatory elements that are active during myogenesis, but are lacking in the zfMSTN-1 promoter. In fact, zfMSTN-2 expression was limited to the early stages of somitogenesis, whereas zfMSTN-1 was expressed throughout embryogenesis. These data suggest that zfMSTN-2 may be more closely associated with skeletal muscle growth and development. They also resolve the previous ambiguity in classification of fish MSTN genes.     

Overexpression of the dominant-negative form of myostatin results in doubling of muscle-fiber number in transgenic medaka (Oryzias latipes)

In addition to altering the phenotypes of gene-modified animals, transgenesis also has the potential to facilitate access to the various mechanisms underlying the development and functioning of specific phenotypes and genes, respectively. Myostatin (MSTN) is implicated in double-muscling when mutated in mammals, indicating that MSTN is a negative regulator of skeletal muscle formation. In order to elucidate the role of an MSTN equivalent in fish muscle formation, we created a transgenic medaka strain that expresses dominant-negative MSTN exclusively in skeletal muscle, d-rR-Tg(OlMA1–C315Y–MSTN–hrGFPII–FLAG). The transgenic fish exhibited increased production of skeletal muscle fibers at the adult stage (hyperplasia), although gross muscle mass was not altered. During embryogenesis, ectopic accumulation and misalignment of muscle fibers, possibly due to muscle-fiber hypertrophy, were observed in the transgenic medaka. Our findings suggest that MSTN function is required for regulating the appropriate growth of skeletal muscle in medaka. Unlike in mammals, MSTN loss-of-function failed to induce double-muscling in medaka, despite the highly conserved nature of MSTN function among taxa.     

Molecular cloning,prokaryotic expression and promoter analysis of squalene synthase gene from Schizochytrium Limacinum

Squalene synthase (SQS) is an important enzyme in the steroid biosynthetic pathways which condenses two molecules of farnesyl pyrophosphate into a squalene. In this study, the gene encoding SQS was isolated from Schizochytrium limacinum and characterized. The full-length cDNA of S. limacinum SQS gene (SlSQS) is 1605 bp in length, it contains a 1293 bp ORF encoding a polypeptide of 430 amino acids. Multiple amino acid sequence alignment showed that the SlSQS protein sequence shared 5 conserved signature domains and a hydrophobic carboxy-terminal part with other known SQS protein sequences. C-terminal-truncated SlSQS was constructed into expression vector pGEX and successfully expressed in Escherichia coli cells. The expressed fusion protein was confirmed to have SQS activity. In addition, a 724 bp promoter region of SlSQS was also cloned and several cis-acting elements were predicted. These results might be helpful to understand the structure and expression regulation of SQS in S. limacinum.     

Molecular Cloning and Expression Analysis of the Myostatin Gene in Sea Perch (<Emphasis Type="Italic">Lateolabrax japonicus</Emphasis>)

Myostatin (MSTN) is a member of the transforming growth factor-β (TGF-β) superfamily that functions as a negative regulator of skeletal muscle development and growth in mammals. However, few reports are available about the structure and function of MSTN in teleost. Here, the MSTN gene was cloned from sea perch (Lateolabrax japonicus) by homology cloning and genomic walking. In the 4873-bp genomic sequence, three exons, two introns, and 5′ and 3′ flanking sequences were identified. The sea perch MSTN gene encodes a 374-amino acid protein, including a signal peptide, conserved cysteine residues, and a RXXR proteolytic cleavage domain. Expression analysis of MSTN revealed that MSTN was highly expressed in eyes, brain, and muscle; intermediately in intestine; and weakly in gill, spleen, liver, and heart. It was demonstrated that MSTN mRNA was highly expressed in embryonic stem cell line (LJES1), but it was undetectable in several types of somatic cell lines from sea perch, including fibroblast-like cell, epithelioid cell, and lymphocyte-like cell. Further, it was demonstrated that the 5′ flanking region of the MSTN gene can drive the expression of green fluorescent protein (GFP) reporter gene in LJES1 cells and transgenic zebrafish (Danio rerio). This is the first report on the expression profile of MSTN gene in various types of cell cultures.     

A SNP in the 5′ flanking region of the myostatin-1b gene is associated with harvest traits in Atlantic salmon (Salmo salar)

Background

Myostatin (MSTN) belongs to the transforming growth factor-β superfamily and is a potent negative regulator of skeletal muscle development and growth in mammals. Most teleost fish possess two MSTN paralogues. However, as a consequence of a recent whole genome-duplication event, salmonids have four: MSTN-1 (?1a and -1b) and MSTN-2 (?2a and -2b). Evidence suggests that teleost MSTN plays a role in the regulation of muscle growth. In the current study, the MSTN-1b gene was re-sequenced and screened for SNP markers in a commercial population of Atlantic salmon. After genotyping 4,800 progeny for the discovered SNPs, we investigated their association with eight harvest traits - four body-weight traits, two ratios of weight traits, flesh colour and fat percentage - using a mixed model association analysis.

Results

Three novel SNPs were discovered in the MSTN-1b gene of Atlantic salmon. One of the SNPs, located within the 5′ flanking region (g.1086C?>?T), had a significant association with harvest traits (p?<?0.05), specifically for: Harvest Weight (kg), Gutted Weight (kg), Deheaded Weight (kg) and Fillet Weight (kg). The haplotype-based association analysis was consistent with this result because the two haplotypes that showed a significant association with body-weight traits, hap4 and hap5 (p?<?0.05 and p?<?0.01, respectively), differ by a single substitution at the g.1086C?>?T locus. The alleles at g.1086C?>?T act in an additive manner and explain a small percentage of the genetic variation of these phenotypes.

Conclusions

The association analysis revealed that g.1086C?>?T had a significant association with all body-weight traits under study. Although the SNP explains a small percentage of the variance, our results indicate that a variation in the 5′ flanking region of the myostatin gene is associated with the genetic regulation of growth in Atlantic salmon.

     

The effect of leader peptide mutations on the biological function of bovine myostatin gene

The growth of muscle fibers can be negatively regulated by bovine myostatin. The first two exons of myostatin gene code for the N-propeptide and its third exon codes for the C-polypeptide. Myostatin is secreted as a latent configuration formed by dimerization of two matured C peptides non-covalently linked with the N terminal pro-peptide. Pro-peptide has two distinct functions in guiding protein folding and regulating biological activity of myostatin. When the structure of the leader peptide is altered via mutations resulting in more tight binding with the mature peptide, myostatin function is inhibited, resulting in the changes of P21 and CDK2 expression levels which are relatedto the regulation of cell cycle. In the present study, the coding region of bMSTN (bovine myostatin) gene was amplified and mutated (A224C and G938A) through fusion PCR, and the mutated bMSTN gene (bMSTN-mut) was inserted in frame into the pEF1a-IRES-DsRed-Express2 vector and transfected into bovine fibroblast cells. The expression levels of bMSTN-mut, P21 and CDK2 (cyclin dependent kinase 2) were examined with qPCR and Western-blotting. Changes in cell cycle after transfection were also analyzed with flow cytometry. The results indicated that leader peptide mutation resulted in down-regulation of P21 expression levels and up-regulation of CDK2 expression levels. The flow cytometry results showed that the proportion of cells in the G0/G1-phase was lower and that of cells in the S-phase was higher in bMSTN-mut transfected group than that in the control group. The proliferation rate of bMSTN-mut transfected cells was also significantly higher than that of the control cells. In conclusion, the studies have shown that the pEF1a-IRES-DsRed-Express2–bMSTN-mut recombinant plasmid could effectively promote the proliferation of bovine fibroblast cells. The site-directed mutagenesis of bMSTN gene leader peptide and in vitro expression in bovine fibroblast cells could be helpful to further the studies of bMSTN in regulating bovine muscle cell growth and development.     

Phylogenesis and Biological Characterization of a New Glucose Transporter in the Chicken (Gallus gallus), GLUT12

In mammals, insulin-sensitive GLUTs, including GLUT4, are recruited to the plasma membrane of adipose and muscle tissues in response to insulin. The GLUT4 gene is absent from the chicken genome, and no functional insulin-sensitive GLUTs have been characterized in chicken tissues to date. A nucleotide sequence is predicted to encode a chicken GLUT12 ortholog and, interestingly, GLUT12 has been described to act as an insulin-sensitive GLUT in mammals. It encodes a 596 amino acid protein exhibiting 71% identity with human GLUT12. First, we present the results of a phylogenetic study showing the stability of this gene during evolution of vertebrates. Second, tissue distribution of chicken SLC2A12 mRNA was characterized by RT-PCR. It was predominantly expressed in skeletal muscle and heart. Protein distribution was analysed by Western blotting using an anti-human GLUT12 antibody directed against a highly conserved region (87% of identity). An immuno-reactive band of the expected size (75kDa) was detected in the same tissues. Third a physiological characterization was performed: SLC2A12 mRNA levels were significantly lowered in fed chickens subjected to insulin immuno-neutralization. Finally, recruitment of immuno-reactive GLUT12 to the muscle plasma membrane was increased following 1h of intraperitoneal insulin administration (compared to a control fasted state). Thus insulin administration elicited membrane GLUT12 recruitment. In conclusion, these results suggest that the facilitative glucose transporter protein GLUT12 could act in chicken muscle as an insulin-sensitive transporter that is qualitatively similar to GLUT4 in mammals.     

Evolutionarily conserved and divergent regulatory sequences in the fish rod opsin promoter

Fish have multiple types and subtypes of opsin genes that are expressed in a highly regulated manner in retinal photoreceptor cells. In the rod opsin proximal promoter region (RPPR) of zebrafish (Danio rerio), the BAT 1 regulatory region contains highly conserved OTX (GATTA) and OTX-like (TATTA) sequences that can be recognized by the mammalian cone–rod homeobox (CRX) protein. However, binding of zebrafish crx to the OTX sequence has remained elusive. In contrast to the BAT 1 region, the Ret 1 region, located approximately 20 bp upstream of the BAT 1 region in mammals, is not conserved in zebrafish. In the Ret 1 region, even the core OTX-like sequence (AATTA sequence in mammals) is destructed. We show in this study that a region between Ret 1 and BAT 1 (denoted IRB, Inter-Ret 1-BAT 1) is highly conserved among fish species. Using electrophoretic mobility shift assay (EMSA), we show that zebrafish crx binds to the conserved OTX sequence and that the fish-specific IRB region specifically binds elements present in both retinal and brain nuclear extracts of zebrafish. These results imply that the regulatory mechanisms of opsin gene expression consist not only of evolutionarily conserved but also of divergent machinery among different animal taxa.     

Neurexophilin 1 Gene Polymorphism in Chickens and Its Variation Among Species

Neurexophilin 1 (nxph1) has been considered a potential candidate marker for sperm storage in chicken sperm storage tubules. In this work, one mutation of chicken nxph1 was detected. We analyzed 18 nxph1 gene sequences from 18 species. The coding sequence length of the zebra fish nxph1 gene is 819 bp; that of the other species is 816 bp. Amino acid alignment analysis revealed that the gene product is a conserved protein, especially in mammals. The sequences of mammals are highly conserved. We found 202 conserved amino acids (70–271), and there were only eight mutations in the remaining 69 amino acids. That level of conservation could be due to the nxph1 gene having been subjected to substantial constraints or strong purifying selection during millions of years of evolution.