Genomic cloning and promoter functional analysis of myostatin-2 in shi drum, Umbrina cirrosa: conservation of muscle-specific promoter activity.

Myostatin (MSTN) is a member of the transforming growth factor-ß superfamily, known as a negative regulator of skeletal muscle development and growth in mammals. In contrast to mammals, fish possess at least two paralogs of MSTN: MSTN-1 and MSTN-2. Here we describe the cloning and sequence analysis of spliced and precursor (unspliced) transcripts as well as the 5' flanking region of MSTN-2 from the marine fish Umbrina cirrosa (ucMSTN-2). In silico analysis revealed numerous putative cis regulatory elements including several E-boxes known as binding sites to myogenic transcription factors. Transient transfection experiments using non-muscle and muscle cell lines showed high transcriptional activity in muscle cells and in differentiated neural cells, in accordance with our previous findings in MSTN-2 promoter from Sparus aurata. Comparative informatics analysis of MSTN-2 from several fish species revealed high conservation of the predicted amino acid sequence as well as the gene structure (exon length) although intron length varied between species. The proximal promoter of MSTN-2 gene was found to be conserved among Perciforms. In conclusion, this study reinforces our conclusion that MSTN-2 promoter is a very strong promoter, especially in muscle cells. In addition, we show that the MSTN-2 gene structure is highly conserved among fishes as is the predicted amino acid sequence of the peptide.

The salmonid myostatin gene family: a novel model for investigating mechanisms that influence duplicate gene fate.

BACKGROUND: Most fishes possess two paralogs for myostatin, a muscle growth inhibitor, while salmonids are presumed to have four: mstn1a, mstn1b, mstn2a and mstn2b, a pseudogene. The mechanisms responsible for preserving these duplicates as well as the depth of mstn2b nonfunctionalization within the family remain unknown. We therefore characterized several genomic clones in order to better define species and gene phylogenies. RESULTS: Gene organization and sequence conservation was particularly evident among paralog groupings and within salmonid subfamilies. All mstn2b sequences included in-frame stop codons, confirming its nonfunctionalization across taxa, although the indels and polymorphisms responsible often differed. For example, the specific indels within the Onchorhynchus tshawytscha and O. nerka genes were remarkably similar and differed equally from other mstn2b orthologs. A phylogenetic analysis weakly established a mstn2b clade including only these species, which coupled with a shared 51 base pair deletion might suggest a history involving hybridization or a shared phylogenetic history. Furthermore, mstn2 introns all lacked conserved splice site motifs, suggesting that the tissue-specific processing of mstn2a transcripts, but not those of mstn2b, is due to alternative cis regulation and is likely a common feature in salmonids. It also suggests that limited transcript processing may have contributed to mstn2b nonfunctionalization. CONCLUSIONS: Previous studies revealed divergence within gene promoters while the current studies provide evidence for relaxed or positive selection in some coding sequence lineages. These results together suggest that the salmonid myostatin gene family is a novel resource for investigating mechanisms that regulate duplicate gene fate as paralog specific differences in gene expression, transcript processing and protein structure are all suggestive of active divergence.

[Identification and characterization of myostatin gene in rough-skinned sculp, Trachidermus fasciatus].

Myostatin is a member of the TGF-beta superfamily and acts as a negative regulator of skeletal muscle growth. The characterization of the myostatin gene and its expression in Trachidermus fasciatus was reported in the current study. A full-length of 2 568 bp myostatin cDNA sequence in T. fasciats was cloned by 5' and 3' RACE, which included a 1 131 bp complete ORF encoding a 376 amino acid peptide, a 106 bp long 5'-UTR and a 1331 bp long 3'UTR. As other MSTN, the putative peptide contains a 22 amino acids long signal peptide, a conserved RARR proteolytic processing site, and 10 conserved cysteine residues in the C terminal of the protein. The Trachidermus fasciatus MSTN has high homology with Umbrina cirrosa, Morone saxatilis, Morone americana, Morone chrysops myostatin while has low homology with mammalian and birds myostatin. The phylogenetic analysis showed that the T. fasciatus myostatin had the closest relationship with U. cirrosa. In the four examined tissues, the myostatin gene was highly expressed in muscle and intestine and weakly expressed in brain and liver. These results suggested that the fish myostatin gene might not only play roles in muscle development but also contribute to other biological functions.