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

<p>Abstract</p> <p>Background</p> <p>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 <it>MSTN</it>: <it>MSTN-1 </it>and <it>MSTN-2</it>. To date, <it>MSTN-2 </it>promoters have been cloned only from salmonids and zebrafish.</p> <p>Results</p> <p>Here we described the cloning and sequence analysis of <it>MSTN-2 </it>gene and its 5' flanking region in the marine fish <it>Sparus aurata </it>(sa<it>MSTN-2</it>). 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 sa<it>MSTN-2 </it>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 sa<it>MSTN-2 </it>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.</p> <p>Conclusion</p> <p>These results show for the first time a substantial degree of polymorphism in both the promoter and first intron of <it>MSTN-2 </it>gene in a perciform fish species which points to chromosomal rearrangements that took place during evolution.</p

All-fish gene constructs for growth hormone gene transfer in fish

In order to develop all-fish expression vectors for microinjection into fertilized fish eggs, we have prepared the following cunstructs: rainbow trout metallothionein a/b and the gilthead seabream growth hormone cDNA (ptMTa-gbsGHcDNA, ptMTb-gsbGHcDNA), carp ß-actin gilthead seabream GH cDNA (pcAßgsbGHcDNA). The inducible metallothionein promoters a and b were cloned from rainbow trout, and the constitutive promoter ß-actin was isolated from carp. The metallothionein promoters were cloned by using the PCR technique. The tMTa contains 430 bp, while the tMTb contains 260 bp (Hong et al. 1992). These two promoters were introduced to pGEM-3Z containing the GH cDNA of Sparus aurata to form ptMTa-gsbGH and ptMTb-gsbGH, respectively. The carp cytoplasmic ß-actin gene was chosen as a source for isolating strong constitutive regulatory sequences. One of these regulatory sequences in pUC118 was Iigated to GH cDNA of S. aurata to form the pcAß-gsbGHcDNA. Expression of the constructs containing the metallothionein promoters was tested in fish cell culture and was found tobe induced effectively by zinc. The ptMTa gsb-GH cDNA construct was microinjected into fertilized carp eggs, and integration in the genome of carp was detected in the DNA isolated from fins at the age of two months

Identification of a novel transforming growth factor-β (TGF-β6) gene in fish: regulation in skeletal muscle by nutritional state

<p>Abstract</p> <p>Background</p> <p>The transforming growth factor-β (TGF-β) family constitutes of dimeric proteins that regulate the growth, differentiation and metabolism of many cell types, including that of skeletal muscle in mammals. The potential role of TGF-βs in fish muscle growth is not known.</p> <p>Results</p> <p>Here we report the molecular characterization, developmental and tissue expression and regulation by nutritional state of a novel TGF-β gene from a marine fish, the gilthead sea bream <it>Sparus aurata</it>. <it>S. aurata </it>TGF-β6 is encoded by seven exons 361, 164, 133, 111, 181, 154, and 156 bp in length and is translated into a 420-amino acid peptide. The exons are separated by six introns: >643, 415, 93, 1250, 425 and >287 bp in length. Although the gene organization is most similar to mouse and chicken TGF-β2, the deduced amino acid sequence represents a novel TGF-β that is unique to fish that we have named TGF-β6. The molecule has conserved putative functional residues, including a cleavage motif (RXXR) and nine cysteine residues that are characteristic of TGF-β. Semi-quantitative analysis of TGF-β6 expression revealed differential expression in various tissues of adult fish with high levels in skin and muscle, very low levels in liver, and moderate levels in other tissues including brain, eye and pituitary. TGF-β6 is expressed in larvae on day of hatching and increases as development progresses. A fasting period of five days of juvenile fish resulted in increased levels of TGF-β6 expression in white skeletal muscle compared to that in fed fish, which was slightly attenuated by one injection of growth hormone.</p> <p>Conclusion</p> <p>Our findings provide valuable insights about genomic information and nutritional regulation of TGF-β6 which will aid the further investigation of the <it>S. aurata </it>TGF-β6 gene in association with muscle growth. The finding of a novel TGF-β6 molecule, unique to fish, will contribute to the understanding of the evolution of the TGF-β family of cytokines in vertebrates.</p

Ontogeny of responsiveness to gonadotrophins and prostaglandin E in the neonatal rat ovary

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