We report the cloning of a new trout myogenic cDNA which encodes helix-loop-helix protein homologous to the myogenic factor myogenin. Northern analyses indicate that trout myogenin (Tmyogenin) transcripts accumulate in large amounts in the myotomal musculature of embryos and frys. In adults, transcripts concentrate within the thin lateral layer of red (slow oxydative) muscle fibres. They are present only in low amounts in white (fast glycolytic) muscle fibres which constitute the major part of the trunk musculature. Using an in vitro myogenesis system, we observed that the trout myogenin encoding gene is not activated until myosatellite cells fuse to generate multinocleated myotubes, indicating that Tmyogenin lies downstream of muscle determination factors. All these observations show that in a major taxinomic group like teleosts, a gene with homology to myogenin exists. Its activation during myogenesis suggests that it acts as a major developmental regulator of muscle differentiation.
We have isolated the cDNA encoding a myogenic factor expressed in embryonic trout muscle by hybridization with a Xenopus MyoD cDNA. Nucleotide sequence analysis and amino acid comparison showed that this cDNA called TMyoD encodes a polypeptide of 276 amino acids with 70% identity to the entire Xenopus MyoD protein and 92% identity within the basic and myc-like region. Results from Northern blotting showed that the corresponding transcript is expressed both in adult and embryonic skeletal musculature and in an in vitro myogenesis system, but is undetectable in cardiac and smooth muscles and in non muscle tissues.Key words: MyoD; Myogenesis; Teleost; (Satellite cell)The MyoD gene identified by substract cloning for myoblast specific RNA is the prototype of a family of master regulators of skeletal myogenesis which includes in vertebrates three other members namely myogenin, Myf5 and MRF4 identified subsequently [1][2][3][4][5]. All these genes encode proteins that share a highly conserved central region termed the basic/helix-loop-helix(B-HLH) domain related to the c-myc superfamily and contain sequences essential for both dimerisation and DNA binding [6]. In multipotential 10T1/2 cells, transfection experiments have shown that forced expression of these exogenous myogenic factors is sufficient to drive them down the muscle differentiation pathway suggesting their functions in myogenic lineage determination [1][2][3][4][5]. In contrast to vertebrates whose genome encodes multiple members of the MyoD family, invertebrates, including Sea urchin [7], C. elegans [8] and Drosophila [9], appear to contain only a single myogenic factor encoding gene. However, the myogenic factor for Sea urchin and C. elegans activates myogenesis in 10T1/2 cells indicating a highly conserved mechanism for muscle genes activation. Myogenic factors have been studied in mammals, amphibians and birds [10], but nothing is known to date in fish. To analyse early developmental events leading to muscle formation in fish, we set out to isolate myogenic regulatory factors from Rainbow trout (Oncorhynchus mykiss) embryos (398 degree days) using a probe which spanned functional domains of the Xenopus MyoD cDNA. For this purpose, a Agtl0 cDNA library was constructed from poly(A) ÷ RNA of the trunk of rainbow trout embryos. The double strands cDNAs synthetized by the method of Gubler et al. [11] were size fractionated by gel filtration on a sepharose 4B column (Pharmacia) and the largest fractions were pooled, inserted into Agtl0 vector (Stratagene) and encapsided using an in vitro packaging kit (Amersham). After amplification of the cDNA library, approximately 5 • 105 plaques were screened at low stringency with a fragment from the Xenopus MyoD cDNA encompassing the B-HLH domain [12]. From 9 positive clones, we identified a single cDNA of 1.5 kb which had two EcoRI fragments of aproximately 1.2 and 0.3 kb. Restriction analysis and sequencing showed that this internal EcoRI site was not situated near a Notl site which is contained in the linker...
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