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Abstract: Two types of the fast skeletal myosin heavy chain (MYH) genes were cloned from a genomic DNA library of carp (Cyprinus carpio L.) and named MYH10 and MYH30, which showed the sequence similarity to the MYH cDNAs predominantly expressed in carp acclimated to 10 and 30 °C, respectively. The 5′-flanking region of about 3 kbp in size each from MYH10 and MYH30 contained various cis-elements to bind to transcriptional regulatory factors such as MyoD family and myocyte enhancer factor 2 (MEF2) family members. To localize functional regions responsible for the MYH gene expression in a temperature-dependent manner, a series of deletion constructs were prepared from the 5′-flanking region, inserted upstream the luciferase gene in a commercially available plasmid, and injected into the dorsal fast muscle of carp acclimated to 10 and 30 °C. The sequence of −1004 to −995 bp with the transcriptional activity in MYH30 was identified as an MEF2 binding site. While the activity given by a sequence of −921 to −824 bp in MYH10 contained only a GATA box, that of the activity of the −1 kbp construct from MYH10 was markedly higher in carp reared at 10 °C than fish reared at 30 °C. On the other hand, no temperature-dependent expressional regulation was observed for MYH30 even with the full-length construct of −3 kbp. The DNA fragment of −921 to −824 bp in MYH10 and MEF2 binding site in MYH30 interacted with nuclear proteins extracted from carp fast skeletal muscle as revealed by electrophoretic mobility shift assay. The signal intensity of a complex formed between the DNA fragment of MYH10 and nuclear extracts from the 10 °C-acclimated carp were higher than those with extracts from the 30 °C-acclimated fish. Although MEF2-binding site in MYH30 could form complex with nuclear extracts from the 30 °C-acclimated carp, the same or stronger signals were detected in complex formed with extracts from the 10 °C-acclimated fish. [Copyright &y& Elsevier]

Three embryonic class II myosin heavy chains (MYHs) were cloned from the common carp (Cyprinus carpio L.), MYHemb1, MYHemb2 and MYHemb3. MYH DNA clones were also isolated from the slow muscle of adult carp acclimated to 10°C (MYHS10) and 30°C (MYHS30). Phylogenetic analysis demonstrated that MYHemb1 and MYHemb2 belonged to the fast skeletal muscle MYH clade. By contrast, the sequence of MYHemb3 was similar to the adult slow muscle isoforms, MYHS10 and MYHS30. MYHemb1 and MYHemb2 transcripts were first detected by northern blot analysis in embryos 61 h post-fertilization (h.p.f.) at the heartbeat stage, with peak expression occurring in 1-month-old juveniles. MYHemb1 continued to be expressed at low levels in 7-month-old juveniles when MYHemb2 was not detectable. MYHemb3 transcripts appeared at almost the same stage as MYHemb1 transcripts did (61 h.p.f.), and these genes showed a similar pattern of expression. Whole mount in situ hybridization analysis revealed that the transcripts of MYHemb1 and MYHemb2 were expressed in the inner part of myotome, whereas MYHemb3 was expressed in the superficial compartment. MYHS10 and MYHS30 mRNAs were first detected at hatching. In adult stages, the expression of slow muscle MYH mRNAs was dependent on acclimation temperature. MYHS10 mRNA was expressed at an acclimation temperature of 10 and 20°C, but not at 30°C. In contrast, MYHS30 mRNA was strongly expressed at all acclimation temperatures. The predominant MYH transcripts found in adult slow muscle and in embryos at hatching were expressed in adult fast muscle at some acclimation temperatures but not others. A MYH DNA clone was isolated from the cardiac muscle of 10°C-acclimated adult fish (MYHcard). MYHcard mRNA was first detected at 61 h.p.f., but strong signals were only observed in the adult myocardium. The present study has therefore revealed a complex pattern of expression of MYH genes in relation to developmental stage, muscle type and acclimation temperature. None of the skeletal muscle MYHs identified so far was strongly expressed during the late juvenile stage, indicating further developmentally regulated members of the MYH II gene family remain to be discovered. [ABSTRACT FROM AUTHOR]

Embryos of the common carp, Cyprinus carpio L., were reared from fertilization of the eggs to inflation of the swim bladder in the larval stage at 18 and 25°C. cRNA probes were used to detect transcripts of the myogenic regulatory factors MyoD, Myf-5 and myogenin, and five myosin heavy chain (MyHC) isoforms during development. The genes encoding Myf-5 and MyoD were switched on first in the unsegmented mesoderm, followed by myogenin as the somites developed. Myf-5 and MyoD transcripts were initially limited to the adaxial cells, but Myf-5 expression spread laterally into the presomitic mesoderm before somite formation. Two distinct bands of staining could be seen corresponding to the cellular fields of the forming somites, but as each furrow delineated, Myf-5 mRNA levels declined. Upon somite formation, MyoD expression spread laterally to encompass the full somite width. Expression of the myogenin gene was also switched on during somite formation, and expression of both transcripts persisted until the somites became chevron-shaped. Expression of MyoD was then downregulated shortly before myogenin. The expression patterns of the carp myogenic regulatory factor (MRF) genes most-closely resembled that seen in the zebrafish rather than the rainbow trout (where expression of MyoD remains restricted to the adaxial domain of the somite for a prolonged period) or the herring (where expression of MyoD persists longer than that of myogenin). Expression of two embryonic forms of MyHC began simultaneously at the 25-30 somite stage and continued until approximately two weeks post-hatch. However, the three adult isoforms of fast muscle MyHC were not detected in any stage examined, emphasizing a developmental gap that must be filled by other, as yet uncharacterised, MyHC isoform(s). No differences in the timing of expression of any mRNA transcripts were seen between temperature groups. A phylogenetic analysis of the MRFs was conducted using all available full-length amino acid sequences. A neighbour-joining tree indicated that all four members evolved from a common ancestral gene, which first duplicated into two lineages, each of which underwent a further duplication to produce Myf-5 and MyoD, and myogenin and MRF4. Parologous copies of MyoD from trout and Xenopus clustered closely together within clades, indicating recent duplications. By contrast, MyoD paralogues from gilthead seabream were more divergent, indicating a more-ancient duplication. [ABSTRACT FROM AUTHOR]

SUMMARY: Common carp (Cyprinus carpio L.) were acclimated to 30°C from 20°C over 24 h and maintained at that temperature for 35 days. Changes in the expression of mRNA transcripts for fast myosin heavy chain (MyHC) isoforms, E12 of the E protein, and myogenic regulatory factors belonging to the MyoD (MyoD, myogenin, myf-5) and myocyte enhancer factor-2 (MEF2A, MEF2C) gene families were investigated in the fast myotomal muscle using northern blot analysis. Myofibrillar Mg2+-ATPase activity gradually declined over 21 days and was significantly different from the starting level after only 3–5 days. Over the same time period there was a dramatic decrease in the mRNA transcripts of the MyHC isoform predominantly expressed in cold-acclimated carp (10°C-type MyHC) and a significant increase in the transcripts of the MyHC isoform predominantly expressed in warm-acclimated carp (30°C-type MyHC). The levels of myogenin mRNA transcripts almost doubled over 3–7 days before declining to 30% of the starting levels for the remainder of the experiment. While the levels of E12 mRNA gradually decreased, MEF2A and MEF2C mRNA transcripts showed a significant decrease over 24 h and then stabilized at the lower levels. In contrast, the levels of MyoD mRNA were not affected by temperature acclimation. The possible role of myogenic regulatory factors in the expression of temperature-specific isoforms of MyHC in common carp is briefly discussed. [ABSTRACT FROM AUTHOR]

Presents information on a study which characterizes cDNA clones encoding the carp myosin regulatory light chain and examines the coordinated expression of mRNA levels for the myosin heavy and light chains following temperature acclimation. Materials and methods used; Results and discussion.