| Budget Amount *help |
¥6,300,000 (Direct Cost: ¥6,300,000)
Fiscal Year 1996: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1995: ¥4,300,000 (Direct Cost: ¥4,300,000)
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| Research Abstract |
Carp, Cyprinus carpio (0.5-0.8kg in body weight), were acclimated to either 10 or 30ﾟC for a minimum of 5 weeks, and their dorsal fast skeletal muscles were used for isolation of light meromyosin (LMM), C-terminal one third of myosin heavy chain. The microsequence analysis after SDS-PAGE revealed that the LMM component from the 10ﾟC-acclimated carp contained an N-terminal amino acid sequence different from that of the 30ﾟC-acclimated carp. DNA fragments encoding an N-terminal region of LMM were amplified by PCR or reverse transcription-PCR,demonstrating that the two acclimated groups further contained several amino acids substituted.
cDNA clones encoding LMM were isolated from cDNA libraries constructed from fast skeletal muscles of carp acclimated to 10 and 30ﾟC,using the above-mentioned DNA as a probe. All clones covered at least the full length of LMM.cDNAs were identified which were the predominant types expressed in 10ﾟC-and 30ﾟC-acclimated fish, as well as an intermediate type present at all acclimation temperature. Northern blot analysis using probes ofthree kinds of DNA fragments from the 3'untranslated region of carp acclimated to 10,20 and 30ﾟC further confirmed the presence of acclimation temperature-specific isoforms. In addition, it was found that mRNA levels of three isoforms were altered in an acclimation temperature-dependent manner. The deduced amino acid sequence identity of LMM isoforms were 95.6% between the 10 and 30ﾟC types and 97.8% between the 10ﾟC and intermediate types. When these were compared with those of homoiotherms, the 30ﾟC-acclimated type was more similar to those of homoiotherms than was the 10ﾟC-acclimated type. These results suggest that the 30ﾟC type of LMM is more thermostable than that of 10ﾟC type.
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