First published online December 14, 2005
Journal of Experimental Biology 209, 188-198 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.01978
Molecular cloning and mRNA expression analysis of carp embryonic, slow and cardiac myosin heavy chain isoforms
Yoshiaki Nihei1,
Atsushi Kobiyama1,*,
Daisuke Ikeda1,
Yosuke Ono1,
Satoshi Ohara1,
Nicholas J. Cole2,
Ian A. Johnston3 and
Shugo Watabe1,
1 Laboratory of Aquatic Molecular Biology and Biotechnology, Graduate School
of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo
113-8657, Japan
2 Division of Cell and Developmental Biology, MSI/WTB Complex, University of
Dundee, Dow Street, Dundee, DDI 5EH, UK
3 Gatty Marine Laboratory, Division of Environmental and Evolutionary
Biology, School of Biology, University of St Andrews, St Andrews, Fife KY16
8LB, UK
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Fig. 1. Comparison of the partial deduced amino acid sequences of carp embryonic
(emb), slow skeletal and cardiac types of myosin heavy chain (MYH) with those
of corresponding regions of carp fast skeletal MYH isoforms. The data on adult
fast muscle MYH sequences were taken from Imai et al.
(1997 ). Slow 10, slow 30 and
10°C fast, 30°C fast refer to the isoforms predominantly expressed at
10°C and 30°C, respectively. I fast is an intermediate type, which is
expressed over a broad range of temperature. Amino acid residues identical to
those of carp MYHemb1 are indicated by dots. Numbers in the right
margin represent amino acid residues from the N terminus. Primer binding
regions of MYHemb1, MYHemb2 and
MYHemb3 are boxed.
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Fig. 3. The neighbor-joining (A, NJ) and maximum parsimony (B, MP) tree based on
deduced amino acid sequences in partial rod domains of MYHs. Carp MYHs
identified in this study (in bold) were compared with H. sapiens: Hs
MYH3 (embryonic), NM_002470; Hs MYH2 (adult fast IIa), AF111784; Hs MYH1
(adult fast IId/x), AF11785; Hs MYH4 (adult fast IIb), AF11783; Hs MYH8
(perinatal), NM_002474; Hs MYH13 (extraocular), AF11782; Hs MYH6
(alpha-cardiac), NM_002471; Hs MYH7 (beta-cardiac), NM_000257; C.
carpio: Cc F10 (adult 10°C-acclimated fast), D89990; Cc Fint (adult
20°C-acclimated fast), D89991; Cc F30 (adult 30°C-acclimated fast),
D89992; D. rerio: Dr myhz1 (embryonic fast), AF180893; Dr myhz2
(embryonic fast), NM_152982; Dr myhc4 (embryonic fast), AY921650; Dr artial
(atrial), AY138982; Dr ventricular (ventricular), AF114427; Dr smyhc1
(embryonic slow), AY921649. MYH sequences of T. rubripes (Tr) were
constructed by using Fugu genomic sequence assembly data version 3.0
(Ikeda et al., 2004).
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Fig. 4. Specificity of probes for northern blot hybridization from eight types of
cDNA clones encoding MYH isoforms of carp. Nylon membranes incorporating
MYHF10, MYHF30, MYHS10, MYHS30,
MYHemb1, MYHemb2, MYHemb3 and
MYHcard cDNA clones were hybridized with probes specific to
MYHS10, MYHS30, MYHemb1,
MYHemb2, MYHemb3 and MYHcard
used for northern blot analysis. S10, S30, slow cDNA, emb1-3 embryonic 1-3
cDNA, cardiac, cardiac cDNA (all at 1, 10 or 100 ng).
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Fig. 5. Changes in the accumulated mRNA levels of carp MYH isoforms during
ontogeny. Total RNAs (20 µg) were electrophoresed in a 1% agarose gel and
transferred on to the nylon membranes, which were then hybridized with
32P-labeled probe. Lanes contain total RNAs from the whole embryos
at 30 h, 42 h, 61 h, and 77 h.p.f., from the whole larvae at hatching (96 h),
from the muscle of juveniles aged 1 and 7 months post-hatching and from the
fast and slow skeletal muscles of thermally acclimated adult fish. An ethidium
bromide-stained gel shows 28S and 18S rRNA. Consensus indicates the membrane
hybridized with the probe, which is supposed to react with all skeletal MYH
mRNA of carp. Each lane contains 5 µg of total RNA.
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Fig. 6. Localization of the transcripts of MYHemb1,
MYHemb2 and MYHemb3 in carp embryos. Carp
embryos were hybridized with probes specific to MYHemb1
(A,B), MYHemb2 (C,D) and MYHemb3 (E,F)
at 77 (A,C,E) and 95 (B,D,F) h.p.f. G and H are higher magnifications of the
caudal region shown in B and F, respectively. (I,J,K) Transverse sections of
B,D,F, respectively, in the regions indicated by arrowheads. Arrows in J
indicate the transcripts of MYHemb2 in the four `corners'
of the trunk.
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Fig. 7. Schematic representation of the mRNA expression patterns of the various
MYHs identified in relation to muscle type, developmental stage and
acclimation temperature. Since northern blot analysis was only performed on
pure muscle types in adults, the tissue-specific expressions in embryonic and
juvenile fish were based on inference (see text for discussion), and therefore
remain a hypothesis. Putative MYH possibly expressed in juveniles are
indicated by question marks.
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© The Company of Biologists Ltd 2006
and ß cardiac, and chicken atrial MYHs.
Comparative data are from the zebrafish
(Yelon et al., 1999