cDNA clones encoding the myosin regulatory light chain (RLC) were isolated from a cDNA library prepared from fast skeletal muscle of the carp Cyprinus carpio L. Two types of cDNA clone encoding carp RLC were found with identical deduced amino acid sequences. The two mRNAs differed in the number of polyadenylation signals prior to the poly(A) tail in the 3′ non-coding region. The two mRNA species, with approximate sizes of 1.4 and 0.8 kilobases, were also observed in northern blot analysis. Carp were acclimated for a minimum of 5 weeks to either 10 °C or 30 °C (14 h:10 h light:dark photoperiod). The total levels of mRNA transcripts coding for the RLC and myosin heavy chain were, respectively, 3.3 and 3.9 times higher in cold-than in warm-acclimated fish. Differences in the levels of RLC in mRNA transcripts were largely due to the concentration of the 1.4 kilobase mRNA species.
Myosin, as well as actin, is a major myofibrillar protein and consists of two heavy chains of approximately 200 kDa and two pairs each of essential (ELC) and regulatory (RLC) light chains of approximately 20 kDa (Harrington and Rodgers, 1984). Myosin is cleaved by limited proteolysis into two functional domains. The head portion, called subfragment-1 (S1), contains ATPase catalytic and actin-binding sites, whereas the tail portion, called the rod, is responsible for thick filament formation. The three-dimensional structure of chicken fast skeletal muscle S1 revealed that two light chains, one each of ELC and RLC, bind noncovalently to a long α-helical chain found at the C-terminal portion of the S1 heavy chain, acting to stabilize the helix (Rayment et al. 1993). It is considered that this portion of myosin, called the neck region, acts as a rigid lever arm during the powerstroke of the crossbridge cycle (Uyeda et al. 1996; Anson et al. 1996).
Eurythermal fish such as carp Cyprinus carpio and goldfish Carassius auratus show changes in myofibrillar ATPase activity following temperature acclimation (Johnston et al. 1975; Heap et al. 1985). Maximum force production, contraction speed and Ca2+-sensitivity are altered by temperature acclimation in isolated muscle fibres (Johnston et al. 1985). Contraction speed is also modified by the type and ratio of ELCs (Crockford and Johnston, 1990).
We reported that the change in myofibrillar ATPase activity was accomplished mainly as a result of the altered expression of myosin isoforms having different ATPase activities (Hwang et al. 1990, 1991; Watabe et al. 1992, 1994; Guo et al. 1994). Recently, we isolated cDNAs encoding three types of carp myosin heavy chain isoforms and showed that the mRNA levels of the three isoforms were dramatically changed following temperature acclimation (Watabe et al. 1995; Imai et al. 1997; Hirayama and Watabe, 1997). Moreover, we also cloned cDNAs encoding two ELC isoforms, LC1 and LC3, from a carp fast muscle cDNA library and showed that the molar ratio of LC1 mRNA to LC3 mRNA was significantly changed following temperature acclimation (Hirayama et al. 1997). Thus, the change in myosin ATPase activity after temperature acclimation seems to be caused by the expression of myosin heavy, and possibly light, chain isoforms.
It is well known that myosin RLC from vertebrate skeletal muscle is quite different from those from vertebrate smooth muscle and non-muscle tissues. The Mg2+-ATPase activity of smooth muscle and non-muscle myosins is regulated by phosphorylation of the RLC. In contrast, contraction of vertebrate skeletal muscle is primarily regulated by binding of Ca2+ to the tropomyosin–troponin complex in thin filaments.
Although skeletal myosin RLC of higher vertebrates has also been demonstrated to be phosphorylated (Perrie et al. 1973), its physiological significance is still ambiguous, unlike the RLC from smooth muscle and non-muscle tissues. In the case of fish, the skeletal muscle RLC has been purified from a few species and its physicochemical and immunological properties have been well studied (Huriaux and Focant 1977; Watabe et al. 1983; Ochiai et al. 1988). Moreover, Yancey and Johnston (1982) showed that the fish RLC could also be phosphorylated. However, no information is available on its primary structure; this information would be useful for understanding the physiological roles of the skeletal muscle RLC in the regulation of muscle contraction.
The aim of the present study was to characterize cDNA clones encoding the carp myosin RLC and to examine the coordinated expression of mRNA levels for the myosin heavy and light chains following temperature acclimation.
Materials and methods
Carp, Cyprinus carpio L. (0.5–0.8 kg in body mass), were acclimated to either 10 or 30 °C for a minimum of 5 weeks under a 14 h:10 h light:dark photoperiod (Heap et al. 1985). Fast skeletal muscle isolated from the dorsal epaxial muscle was used in all experiments.
Screening of the cDNA library
Fast skeletal muscle cDNA libraries of carp acclimated to 10 and 30 °C (Imai et al. 1997; Hirayama et al. 1997; Hirayama and Watabe, 1997) were screened using anti-skipjack 5,5′-dithio-bis-2-nitrobenzoic acid light chain (RLC) antiserum (Ochiai et al. 1989) using the method of Sambrook et al. (1989). XL1-Blue infected with λZAP II cDNA library was cultured on an agar plate at 42 °C for 4 h, and a nitrocellulose filter containing 10 mmol l−1 isopropyl-β-D-thiogalactopyranoside was then overlaid on the plate and incubated at 37 °C for 4 h. The nitrocellulose filter replica was screened using anti-skipjack RLC antiserum. In vivo excision was carried out according to the manufacturer’s instructions (Stratagene).
DNA sequence analysis
Various restriction fragments of carp RLC cDNA clones were subcloned into pBluescript II using Escherichia coli strain MV1190 as a host bacterium. Sequencing was performed using Dye Deoxy terminator cycle sequence kits with a DNA sequencer (model 373S; Perkin Elmer).
Northern blot analysis
Carp acclimated to 10 and 30 °C were killed, and total RNA was isolated from fast skeletal muscle with a solution containing Isogen, a guanidine-isothiocyanate-based reagent (Nippon Gene) (five fish per acclimation temperature). The concentration of RNA was determined from the absorbance at 260 nm using a Shimadzu UV-160 spectrophotometer. Total RNA (5 μg) was size-fractionated by electrophoresis on a 1.2 % agarose gel in 0.2 mol l−1 Mops (pH 7.0) containing 18 % formamide, 0.05 mol l−1 sodium acetate and 5 mmol l−1 EDTA, and capillary-transferred from the gel to a nylon membrane. pLC2 10-2 (see Fig. 1) was radioactively labelled in the presence of [α-32P]dCTP with a random primer (DNA-labelling kit ver. 2; Takara) and used as a probe for mRNA encoding the regulatory light chain. The oligonucleotide probe 5′-dGGTTGAAGAACTGTTGCAGTTTCTC-3′, which could recognize all three carp myosin heavy chain isoforms (Hirayama and Watabe, 1997), was also synthesized and labelled with [γ-32P]ATP using a Megalabel DNA 5′ end-labelling kit (Takara). This nucleotide sequence region encoding the long α-helix is well conserved among various myosins (Patterson and Spudich, 1996). Hybridization was performed according to Church and Gilbert (1984). The membrane was prehybridized at 65 °C for 14 h in a solution containing 250 mmol l−1 Na2HPO4 (pH 7.2), 1 mmol l−1 EDTA and 7 % SDS. The blot was then hybridized at 65 °C for 18 h in the same solution containing 32P-labelled cDNA probes. After washing with 0.5×SSC (1×SSC is 0.15 mol l−1 sodium chloride, 0.015 mol l−1 sodium citrate) containing 0.1 % SDS at 65 °C, the blot was exposed to X-ray films for 24 h using an intensifying screen. The hybridized membrane was scanned with a Fujix Bas 1000 computerized densitometer scanner and quantified using the recommended program in the range where there was a linear relationship between the amount of 32P incorporated and the scanned values.
A Student’s t-test was employed to compare differences between values for northern blot analysis between carp acclimated to 10 and 30 °C.
Results and discussion
Cloning of myosin RLC cDNA from carp fast skeletal muscle When 5×104 plaques of fast skeletal muscle cDNA libraries from carp acclimated to either 10 or 30 °C were screened with anti-skipjack myosin RLC antiserum, approximately 20 clones were obtained from each of the libraries. Agarose gel electrophoresis together with partial nucleotide sequence analysis revealed that these clones could be separated into two types according to their size. Fig. 1 shows the restriction maps of representative cDNA clones from both types. The two cDNA clones, pLC2 10-2 and pLC2 10-4, had 840 and 1395 nucleotides, respectively, and contained the entire coding region as well as the 3′ non-coding region including poly(A) tails (Fig. 2). Comparison of the nucleotide sequence between the two clones revealed that there were 17 nucleotide differences in corresponding regions. In addition, we detected multiple allelic variants for both shorter and longer mRNAs encoding RLCs in the cDNA library constructed from one carp individual (data not shown). We also found several variations in DNA nucleotide sequences for homologous clones of carp myosin heavy and essential light chains even from one individual (our unpublished data). Although it is difficult to explain the existence of such variations, they may be caused by the tetraploid origin of carp. It has been claimed that duplicate loci appear to remain expressed for millions of years in the case of carp somatotropin (Larhammar and Risinger, 1994).
However, the deduced amino acid sequences of the two clones were identical. Interestingly, pLC2 10-4 had three polyadenylation signals which were arrayed in tandem in the 3′ non-coding region with the poly(A) tail added downstream of the third putative polyadenylation signal. pLC2 10-2 had a polyadenylation signal at the position corresponding to the first site in pLC2 10-4. Therefore, the two cDNAs are probably produced from a single gene by the alternative use of three polyadenylation signals. The other possibility is that the two types of mRNA are transcribed from their respective genes, which have been duplicated during evolution from a single gene. Although most eukaryotic mRNAs possess a single polyadenylation signal, a number of examples of mRNAs with multiple tandem poly(A) sites have been reported (Edwards-Gilbert et al. 1997).
Northern blot analysis of carp fast skeletal muscle myosin RLCs
To investigate the mRNA levels of carp RLCs following temperature acclimation, we performed northern blot analyses using the 32P-labelled pLC2 10-2 cDNA clone as a probe. Hybridization patterns with total RNAs from the fast skeletal muscle of carp acclimated to 10 and 30 °C are shown in Fig. 3A. Two bands appeared in all the samples tested. The sizes of the two RNA bands were approximately 1.4 and 0.8 kb. It seems that the two transcripts corresponded to the two types of carp RLC mRNAs described above, since only the slower-migrating band was hybridized with a probe derived from 866–1333 nucleotides of pLC2 10-4 and specific to the longer mRNA (data not shown). Interestingly, the molar ratio of the two transcripts was dependent on the acclimation temperature of the fish. The ratio of the longer to the shorter mRNA was 1.59±0.20 (mean ± S.E.M., N=5) for the 10 °C-acclimated carp and 0.69±0.12 (N=5) for the 30 °C-acclimated fish (P<0.0001). Although it has been reported that different forms of mRNA encoding the same protein have different stabilities and may not be translated with the same efficiency (Edwalds-Gilbert et al. 1997), changes in the ratio of the two carp myosin RLC mRNAs could result in altered protein levels. Moreover, the sum of two mRNA signals, based on the content of ribosomal RNA and measured with a Fujix Bas 1000 scanner, was significantly different between the 10 °C- and 30 °C-acclimated carp (P<0.001). Fish acclimated to 10 °C showed approximately 3.3-fold higher mRNA levels than 30 °C-acclimated fish. The level of the longer mRNA in the 10 °C-acclimated carp was 4.2 times higher than in the 30 °C-acclimated carp (P<0.001). However, the concentration of the shorter mRNA was independent of acclimation temperature (P>0.05). These results indicate that changes in the expression of RLC transcripts in the carp following temperature acclimation are primarily regulated by the longer mRNA species.
Northern blot analysis of carp fast skeletal muscle myosin heavy chain
We have reported previously that the composition of three types of fast skeletal myosin heavy chain isoform at the mRNA level also changed following temperature acclimation in carp (Imai et al. 1997; Hirayama and Watabe, 1997). In the present study, the accumulated levels of mRNAs encoding all three isoforms was 3.9 times higher in the 10 °C-acclimated than in the 30 °C-acclimated carp (P<0.001) (Fig. 3B). Thus, mRNA transcript levels of both the RLC and myosin heavy chain increased to a similar degree with cold-acclimation. The changes in mRNA transcript levels may reflect the altered composition of myosin heavy chain isoforms. To assess the physiological significance of our results, we are now performing additional experiments to obtain data on the time course of cold adaptation, the developmental sequence and the expression pattern in different muscle types.
Comparison of the deduced amino acid sequence of carp fast skeletal muscle RLC with those from other vertebrates
Myosin light chains are members of the EF hand superfamily, which includes calmodulin and troponin-C, consisting of four EF-hand-like domains (Trybus, 1994). Loop I in the first EF hand motif of RLC retains the ability to bind to a metal ion, and this site is probably occupied by Mg2+in vivo (daSilva and Reinach, 1991). Fig. 4 compares the amino acid sequence of carp fast skeletal muscle RLC with those from other vertebrate fast and smooth muscles. Carp fast skeletal muscle RLC showed 84.0 % sequence identity with those from chicken (Reinach and Fischman, 1985), rat (Nudel et al. 1984) and rabbit (Maeda et al. 1990) fast skeletal muscle, but only 51.7 % identity with that from chicken smooth muscle (Messer and Kendrick-Jones, 1988). It was noted that the amino acid sequences of helices D and H were identical between fast skeletal muscle RLCs. Moreover, the primary structures of loops I, II and IV were well conserved between skeletal muscle RLCs. In addition, it is known that the skeletal muscle RLC of higher vertebrates is phosphorylated by skeletal muscle myosin light chain kinase both in vivo and in vitro at Ser-15 (Perrie et al. 1973; Sweeney and Stull, 1990). This serine residue is also conserved in carp skeletal muscle RLC (Fig. 4). Although the phosphorylation site has not yet been identified, fish RLC is also phosphorylated both in vivo and in vitro (Yancey and Johnston, 1982). These observations suggest that carp RLC may also be phosphorylatable by skeletal muscle myosin light chain kinase at this serine residue.
S.W. was supported by Grant-in-Aids for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan, and from the Asahi Glass Foundation, Japan. Y.H. was supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists.