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Physiol. Genomics 29: 201-214, 2007. First published January 16, 2007; doi:10.1152/physiolgenomics.00078.2006 Free Article
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Received 2 May 2006; accepted in final form 6 January 2007.
Physiological Genomics 29:201-214 (2007)
1094-8341/07 $8.00 © 2007 American Physiological Society

Fast skeletal muscle myosin heavy chain gene cluster of medaka Oryzias latipes enrolled in temperature adaptation

Chun-Shi Liang 1, Atsushi Kobiyama 1, Atsushi Shimizu 2, Takashi Sasaki 2, Shuichi Asakawa 2, Nobuyoshi Shimizu 2 and Shugo Watabe 1

1 Laboratory of Aquatic Molecular Biology and Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan
2 Department of Molecular Biology, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
To disclose mechanisms involved in temperature acclimation of fish muscle, we subjected eurythermal fish of medaka Oryzias latipes to cloning of myosin heavy chain genes (MYHs). We cloned cDNAs encoding fast skeletal muscle myosin heavy chain (MYH) isoforms from cDNA libraries of medaka acclimated to 10 and 30°C and observed that different MYH cDNA clones are expressed in the two temperature-acclimated fish. Subsequently, we isolated several overlapping MYH contigs by shotgun cloning strategy from a medaka genomic library. Contig assembly of the complete medaka MYH (mMYH) locus of 219 kbp revealed a cluster of tandemly arrayed 11 mMYHs, in which eight genes are actually transcribed, with the remaining three being pseudogenes. Expression analysis of the transcribed genes revealed that two genes were each highly expressed in medaka acclimated to 10 and 30°C, whereas comparatively lower expression levels of the three genes were exclusively observed in medaka acclimated to 30°C. cDNAs of the remaining genes were too underrepresented in the libraries to determine the expression levels, and the transcripts could only be obtained by reverse transcription-polymerase chain reaction. Deduced amino acid sequences in the loop 1 and loop 2 regions of mMYHs were highly variable, suggesting that these isoforms were functionally different. The present findings consolidate our knowledge on teleost MYH multigene family and would provide further insight into the mechanisms by which expressions of individual MYH molecules are fine-tuned with environmental temperature fluctuations with further functional analysis of the genes concerned.

cDNA library; genomic bacterial artificial chromosome library; temperature acclimation; myofibrillar Mg2+-ATPase activity


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
MYOSINS ARE ACTIN-BASED MOLECULAR motors that convert chemical energy released from the hydrolysis of ATP into mechanical force in eukaryotic cells. Numerous myosins comprising 18 classes (48, 52) have been described, including a subgroup of class II that contains sarcomeric myosins functionally to drive muscle contraction. Class II or conventional two-headed myosin is a heterohexamer consisting of two heavy chains (MYHs) of ~200 kDa and four light chains of ~20 kDa. The NH2-terminal half of each MYH folds into a globular head, termed subfragment-1 (S1), which contains an ATP- and an actin-binding site (30). The COOH-terminal halves of two heavy chains form an {alpha}-helical coiled coil structure, a rod. Rods can be separated further into two fragments by limited proteolysis; an NH2-terminal half called subfragment-2 (S2) and a COOH-terminal half of light meromyosin (LMM) (11). Furthermore, tryptic digestion of S1 results in the formation of 25-, 50-, and 20-kDa fragments from the NH2 to COOH termini, which are linked by two variable regions termed surface loops 1 and 2 (31, 44). Loop 1, which connects the 25-kDa and 50-kDa tryptic fragments of S1, is closely associated with the ATP binding site and likely tunes the rate constant for ADP release (31), whereas loop 2 connects the 20-kDa and 50-kDa tryptic fragments of S1 and is one of the actin-binding sites, possibly regulating Vmax of actin-activated Mg2+-ATPase activity (31). Both the length and amino acid sequence of the two surface loops can vary between different myosin types (10). This diversity is thought to play a crucial role in determining speed of contraction and motor function in a particular category of muscle (44, 50).

Mammalian sarcomeric MYHs are encoded by multigene families (52, 57). Eight sarcomeric MYHs including two cardiac (MYH-{alpha}, MYH-ß) and six skeletal (embryonic MYH, perinatal MYH, MYH-IIa, MYH-IIb, MYH-IIx/d, extraocular MYH) form tightly linked clusters on human chromosomes 14 and 17, respectively, and on mouse chromosomes 14 and 11, respectively (40). Transcriptional regulation of the MYH family is complex, with individual genes differentially expressed during development and in response to hormonal, physiological, and other signals (42, 55, 58). Recent progress on the total genome studies revealed the full sequences of various types of MYH from human (6), torafugu pufferfish Takifugu rubripes (18), and zebrafish Danio rerio (32). Among invertebrates, the full-length MYH sequences have been reported for nematode Caenorhabditis elegans body wall (25), fruit fly Drosophila melanogaster flight skeletal (9), and scallop Argopecten irradians striated and smooth (36) muscles. The organization of vertebrate MYH is markedly different from that of invertebrates, although MYHs from both vertebrates and invertebrates have consensus DNA nucleotide and deduced amino acid sequences in the exon regions. Interestingly, Drosophila skeletal (9) and scallop striated and smooth (36) muscle MYHs are transcribed into different mRNAs by alternative splicing from a single gene.

Eurythermal temperate fish are able to survive over a wide range of water temperature ranging from near zero to >30°C throughout the year while maintaining their normal physiological and biochemical processes in vivo by temperature acclimation. Goldfish Carassius auratus acclimated to different experimental temperatures show unique temperature-performance curves of maximum cruising speed. In general, swimming speed increases at low temperatures and decreases at high temperatures following cold acclimation, and the opposite responses are observed following acclimation to warm temperatures (8). Biochemical evidence for such physiological temperature acclimation was disclosed by Johnston et al. (23), who showed that goldfish change myofibrillar Mg2+-ATPase activity following temperature acclimation. Heap et al. (12) observed that common carp Cyprinus carpio reversibly change this myofibrillar catalytic function within 5 wk, suggesting that protein synthesis or the turnover of myofibrillar components is involved in the process. Later, a series of studies from our group showed that common carp express a number of fast skeletal muscle MYH isoforms in association with temperature change. We have characterized three temperature-specific isoforms of common carp MYH, 10°C, 30°C, and intermediate type (I-type), at both the DNA and protein levels (13, 20, 55). The former two types of isoforms were predominantly expressed in common carp acclimated to respective temperatures, while the I-type isoform was expressed in fish acclimated to 10 and 20°C. Grass carp Ctenopharyngodon idellus also undergo an adaptation to fluctuating environmental temperatures by selectively expressing different fast skeletal MYH isoforms as do common carp (45). We also provided evidence that these temperature-specific isoforms of MYH are not alternatively transcribed forms of a single MYH; rather they are the products of a multigene family. While we have recently determined the complete genome sequence of the common carp I-type MYH (33), the genome organization of the rest MYH isoforms still remains to be elucidated.

Medaka Oryzias latipes is also a temperate eurythermal fish like goldfish, common carp, and grass carp. They inhabit a wide range of temperatures from 4°C to 40°C (21). Its small genome size has been reported to be ~800 Mb (21), which is one-half and one-fourth of those of zebrafish and human, respectively. Besides, medaka is a useful animal for molecular biological and genetic research in that it has a short generation time, transparent egg, a large genetic diversity within species, and inbred strains (34). Moreover, medaka is easy to rear. Therefore, medaka offers an appropriate model for understanding the genome not only of teleost fish but also those of higher vertebrates. Fortunately, medaka genomic bacterial artificial chromosome (BAC) library has recently been constructed (29) and is now available for comprehensive research on multiple genes such as MYH with the functions and expressions, which change in a temperature acclimation-dependent manner.

The objective of the present study was to isolate cDNA and genomic clones encoding fast skeletal muscle MYH. As a result, we isolated 11 types of MYHs, revealing that they are tandemly arrayed within 219 kbp.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Fish strain.
Adult medaka of the HNI inbred strain (0.3–0.4 g in body wt) (16) were reared at 20°C in a 17-liter aquarium for at least 5 wk. For acclimation experiments, about 10 individuals were put into the aquarium, and temperature was subsequently raised to either 10 or 30°C in 1 day and maintained for a minimum of 5 wk under a 14:10-h light-dark photoperiodic regime. Heap et al. (12) reported that changes in myofibrillar ATPase activity following temperature transfer were apparent after 1 or 2 wk and a steady state was achieved after 4 or 5 wk. Therefore, in our previous studies as well as the present one, fish were acclimated for a minimum of 5 wk. We observe no apparent differences in feed intake and swimming activities between 10°C- and 30°C-acclimated medaka after acclimation for 5 wk. The fast skeletal muscle was used to prepare myofibrils, to construct cDNA libraries, and to synthesize first-strand cDNA. We carefully removed slow muscle tissue from fast skeletal muscle tissue.

Preparation and measurement of myofibrillar Mg2+-ATPase activity.
Myofibrils were prepared with a medium containing 25 mM KCl, 0.1 mM dithiothreitol, and 39 mM borate buffer (pH 7.0) (53), and their protein concentrations were determined with the BCA Protein Assay Kit (Pierce, Rockford, IL). Myofibrillar Mg2+-ATPase activity was measured at 25°C by the malachite green method for Pi released (28). Reaction mixtures contained 20 mM 3-(N-morpholino)-propanesulfonic acid-NaOH (pH 7.4), 30 mM KCl, 4 mM MgCl2, 0.1 mM CaCl2, and 0.04 mg/ml myofibril. The reaction was initiated by the addition of ATP to 1 mM following a 2-min incubation at 25°C without ATP. Aliquots of 100 µl were taken from each reaction mixture at 5-min intervals up to 30 min and mixed with 100 µl of ice-cold 0.6 M perchloric acid to stop the reaction. Student's t-test was employed to compare differences in data for myofibrillar Mg2+-ATPase activity between medaka acclimated to 10 and 30°C.

Construction of cDNA library.
Total RNA was prepared from the fast skeletal muscle of medaka acclimated to either 10 or 30°C using an ISOGEN system (Nippon Gene, Tokyo, Japan). mRNAs were prepared from 2 µg of total RNA using Oligotex-dT30 mRNA Purification Kit (Takara, Tsu, Japan) followed by construction of cDNAs with a TimeSaver cDNA Synthesis Kit (Amersham Biosciences, Buckinghamshire, UK). cDNAs thus obtained were inserted into {lambda}ZAPII phage vector according to the manufacturer's instruction (Stratagene, La Jolla, CA).

Screening of cDNA library and in vivo excision.
First-strand cDNA was synthesized from 1 µg of total RNA prepared from dorsal skeletal muscle of medaka using a SuperScript First-Strand Synthesis System (Invitrogen, Carlsbad, CA) according to the manufacturer's instruction. A 438-bp cDNA fragment used as a DNA probe specific to medaka MYH (mMYH) was amplified by polymerase chain reaction (PCR) with a primer pair, MHC3F and mMYH3'R (Table 1), which were designed based on the data reported for common carp fast skeletal isoforms of LMM (20). PCR was performed for 30 cycles using Ex Taq DNA polymerase (Takara). Each thermal cycle consisted of denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 1 min, with a final extension step at 72°C for 10 min. The amplified cDNA fragments were labeled with digoxigenin (DIG) DNA Labeling and Detection Kits (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instruction. We carried out in vivo excision of clones from {lambda}ZAPII libraries according to the methods of Short et al. (39) and subjected the 3'-regions of excised clones to nucleotide sequencing using an ABI PRISM 373A DNA sequencer after labeling DNAs with Dye-Deoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA).


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  		Table 1. Nucleotide sequences of primers used for PCR amplifications

 
Phylogenetic analysis.
The deduced amino acid sequences of cDNAs, which encoded a part of LMM in mMYHs, were aligned with those of common carp using CLUSTAL W (47). The starting point of the alignment corresponded to Lys-1814 in LMM of common carp 10°C-type MYH (13). The alignment was used to generate a phylogenetic tree by the neighbor-joining method with a bootstrap value from 1,000 replicates (38). Amino acid sequences of MYHs were deduced from cDNA nucleotide sequences retrieved from DDBJ/EMBL/GenBank databases (accession numbers in parenthesis) for adult 10°C-type fast skeletal (carp-10; D89990), adult I-type fast skeletal (carp-I; D88991), adult 30°C-type fast skeletal (carp-30; D89992), embryonic fast skeletal type 1 (carp-ebm1; AB104622), embryonic fast skeletal type 2 (carp-emb2; AB104623), adult 10°C-type slow skeletal (carp-slow10; AB104625), adult 30°C-type slow skeletal (carp-slow30; AB104626), embryonic slow skeletal type MYH (carp-emb3; AB104624), and carp-cardiac (AB104627). Details of common carp embryonic, slow skeletal and cardiac MYHs are described in Ref. 35.

In addition, to determine the relationship of medaka MYH pseudogenes and those that were proven to be transcribed, a phylogenetic tree was constructed for the partial cDNA nucleotide sequences corresponding to exon 18–22.

3'-Rapid amplification of cDNA ends.
First-strand cDNA was synthesized from total RNA extracted from dorsal skeletal muscle of medaka acclimated to 20°C using a 3'-rapid amplification of cDNA ends (3'-RACE) System (Invitrogen). PCR was performed as described above with a mMYH-specific sense primer mMYH-F designed from the nucleotide sequences of mMYHs obtained from the cDNA libraries of the 10°C- and 30°C-acclimated medaka and an abridged universal amplification primer, AUAP, contained in the kit (Table 1). Amplified DNA fragments were subcloned into pGEM-T vector (Promega, Madison, WI) using Escherichia coli strain JM 109 (Promega) as a host bacterium, and nucleotide sequencing on both strands was performed as mentioned above.

Screening of medaka genomic BAC library.
A high-density replica membrane of a medaka HNI inbred strain genomic BAC library (29) was used for screening of mMYH. The BAC library was screened by colony hybridization with another mMYH-specific cDNA probe of 463 bp, which was amplified by PCR as mentioned above with a primer pair of MHC3F and MYH-10R (Table 1). The cDNA probe corresponded to the entire lengths of exons 38–40 flanking partially in both sides by exons 37 and 41 of carp I-type MYH (33). The cDNA probe was labeled with DIG-11-dUTP using DIG-High Prime DNA Labeling and Detection Starter Kit II (Roche) according to the protocol supplied by the manufacturer. Purified BAC DNA (1 µg) from each positive clone was digested with HindIII and subjected to Southern blot hybridization with the same probe described above to confirm the presence of positive inserts and to examine whether the clones contained the complete mMYH cluster.

BAC clone sequence analysis.
The shotgun strategy was employed for sequencing selected BAC clones. BAC DNA was hydrodynamically sheared with a nebulizer to obtain DNA fragments with an average size of 2–3 kbp, which were ligated into pUC118 vector (Takara). Each BAC clone was sequenced at a sevenfold coverage using ~1,150 and 1,350 shotgun clones from BAC clones 70M19 and 36I15, respectively, using the BigDye Terminator v3.1 Cycle Sequencing Kit with an ABI PRISM 377 or 3700 DNA sequencer (Applied Biosystems). To assemble the individual shotgun sequences into contigs, we used the computer programs Phred, Phrap, and Consed for base calling, assembly of sequences, and viewing and editing analysis, respectively. The gaps between BAC clones were closed by a combination of BAC walking, directed PCR, and further sequencing of individual clones.

Analysis of genomic BAC clone sequences.
The two genomic sequences of the BAC clones, 70M19 and 36I15, were compared by dot-matrix program after masking the repetitive elements by Repeat-Masker2 program (http://www.repeatmasker.org). The complete exon-intron organization of common carp I-type MYH (33) was used as a reference to predict the exon-intron organizations as well as the splice donor and acceptor sites in the genomic sequences of mMYHs. Loop 2 and the 5'- and 3'-regions were determined referring to the cDNA sequences obtained by RT-PCR and 5'- and 3'-RACE. The deduced amino acid sequences from the predicted exons were also analyzed by using BLASTP and BLASTX (1).

5'-RACE.
5'-RACE was employed to identify the transcription start site of mMYHs using a 5'-RACE System (Invitrogen) according to the instructions of the manufacturer. First-strand cDNA was synthesized with a gene-specific primer mMYH-E7R, while PCR amplification was performed with AUAP contained in the kit and another gene-specific antisense primer mMYH-E5R (Table 1). In a parallel experiment, primers mMYH-E7R and mMYH-E5R were replaced with primers mMYH-1,11-E7R and mMYH-1,11-E4R, respectively, to increase the specificity for mMYH-1 and mMYH-11. Amplified DNA fragments were subcloned and completely sequenced as described previously.

RT-PCR.
RT-PCR was carried out to amplify the regions encoding loop 2 from different isoforms of mMYH to determine exon-intron organizations. First-strand cDNA synthesized from total RNA of medaka dorsal skeletal muscle was used as a template. Two pairs of gene-specific primers (loop2-F, loop2-R, mMYH-1,2-loop2-F, and mMYH-1,2-loop2-R) designed from the mMYH sequences determined in the present study were used in PCR to amplify cDNAs encoding the loop 2 region (Table 1). Primers loop2-F and loop2-R were used to amplify the regions encoding loop 2 of mMYH-3 to mMYH-11, whereas mMYH-1,2-loop2-F and mMYH-1,2-loop2-R were used to amplify the same from mMYH-1 and mMYH-2 (Table 1). PCR and other experimental conditions were the same as described above. On the other hand, the exon-intron organizations around the regions encoding loop 1 of mMYHs were revealed by comparison with common carp fast skeletal MYH cDNAs (13). RT-PCR was also performed for analyzing the expression patterns of mMYH-4, mMYH-5, and mMYH-10 from medaka acclimated to 10 and 30°C using first-strand cDNA from respective fish as a template and several gene-specific primers as shown in Table 1.

Comparison of the 5'-flanking sequences of mMYHs with common carp MYH10 and MYH30.
The 5-kbp regions in the 5'-flanking sequence of mMYHs were compared with those of common carp MYH10 (AB201260) and MYH30 (AB201261) by a dot-matrix program (41).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Myofibrillar Mg2+-ATPase activity.
We examined myofibrillar Mg2+-ATPase activities of the 10°C- and 30°C-acclimated medaka, referring to the procedure for goldfish (23), common carp (12), and grass carp (46). Although we did not determine the activity in the 20°C-acclimated medaka, it has been reported that myosin from common carp acclimated to 20°C showed intermediate properties between those of the 10°C- and 30°C-acclimated myosins (54) The activities of the 10°C- and 30°C-acclimated medaka were 1.10 ± 0.06 and 0.94 ± 0.05 µmol Pi min–1mg–1, respectively. The activity was significantly higher in the 10°C- than 30°C-acclimated medaka (P < 0.05).

Cloning of mMYH cDNA clones.
cDNA libraries from the 10- and 30°C-acclimated medaka skeletal muscles were screened with a DNA probe specific to mMYH, which yielded a number of positive clones (Fig. 1). Subsequent nucleotide sequencing of the clones revealed the existence of seven different types of mMYH encoding a partial LMM region (Fig. 2). These cDNA clones were named m-10-1, m-10-2, and m-30-1 through m-30-5 (Figs. 1, 2). Four types of cDNA clones, m-10-1, m-10-2, m-30-1, and m-30-2, were obtained from the 10°C-acclimated medaka cDNA library, whereas all of the seven types were obtained from the 30°C-acclimated medaka cDNA library (Fig. 1). Alignments of the nucleotide and deduced amino acid sequences of the cDNAs as carried out by CLUSTAL W (47) are shown in Figs. 2 and 3, respectively. The most conspicuous differences among the seven types of cDNA clones were observed with the sequences in the 3'-untranslated region (3'-UTR) (Fig. 2). The stop codons of mMYHs were found in the position as that of carp-10 and carp-30 (20), except for m-30-3 and m-30-5 cDNAs. While m-30-3 cDNA had a sequence of six nucleotides shorter to the stop codon, m-30-5 cDNA contained three nucleotides more. M-10-1, m-10-2, m-30-1, and m-30-2 cDNAs showed similar sequences in the region of the stop codon to a putative polyadenylation signal AATAAA (3), whereas the sequences of m-30-3, m-30-4, and m-30-5 cDNAs were markedly different from each other and also from the remainders. In addition, m-30-5 cDNA contained an unusual variant TATAAA (3) as a putative polyadenylation signal. Reflecting the above sequence characteristics, m-10-1, m-10-2, m-30-1, and m-30-2 cDNAs showed a high nucleotide sequence identity of not less than 95% to each other and were significantly different from m-30-3, m-30-4, and m-30-5 cDNAs with 76–86% identity (Table 2). The identity to each other among the latter clones ranged from 77 to 83%. The nucleotide identities of MYH cDNAs between medaka and common carp were in the range of 63–73%. Interestingly, identical amino acid sequences were deduced for m-10-1 and m-10-2 cDNAs and for m-30-1 and m-30-2 cDNAs (Fig. 3, Table 2). The former two cDNAs showed 96% identity to the latter two cDNAs. While these four cDNAs showed identifies of 85–95% to m-30-3, m-30-4, and m-30-5 cDNAs, the latter three cDNAs showed 85–90% identify to each other. Thus m-30-5 cDNA was most strikingly different from other mMYH cDNAs. Amino acid variations between medaka and common carp cDNAs were observed widely (Fig. 3) and the identity ranged in 81–89% (Table 2).


Figure 1
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  		Fig. 1. Frequencies of fast skeletal medaka myosin heavy chain (mMYH) cDNA clones in the libraries constructed from fast skeletal muscles of medaka acclimated to 10 and 30°C. The numbers indicate those of cDNA clones encoding mMYHs randomly screened from the 10°C- and 30°C-acclimated medaka cDNA libraries. A highly conserved region among cDNA clones was used as a probe for the screening from both cDNA libraries.

 

Figure 2
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  		Fig. 2. Locations of PCR primers to amplify a probe for screening cDNA libraries for fast skeletal mMYHs, and aligned nucleotide sequences determined by 3'-rapid amplification of cDNA ends (RACE) in 3'-translated and untranslated regions for 7 types of cDNA clone together with corresponding sequences of common carp. A: schematic representation of the MYH molecule at the top for orientation purposes. The gray bar represents a location of the probe used to screen medaka cDNA libraries. The locations of primers (MHC3F, mMYH3'R, and AUAP) are shown below. Abbreviations used are: S1, myosin subfragment-1; S2, myosin subfragment-2; LMM, light meromyosin; AUAP, abridged universal amplification primer. B: comparison of the partial nucleotide sequences of cDNA clones encoding mMYH with those of common carp. Carp-10, carp-I, and carp-30 represent the 10°C, I, and 30°C types of common carp adult fast skeletal MYHs, respectively. Data cited are carp-10, carp-I, and carp-30 from Imai et al. (20). Nucleotides identical to those of medaka m-10-1 MYH cDNA are shown by dots, and gaps are represented by dashed lines. The stop codons are shaded, and polyadenylation signals are boxed.

 

Figure 3
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  		Fig. 3. Aligned deduced amino acid sequences of COOH-terminal regions of fast skeletal LMM of medaka MYH encoded by 7 types of cDNA clone together with those of common carp. Amino acids identical to those of medaka m-10-1 MYH are shown by dots. Refer to the legend of Fig. 2 for carp-10, carp-I, and carp-30, respectively.

 

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  		Table 2. Comparison of nucleotide and deduced amino acid sequences of mMYHs encoding a partial LMM region of fast skeletal muscle from thermally acclimated medaka and common carp (%)

 
Phylogenetic tree of mMYHs.
The deduced amino acid sequences corresponding to a part of LMM of mMYHs were performed by neighbor-joining method with the same region in common carp MYHs (Fig. 4). Fast skeletal MYHs were paraphylic from slow and cardiac MYHs in common carp. Furthermore, fast skeletal MYHs in common carp were separated into two clades, one for the adult and the other for the embryonic type. mMYHs were more closely related to those of common carp adult fast skeletal MYHs than to those of embryonic, slow skeletal, and cardiac MYHs, suggesting that the temperature-dependent isoforms of mMYH belong to the adult fast skeletal muscle type.


Figure 4
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  		Fig. 4. Phylogenetic tree based on the amino acid sequences in the partial LMM region of fast skeletal MYH from medaka and common carp. Deduced amino acid sequences of MYHs were aligned with CLUSTAL W, and the tree was constructed by the neighbor-joining method. The bootstrap values from a 1,000-replicate analysis are given at the nodes in percentages. The evolutionary distance between 2 sequences was obtained by summing the length of the connecting branches along the horizontal axis according the scale at the bottom. See MATERIALS AND METHODS for abbreviations.

 
Expression of mMYH transcripts associated with temperature acclimation.
To determine the difference in the mRNA levels of mMYHs associated with temperature acclimation, we performed Northern blot analysis and RNase protection assay (data not shown). The RNase protection assay is a specific, sensitive, and qualitative method for the detection and quantitation of specific mRNAs. It is at least 10-fold more sensitive than Northern blot analysis and is more accurate and direct than RT-PCR analysis (19). However, it was difficult for mMYHs due to their high sequence homology (Table 2, Figs. 1 and 2), especially among m-10-1, m-10-2, m-30-1, and m-30-2 cDNAs, even in 3'-UTR. Therefore, we randomly screened cDNA libraries from the 10°C- and 30°C-acclimated medaka and determined the abundance of mMYH cDNA clones. As shown in Fig. 1, a total of 31 cDNA clones from the 10°C-acclimated medaka cDNA library were analyzed, out of which 16 and 13 clones belonged to m-10-1 and m-10-2 cDNAs, respectively. One clone each for m-30-1 and m-30-2 cDNAs was obtained, whereas no clones corresponding to m-30-3, m-30-4, and m-30-5 cDNAs were obtained from the 10°C-acclimated medaka. On the other hand, analysis of 42 cDNA clones from the 30°C-acclimated medaka cDNA library yielded the highest number of clones for m-30-1 cDNA (17) followed by 11 for m-30-2 cDNA and 7 for m-10-1 cDNA. Two to four clones were obtained for the remaining cDNAs. These results indicate that m-10-1 and m-10-2 cDNAs were predominantly expressed in the 10°C-acclimated medaka, whereas m-30-1 and m-30-2 cDNAs were dominant in the 30°C-acclimated medaka. Clones corresponding to m-30-3, m-30-4, and m-30-5 cDNAs were rarely found in the cDNA library from either the 10°C- or 30°C-acclimated medaka.

In a parallel experiment, we performed 3'-RACE for medaka acclimated to 20°C and isolated 27 cDNAs. Subsequent nucleotide sequencing revealed the existence of 15 for m-10-1 cDNA, 11 for m-10-2 cDNA, and only one for m-30-2 cDNA.

Characterization of genomic DNA clones.
To determine the genomic sequences of mMYH, the genomic BAC library constructed for medaka HNI strain was screened using a cDNA probe specific to mMYH, yielding 28 putative clones. To confirm the existence of positive inserts, we carried out Southern blot hybridization using the same probe as described above (Fig. 5). As a result, 20 out of 28 clones appeared to contain mMYH. It is likely that homologous loci existed that retained a sufficient level of DNA sequence homology so that most restriction fragments showed almost the same profiles, suggesting that mMYHs formed a cluster within a single contig. Since all mMYHs were not contained in a single BAC clone we chose clones 70M19 and 36I15 for further studies.


Figure 5
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  		Fig. 5. Location of a mMYH specific-cDNA probe and Southern blot patterns in screening of medaka genomic bacterial artificial chromosome (BAC) library for fast skeletal mMYHs. A: schematic representation of MYH mRNA at the top for orientation purposes and the exon-intron structure of carp I-type MYH (33) at the bottom. Exons are represented by black boxes, and introns are represented by bars. Amino acid sequences around major proteolytic sites of MYH are connected to their coding region by dashed lines. The gray bar represents a location of the probe used to screen medaka genomic BAC library. The locations of primers, MHC3F and MYH-10R, are shown below. Refer to the legend of Fig. 2 for abbreviations used. B: Southern blot patterns for fast skeletal mMYHs in genomic BAC clones. BAC clones screened from the medaka BAC library using a cDNA probe from the 3'-translated and untranslated region of mMYH were digested with HindIII, subjected to Southern blot analysis, and hybridized with the mMYH-specific cDNA probe described above. The boxed clones of 70M19 and 36I15 were subjected to sequencing.

 
A shotgun strategy revealed that the sizes of genomic BAC clones 70M19 and 36I15 were 164.3 and 167.5 kbp, respectively. The sequences of the two clones were compared by a dot-matrix program, and their relative position was determined (data not shown). Because the two BAC clones showed an identical sequence of 35.8 kbp, this region duplicated was corrected, and finally a combined sequence of 296 kbp was obtained (Fig. 6). A BLAST search on the 70M19-36I15 sequence demonstrated that Notch3 existed in 45 kbp upstream the first mMYH. Notch3 is known as a member of the Notch gene family that is expressed in a variety of tissues during development (26). Torafugu also contain Notch3 upstream a MYH cluster (18). Subsequent analysis of the sequence revealed that the region of 67.3–286 kbp contained a cluster of tandemly arrayed 11 mMYHs of the same transcriptional direction, which were sequentially annotated from the 5'-end as mMYH-1 through mMYH-11. All mMYHs except mMYH-8 were 10–12 kbp in genome size in agreement with that of common carp I-type MYH (33). Interestingly, the size of mMYH-8 was only ~2 kbp, suggesting that it encodes no functional protein. The genomic sequences thus obtained were compared with the sequences of mMYH cDNAs isolated from the 10°C- and 30°C-acclimated medaka cDNA libraries. As a result, mMYH-1, mMYH-2, mMYH-3, mMYH-6, mMYH-7, mMYH-9, and mMYH-11 corresponded to m-30-4, m-30-3, m-10-1, m-10-2, m-30-1, m-30-2, and m-30-5 cDNAs, respectively. These nucleotide sequences have been registered into the DDBJ/EMBL/GenBank databases with the following accession numbers: mMYH-1, AB258222; mMYH-2, AB258223; mMYH-3, AB258224; mMYH-4, AB258219; mMYH-5, AB258221; mMYH-6, AB258220; mMYH-7, AB258225; mMYH-8, AB258217; mMYH-9, AB258226; mMYH-10, AB258218; and mMYH-11, AB258227.


Figure 6
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  		Fig. 6. Schematic diagram of genomic clone 70M19-36I15. The sequence of 70M19-36I15 is indicated along a bar. Black, white, and shaded boxes indicate functional mMYH, pseudogene, and notch3 (26), respectively. Arrows indicate the direction of transcription.

 
Exon-intron organization of mMYHs.
As shown in Fig. 7, exon-intron organizations of mMYHs were analyzed referring to those described for common carp I-type MYH (33). Consensus rules for splice junctions (5) were taken into consideration in determining the 5'- and 3'-boundaries of individual exons. In addition, we used Genscan (http://genes.mit.edu/GENSCANinfo.html/) to identify exon-intron boundaries in mMYHs and confirmed these results by visually aligning sequences with the intron splice consensus rule in regions flanking exons. The 5'- and 3'-UTR of mMYHs were determined by RACE (data not shown) as well as by comparing cDNA sequences obtained from the 10°C- and 30°C-acclimated medaka cDNA libraries (see Fig. 2). Each mMYHs contained a total of 41 exons distributed over 10–12 kbp (Fig. 7). While the 5'-UTR of 55–81 bp was split by two introns, the third exon contained the 5'-UTR of 14–19 bp, followed by 38 exons consisting of 5,805–5,817 bp after the translation start site, which were supposed to be translated into 1,935–1,939 amino acids. The last exons encoded 3–6 COOH-terminal amino acids followed by a stop codon and a 3'-UTR of 85–104 bp. An ATTAAA variant of the consensus polyadenylation signal (3) was found 67–82 bp downstream the stop codon.


Figure 7
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  		Fig. 7. The genomic structure of mMYHs. Exons and introns are represented by boxes and bars, respectively. Gray structures show pseudogenes, and asterisks and filled circles (•) show the initiation and stop codons, respectively. A filled square ({blacksquare}) shows the stop codon inserted in mMYH-4.

 
S1, S2, and LMM regions were encoded by exons 3–22, 22–29, and 29–41, respectively (data not shown). None of the domains’ boundaries corresponded exactly to exon junctions, thus the above relationship between exons and the three MYH domains is of an approximate evaluation. The lengths of exons and introns from mMYHs were compared with the corresponding regions of mammalian MYHs. The total length of mMYHs was about half of that from the mammalian counterparts due to shorter introns in the former (data not shown). However, the total length of the exons was almost the same between medaka and mammal MYHs as also reported previously for common carp I-type MYH (33).

Features of mMYH-4, mMYH-5, and mMYH-10.
While a stop codon was found in exon 18 of mMYH-4, exon 41 was completely missing in mMYH-10. Furthermore, mMYH-8 had only ~2 kbp in genome size from exons 18–25 and exons 23–25 were incomplete, suggesting that the three mMYHs encode no functional proteins. To confirm that mMYH-4 and mMYH-10 are pseudogenes and that mMYH-5, having a complete exon-intron structure but not found in the cDNA libraries, is expressed in medaka, RT-PCR was performed with gene-specific primers with the 10°C- and 30°C-acclimated medaka first-strand cDNAs as templates (Fig. 8). As expected, no PCR product could be obtained for mMYH-4 or mMYH-10, confirming that the two mMYHs are pseudogenes. On the other hand, we obtained the transcripts of mMYH-5 from both 10°C- and 30°C-acclimated medaka, indicating that the gene was a new functional one, although its expression level was too weak to be represented in the cDNA libraries. The nucleotide sequence of this cDNA, which we termed as a new type, matched to exactly that of mMYH-5.


Figure 8
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  		Fig. 8. Expression analysis of mMYH-4, mMYH-5, and mMYH-10 by RT-PCR. A–C: locations of gene-specific primers for mMYHs with arrows; D–F: electrophoretic patterns of PCR products. PCR was performed using either cDNA (lanes 1, first-strand cDNA from the 10°C-acclimated medaka; lanes 2, first-strand cDNA from the 30°C-acclimated medaka) or genomic BAC DNA (lanes 3, BAC clone 36I15). M, molecular markers.

 
Figure 9 shows the phylogenetic tree constructed with the partial cDNA nucleotide sequences corresponding to exon 18–22. The pseudogene of mMYH-4 founded the same clade with mMYH-3/m-10-1, which were predominantly expressed in medaka acclimated to 10°C as described above, whereas the pseudogene of mMYH-8 forms the same clade with mMYH-3/m-10-1, mMYH-4 (pseudogene), and mMYH-6/m-10-2 but was paraphylic from the latter three genes. The last pseudogene of mMYH-10 was monophylic with mMYH-2/m-30-3, which was marginally expressed in medaka acclimated to 30°C.


Figure 9
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  		Fig. 9. Phylogenetic tree based on the cDNA nucleotide sequences in the region of exon 18–22 for fast skeletal MYHs from medaka and common carp. Refer to the legend of Fig. 4 for further details.

 
Comparison of nucleotide and deduced amino acid sequences in the coding region of mMYHs with those of common carp fast skeletal MYH cDNAs.
The nucleotide and deduced amino acid sequences of mMYHs were compared with those of common carp (13), for which the full-length cDNA have been reported (Table 3). The present genes of mMYH-3/m-10-1, mMYH-5/new, mMYH-6/m-10-2, mMYH-7/m-30-1, and mMYH-9/m-30-2 showed a high nucleotide sequence identify of not less than 97% and were different from genes mMYH-1/m-30-4, mMYH-2/m-30-3 and mMYH-11/m-30-5. The nucleotide sequence identity among the latter three genes was 93–96%, whereas the deduced amino acid sequence identity was 93–99%. The nucleotide and deduced amino acid identity among medaka and common carp were in the ranges of 83–84% and 88–92%, respectively.


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  		Table 3. Nucleotide and deduced amino acid sequence identities between fast skeletal MYH cDNA clones isolated from thermally acclimated medaka in comparison whth those of common carp (%)

 
Figure 10 shows the deduced amino acid sequences around loops 1 and 2 of mMYHs, together with those of common carp fast skeletal MYHs. Although mMYHs were generally well conserved as shown in Table 3, the primary structures around loop 1 and 2 were clearly different among the 8 isoform types, suggesting their different roles in motor function. Deduced amino acid sequence of loop 1 was identical among mMYH-2/m-30-3, mMYH-3/m-10-1 and mMYH-6/m-10-2, whereas the loop 1 sequences of mMYH-5/new was identical to that of mMYH-7/m-30-1. Comparison of the former three with the latter two mMYHs showed only one amino acid variation from each other, and these five mMYHs were very different from mMYH-1/m-30-4, mMYH-9/m-30-2, and mMYH-11/m-30-5. As for loop 2, mMYH-5/new, mMYH-7/m-30-1, and mMYH-9/m-30-2 showed the same sequence, whereas the sequence was identical for mMYH-3/m-10-1 and mMYH-6/m-10-2. These five mMYHs contained three amino acid variations and were markedly different from mMYH-1/m-30-4, mMYH-2/m-30-3, and mMYH-11/m-30-5. Amino acid sequences both in loops 1 and 2 were very different between medaka and common carp.


Figure 10
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  		Fig. 10. Deduced amino acid sequences around loop 1 (A) and loop 2 (B) regions of mMYHs along with those of common carp. Refer to the legend of Fig. 2 for carp-10, carp-I, and carp-30, respectively. Amino acid residues identical to those of medaka mMYH-3 are shown by dots, and gaps are represented by dashed lines. The two loop sequences are boxed.

 
Comparison of the 5'-flanking regions of mMYHs with common carp MYH10 and MYH30.
We performed the dot-matrix (41) analysis using the 5'-flanking sequences of 5 kbp of mMYHs with those of 4.8 and 3 kbp from common carp MYH10 and MYH30 (Kobiyama et al., Ref. 27), respectively, retrieved from the database (AB201260; AB201261) (Fig. 11). While MYH10 and MYH30 showed high sequence identity area of ~2 kbp, no such identity was found between common carp and medaka mMYHs. On the other hand, mMYHs could be roughly divided into two groups based on the sequence identity in their 5'-flanking regions. The first group comprising mMYH-3/m-10-1, mMYH-4/pseudogene, mMYH-5/new, mMYH-6/m-10-2, mMYH-7/m-30-1, and mMYH-9/m-30-2 had a high sequence identity area of ~2.5–5 kbp, whereas the remaining mMYHs had unique sequences in their 5'-flanking regions. It should be mentioned that transcripts of these mMYHs, except mMYH-4/pseudogene and mMYH-5/new, were predominantly expressed in medaka acclimated to 10 or 30°C described previously. mMYH-9/m-30-2, which was predominantly expressed in medaka acclimated to 30°C with high nucleotide and deduced amino acid sequence identities in the translated region and 5'-UTR with MYH-3/m-10-1, mMYH-5/new, mMYH-6/m-10-2, and mMYH-7/m-30-1, showed a high sequence identity only in the 5'-flanking region of ~2.5 kbp. mMYH-4/pseudogene also showed a high sequence identity with the above five genes in the 5'-flanking region of ~2.5 kbp. Besides, mMYHs, which include less expressed genes in medaka acclimated to both 10 and 30°C, mMYH-1/m-30-4, mMYH-2/m-30-3, and mMYH-11/m-30-5, had unique 5'-flanking regions and showed no homology with each other. mMYH-10/pseudogene also had unique sequences in 5'-flanking region. These results suggest that the sequence identity in the 5'-flanking regions of mMYHs is closely related to their expression patterns, that is to say, genes sharing high sequence identity in the 5'-flanking regions are more likely to be highly induced upon temperature acclimation than those showing less identity to be differently regulated.


Figure 11
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  		Fig. 11. Dot matrix in a 5-kbp region for 5'-flanking sequences of mMYHs along with those of MYH10 and MYH30 from common carp. MYH10 and MYH30 represent genes expressed predominantly in common carp acclimated to 10 and 30°C (20). Diagonal lines represent the high sequence homology.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the natural environment of fish, seasonal changes are associated with three major alterations in physical conditions: day length, oxygen content, and water temperature, each of which could function as an external trigger for the expression of MYHs (51). However, the acclimation experiment in the laboratory under constant photoperiod and oxygen tension in water showed that temperature is a sufficient trigger for the change of MYHs expression (45).

In the present study, fast skeletal myofibrillar Mg2+-ATPase activity was determined from the 10°C- and 30°C-acclimated medaka. The activity was 1.2-fold higher in the 10°C- than 30°C-acclimated medaka. These results indicate that medaka also express different MYH isoforms with different Mg2+-ATPase activity as in the case of common carp and grass carp acclimated to altered environmental water temperatures. However, it has been reported that myofibrillar Mg2+-ATPase activity in the 10°C-acclimated common carp was 1.7-fold higher than that in the 30°C-acclimated common carp (15). A similar result was also obtained with another cyprinid fish, grass carp (46). Thus, the changes in the activity for thermally acclimated medaka were relatively small, implying that the mechanisms involved in temperature acclimation of medaka is slightly different from those of common carp and grass carp. Alternatively, medaka fast skeletal myofibrils might have been still contaminated with slow skeletal myofibrils, which usually have ATP activity lower than that of fast skeletal myofibrils (22). In the case of mammals, skeletal muscle fibers are classified into one slow-twitch (type I) and two fast-twitch (types IIA and IIB) fiber types (37). These fibers contain different MYHs, which are responsible for their different ATPase activity and speed of contraction in vivo. Medaka may have a similar set of different fiber types, responding to environmental temperature fluctuation differently from other eurythermal fish.

Various types of MYH cDNA were isolated from medaka cDNA libraries and examined for their expression patterns associated with temperature acclimation. It has been reported that the accumulated mRNA levels of the 10°C-type MYH predominantly expressed in common carp acclimated to 10°C are sixfold higher than those in the 30°C-acclimated common carp, whereas the accumulated mRNA level of the 30°C-type MYH in common carp acclimated to 30°C was 11-fold higher than that in the 10°C-acclimated fish (20). Similar temperature acclimation has been reported for grass carp (45).

Imai et al. (20) reported that the 20°C-acclimated common carp expressed both the 10°C- and I-type MYHs. The I-type MYH of grass carp was more expressed in fish acclimated to 20°C (45). In the present study, the 20°C-acclimated medaka predominantly expressed the 10°C-type MYHs, but no I-type MYH was obtained. These results indicate that the temperature acclimation system of fish fast skeletal muscle is slightly different among fish species, although the three fish certainly change skeletal MYH isoforms in response to temperature fluctuation.

The HNI inbred strain medaka genomic BAC library was screened for mMYH using a specific mMYH cDNA probe. The BAC libraries also screened with another DNA probe of ~3 kbp gave a positive region spanning to 290–293 kbp of 70M19-36I15 (data not shown). Purified BAC DNAs from each positive clone were subjected to Southern blot hybridization with the mMYH-specific cDNA probe described above to confirm the presence of positive inserts. The DNAs were subsequently determined for the insert size by digestion with NotI followed by pulsed-field gel electrophoresis and end-sequenced to examine whether another new mMYHs would exist out of the region of 70M19-36I15. As a result, no new adult fast skeletal mMYHs were found in the ranges of 70 kbp of a 5'-region and 90 kbp of a 3'-region neighboring 70M10-36I15 (data not shown). Therefore, totally 11 mMYHs including three pseudogenes were tandemly arrayed in a small region of 219 kbp. Such cluster formation has been reported for fast skeletal mMYHs in pufferfish (18), zebrafish (32), and mammals (6). However, the number of mammalian fast skeletal MYHs in one cluster is restricted to at most six, even including embryonic types, indicating that medaka contain much more adult fast skeletal MYHs in a single cluster. It is also noted that three pseudogenes of MYH are contained in this cluster. All mMYHs including the three pseudogenes were oriented in the same transcriptional direction (see Fig. 6). Intergenic regions ranging from 0.9 to 22.6 kbp were consistently shorter than those of 4.5 to 60 kbp found in six mammalian skeletal MYHs (56, 57). Similar compact intron origination is also found for other genes in teleost. Elgar et al. (7) have reported that the lengths of the genes encoding tuberous sclerosis complex 2 (TSC2) and dystrophin genes of torafugu pufferfish T. rubripes, whose genomic size is 365 Mb (2), are much shorter than those of rat and human counterparts. This pufferfish has various tropomyosin (TPM) genes (TPM1-TPM4), again with much reduced sizes compared with those of human counterparts in spite of duplication of TPM1 and TPM4 in pufferfish (17, 49). For example, torafugu pufferfish TPM1-1 and TPM1-2 are about four and five times smaller than human TPM1, respectively. Thus, our findings in this study on medaka contribute to a growing understanding of genomic organization in teleost MYHs relative to corresponding mammalian genes.

The 5'-flanking region of mMYHs included muscle-specific transcription factor binding sites such as myocyte enhancer factor 2 (MEF2) binding site (14), nuclear factor of activated T cells (NFAT) binding site (24), and E box (CANNTG) (4) (data not shown). MEF2 is a member of the MEF2 transcriptional factor family (14), which interacts with the A/T-rich sequence element to directly promote transcriptional activation of the target gene exclusively in muscle cells (4). NFAT is a transcription factor regulated by calcineurin, which dephosphorylates NFAT, resulting in nuclear import and subsequent activation of NFAT-dependent promoters (24). On the other hand, E box is one of the sequence motifs recognized as a critical regulatory component in muscle gene expression (43). E boxes are the binding sites of the basic helix-loop-helix transcription factors collectively referred to as myogenic regulatory factors (MRFs) including MyoD, Myf5, myogenin, and MRF4.

To localize functional regions of MYHs responsible for its expression in a temperature-dependent manner in our previous study using common carp, a series of deletion constructs was prepared from the 5'-flanking region, inserted upstream from the luciferase gene in a commercially available plasmid, and injected into the dorsal fast muscle of carp acclimated to 10 and 30°C (27). Promoter activity analysis showed that –1 kbp of MYH10, the gene predominantly expressed in the 10°C-acclimated fish, contained the region responsible for a temperature-dependent gene expression. However, we could not identify the region that may regulate temperature-sensitive expression of MYH30, the gene predominantly expressed in the 30°C-acclimated fish, although an MEF2 binding site was shown to be implicated for expression of MYH30 in the 30°C-acclimated carp. We also found MRF binding sites in the proximal promoter region in mMYHs and demonstrated that the MEF2 binding site is crucial for a temperature-dependent expression of mMYHs (details will be described elsewhere). It is interesting to disclose how so many adult fast skeletal mMYHs occurring in one cluster are expressed in a temperature-dependent manner. It is also interesting to know how medaka have evolved to form a cluster containing so many MYHs.

In conclusion, we demonstrated that medaka contain a gene pool of eight transcriptionally active fast skeletal MYHs and that the fish adapt to fluctuating temperatures by fine-tuning the transcriptional switches of these isoforms, the precise mechanism of which awaits further elucidation.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The present study was partly supported by Grant-in-Aids for Exploratory Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, No. 16658085, and for Scientific Research (S) from Japan Society for the Promotion of Science, No. 14104008.


   ACKNOWLEDGMENTS
 
We express our sincere thanks to Dr. M. N. Ahsan, University of Khulna, Bangladesh, for critical reading of the manuscript. We also thank Drs. S. Kinoshita and D. Ikeda, The University of Tokyo, for invaluable advice.

Present address for A. Kobiyama: Laboratory of Aquatic Microbiology, School of Fisheries Sciences, Kitasato University, Ofunato, Iwate 022-0101, Japan.


   FOOTNOTES
 
Address for reprint requests and other correspondence: S. Watabe, Laboratory of Aquatic Molecular Biology and Biotechnology, Graduate School of Agricultural and Life Sciences, The Univ. of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan (e-mail: awatabe{at}mail.ecc.u-tokyo.ac.jp).

Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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