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Diversity of the pufferfish Takifugu rubripes fast skeletal myosin heavy chain genes
Comparative Biochemistry and Physiology, Part D 1 (2006) 28 – 34 www.elsevier.com/locate/cbpd
Review
Diversity of the pufferfish Takifugu rubripes fast skeletal myosin heavy chain genesB Shugo Watabe *, Daisuke Ikeda Laboratory of Aquatic Molecular Biology and Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo 113-8657, Japan Received 16 April 2005; received in revised form 23 October 2005; accepted 6 December 2005 Available online 2 February 2006
Abstract Myosin is a highly conserved, ubiquitous actin-based molecular motor that is distributed as diverse as from prokaryotes to mammalian tissues. Among various types in the myosin family proteins, class II, also called sarcomeric, myosin is a classical, conventional molecule that has been extensively studies on its functional and structural properties. It consists of two heavy chains (MYH) of about 200 kDa and four light chains of about 20 kDa. The exon – intron organization was determined for the major subunit of MYH, which contains ATP-hydrolysis and actin-binding sites, from torafugu (tiger pufferfish) Takifugu rubripes fast skeletal muscles. Comprehensive investigation for fast skeletal MYHs based on the fugu (torafugu) genome database and subsequent construction of their physical map revealed that torafugu contains at least 8 putative skeletal MYHs. Furthermore, genomic structural analysis revealed that skeletal MYHs are not clustered in a single locus, but rather spread to at least four loci, with two of them locating at the mammalian syntenic regions. Such arrangement of torafugu MYHs are in a marked contrast to mammalian fast skeletal MYHs that are clustered in a single locus. These data suggest that an ancient segmental duplication or whole-genome duplication occurred in fish lineage as in many other reported torafugu genes. D 2005 Elsevier Inc. All rights reserved. Keywords: Exon – intron organization; Fast skeletal muscle; Gene duplication; Myosin heavy chain gene; Physical map; Takifugu rubripes; Torafugu
Contents 1. Introduction. . . . . . . . . . . . . . . . . . . . . . . 2. Myosin heavy chain isoforms and muscle development 3. Myosin heavy chain genes . . . . . . . . . . . . . . . 4. Genomic structural analysis of torafugu MYH cluster . 5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .
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i This paper was presented at the TODAI International Symposium on Functional Genomics of Pufferfish Recent Advances and Perspective, The University of Tokyo, Yayoi Auditorium, Tokyo, Japan, 3rd – 6th Nov 2004. Abbreviations: I type, carp intermediate-type myosin heavy chain isoform; LMM, light meromyosin; MYH, myosin heavy chain; S1, myosin subfragment-1; S2, myosin subfragment-2. * Corresponding author. Tel.: +81 3 5841 7520; fax: +81 3 5841 8166. E-mail address: [email protected] (S. Watabe).
1744-117X/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cbd.2005.12.001
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1. Introduction The pufferfish Takifugu rubripes is called torafugu in Japanese, where ‘‘tora’’ means tiger, after its striped skin pattern. The muscle part is edible in Japan, which is one of the most delicious seafood dishes. Myosin accounts for about 50% in total muscle proteins and thus it is reasonably
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predicted that myosin properties affect the quality of muscle in animals consumed as protein resources. For example, these determine postmortem changes and shelf life of fish. Besides such industrial demands, the expression of skeletal myosin isoforms is closely associated with muscle development and recruitment of muscle fibers and possibly nervous fibers as well. Thus it is important to characterize skeletal myosin isoforms both in the viewpoints of fundamental and practical purposes. Recent progress in molecular biological techniques has enabled cloning genes encoding myosin heavy chains (MYH) and light chains from fish. Subsequently the myosin molecule is now more deeply understood at the functional and structural levels. Comparing genome sequences and structures between vertebrates of different lineages is critical in understanding functions, mechanisms involved in transcriptional regulation, and molecular evolution of genes. The divergence of mammals from fish is far older, thus it is a good challenge to disclose the physical map of fish skeletal MYHs. However, in contrast with the mammalian skeletal MYH organization, little has been known about fish even for the total number of the genes (Kikuchi et al., 1999). Interestingly, retaining a gene repertoire similar to human, the torafugu genome size is about 400 Mb, i.e. 7.5 times smaller than that of human (Brenner et al., 1993). The torafugu genome is thus ideal for comparative genomics due to consequent small sizes of introns and intergenic regions (Elgar et al., 1996; Venkatesh et al., 2000) and thus the whole genome sequencing project has been carried out (Clark et al., 2001; Aparicio et al., 2002). Using the draft sequence assembly data with merits from its compactness, we have recently analyzed torafugu MYHs through in silico data mining (Ikeda et al., 2004).
2. Myosin heavy chain isoforms and muscle development lineage Myosin is highly conserved, ubiquitous actin-based motor protein that drives a wide range of motile processes in eukaryotic cells. It consists of various types divided into 9 –11 classes. Conventional type class II includes the extensively studied group of sarcomeric myosins (Berg et al., 2001). It is also the most abundant protein in the contractile apparatus as described above, essential for the contractile process. Sarcomeric myosin consists of two MYHs of about 200 kDa and four light chains of about 20 kDa (Harrington and Rodgers, 1984) (Fig. 1). An Nterminal half of each heavy chain folds into globular head, called subfragment-1 (S1), with two light chains. In contrast, C-terminal halves of two heavy chains, called rod, fold into a-helical coiled-coil structure and have ability to form thick filaments. Myosin is organized in situ into thick filaments in which the heads of the myosin molecules protrude from the thick filaments surface and form cross-bridges with actin-containing thin filaments. Each cross-bridge is thought to be composed of two
29
Essential light chain Regulatory light chain
HMM
LMM
S1
Rod S2 hinge
Fig. 1. The structure of sarcomeric myosin molecule. Abbreviations used are: HMM, heavy meromyosin; LMM, light meromyosin; S1, subfragment1; S2, subfragment-2.
myosin heads, or S1 units, and each of these heads contains a site for ATP hydrolysis and a site for interaction with actin. S1 contains functionally important regions called loop 1 and loop 2. Loop 1 is located near the nucleotide-binding pocket and likely to tune the rate constant for ADP release, whereas loop 2 is one of the actin-binding sites (Goodson et al., 1999). In mammalian skeletal muscles, members of MYH multigene family are expressed in a complex, sequential fashion during development (Sellers and Goodson, 1995). Embryonic, neonatal and adult isoforms are predominantly expressed during respective developmental stages. Furthermore, different isoforms are expressed in fast and slow skeletal muscle fibres and also in cardiac muscles besides skeletal muscles, eight sarcomeric MYHs including six skeletal and two cardiac, in mammals. Two developmental isoforms, embryonic (MYH3) and perinatal (MYH8) MYHs, belong to the fast skeletal type and are expressed during pre- and postnatal development of skeletal muscle, respectively (Lyons et al., 1990). Three adult skeletal muscle isoforms, types IIa (MYH2), IIb (MYH4) and IId/x (MYH1), are expressed primarily in fast fibres. Extraocular MYH (MYH13) is expressed in extrinsic eye muscle, whereas two cardiac types, h (also known as type I, MYH7) and a (MYH6), are expressed predominantly in heart and the h type is additionally expressed in slow fibres. Totally six genes encoding the fast skeletal MYH isoforms are found in tightly linked clusters on chromosomes 17 and 11 in human and mouse, respectively, and their genome organization is highly conserved in spite of their divergence approximately 75 – 110 million years ago. The physical order of these genes from the 5V end is embryonic, IIa, IId/x, IIb, perinatal and extraocular, but, unlike many clustered gene families, does not necessarily reflect the known temporal expression patterns (Weiss et al., 1999; Shrager et al., 2000). However, this evidence suggests that the physical organization of the clustered MYHs is significant for the regulation of their expression patterns. Although differential expression of muscle structural proteins including the class II MYH is a major contributing factor in the diversity of skeletal muscle
30
S. Watabe, D. Ikeda / Comparative Biochemistry and Physiology, Part D 1 (2006) 28 – 34
named 10 -C and 30 -C types, since they were predominantly contained in carp acclimated to their respective habitat temperatures (Imai et al., 1997). Another one defined as the intermediate type (I type) has an intermediate feature for both DNA nucleotide and deduced amino acid sequences between those of the 10 -C and 30 -C types. Most striking differences between the three carp MYH isoforms were found in the sequences in the two S1 loop regions (Hirayama and Watabe, 1997; Watabe, 2002). A considerable number of sarcomeric MYHs have been determined in both vertebrates and invertebrates. The complete sequences have been reported in vertebrates for several sarcomeric MYHs from rat (Rattus norvegicus) (Strehler et al., 1986) and chicken (Gallus gallus) embryonic (Molina et al., 1987), human (Homo sapiens) h cardiac (Jaenicke et al., 1990) and quail (Coturnix japonica) slow muscles (Nikovits et al., 1996). Among the invertebrates, the full-length MYHs have been reported for nematode (Caenorhabditis elegans) body wall (Karn et al., 1983), fruit fly (Drosophila melanogaster) skeletal (George et al., 1989) and scallop (Argopecten irradians) striated and smooth (Nyitray et al., 1994) muscles. The organization of vertebrate MYHs differs from that of invertebrates, although MYHs from both vertebrate and invertebrate have consensus DNA nucleotide and deduced amino acid sequences in the exon regions (Fig. 2). Interestingly, D. melanogaster skeletal (George et al., 1989) and scallop striated and smooth (Nyitray et al.,
fibers, this article is focused mostly on fast skeletal MYHs including those of embryonic type.
3. Myosin heavy chain genes Carp Cyprinus carpio has been used as a model fish for physiological and biochemical studies. Myosin is not exceptional and it has been revealed that its fast skeletal muscle contains sarcomeric myosin structurally similar to those of mammals (Watabe et al., 1998). Previously, Gerlach et al. (1990) and Kikuchi et al. (1999) have reported MYH gene multiplicity for carp. Kikuchi et al. (1999) also demonstrated 29 non-overlapping k clones which encoded skeletal or cardiac muscle type. Carp are known to tolerate broad seasonal temperatures and express temperature-related MYH isoforms in adult fish at both the protein and mRNA levels. Different MYH isoforms have different actin-activated Mg2+-ATPase activities (Guo et al., 1994). An isoform from low temperature exhibits a high ATPase activity, and vice versa for a high temperature isoform. It is well known that temperature acclimation of eurythermal fish such as goldfish Carassius auratus improve their swimming ability at their respective temperatures (Fry and Hart, 1948). We have isolated three types of cDNA clones encoding fast skeletal MYHs from thermally acclimated carp (Watabe et al., 1995; Imai et al., 1997). Two of the three types were Rat emb Exon1
Exon41
Chicken emb Exon1
Exon40
Carp Exon1
Exon41
Torafugu Exon3
Exon41
Nematode
5kbp Exon1
Exon9
Fruit fly Exon1
ab Exon3
a bcd Exon7
abc Exon9
ea b c d Exon11
ab Exon15
Exon19
Fig. 2. The structure of carp and torafugu MYHs in comparison with those of rat, chicken, nematode and fruit fly MYHs. Exons are represented with black boxes. Alternative exons in the fruit fly gene are alphabetically represented. Carp and torafugu represent adult fast skeletal MYHs of MYH I from carp (Cyprinus carpio) (Muramatsu-Uno et al., 2005) and M743 MYH from torafugu (Takifugu rubripes) (unpublished data), respectively. Rat emb and chicken emb represent rat (Rattus norvegicus) (Strehler et al., 1986) and chicken (Gallus gallus) (Molina et al., 1987) embryonic MYHs, respectively. Nematode and fruit fly represent the body wall muscle MYH from Caenorhabditis elegans (Karn et al., 1983) and flight muscle MYH from Drosophila melanogaster (George et al., 1989), respectively.
S. Watabe, D. Ikeda / Comparative Biochemistry and Physiology, Part D 1 (2006) 28 – 34
1994) muscle genes express different MYH mRNAs by alternative splicing from a single gene. Recent progress of the complete genome studies revealed the full sequences of various types of myosin heavy chain genes not only for human (Desjardins et al., 2002) but also torafugu (Ikeda et al., 2004; McGuigan et al., 2004) and zebrafish (Danio rerio) (McGuigan et al., 2004). Very recently, the full sequence of MYH isolated from a genomic library has been also determined for fast skeletal muscle myosin from carp (Muramatsu-Uno et al., 2005). The full length of carp MYH I, which encodes the intermediate-type MYH, was sequenced and totally 41 exons were distributed over 11 kb (Fig. 2). The total length of carp MYH is about half that of mammalian counterparts, mostly duet to the differences in the size of introns. The torafugu fast skeletal MYH shows the same exon –intron organization as and is smaller than the carp MYH I (Ikeda et al., 2004; McGuigan et al., 2004; Muramatsu-Uno et al., 2005). We searched the torafugu genome database using carp MYHI as a probe and found 16 regions which contained the complete or partial sequences of the skeletal MYHs. Although torafugu skeletal MYHs distributed to 38 CDS, only 4 out of 16 regions contained the full-length skeletal MYHs. The sequences of 12 genes were incomplete either due to the presence of gap or location flanking the mayffolds. To avoid overestimation of the gene number of torafugu skeletal MYH, we divided MYHs at the CDS level and counted them in every CDS, concluding that torafugu contains at least 8 skeletal MYHs. To investigate the relationship among fish MYHs including those of torafugu together with human MYHs, a phylogenetic tree was constructed by the neighbor-joining method (Fig. 3). The data of torafugu MYHs are based on the deduced amino acid sequences encoded by coding sequences (CDS) 1 – 8 which are common to MYHs of M454, M743, M939, M1034, M1876, M2528-2 and M6536. Unfortunately, M86 and M2528-1 MYHs lacked CDSs 1 –5 and 1– 14, thus these MYHs could not be used for the present analysis. While torafugu M743 and M2528-2 MYHs were expressed in embryos and adult slow muscle fibers, respectively, MYHs of M454, M1876, M1034, M6536 and M939 were expressed in adult fast skeletal muscle fibers (unpublished data). These seven skeletal muscle type MYH genes including that of an embryonic type were compared in the phylogenetic tree with those from other fish, where the full-length open reading frames are available. Interestingly, torafugu embryonic type M743 MYH formed a cluster with adult fast muscle type MYHs from white croaker (Pennahia argentata) (Yoon et al., 2000), walleye pollack (Theragra chalcogramma) (Togashi et al., 2000), and chum salmon (Oncorhynchus keta) (Iwami et al., 2002), together with embryonic type MYH of zebrafish (McGuigan et al., 2004). These results suggest that MYHs from white croaker, walleye pollack, and chum salmon are possibly those of an ancestor type of fast skeletal MYH. Torafugu M454 MYH, which is expressed in adult
31
Fig. 3. Phylogenetic tree constructed by the neighbor-joining method using the deduced amino acid sequences encoded by CDS 1 – 8 for carp and torafugu MYHs and fast skeletal MYHs from various teleosts. The amino acid sequences of MYHs were deduced from cDNA nucleotide sequences with accession numbers in the DDBJ/EMBL/GenBank databases for zebrafish (Danio rerio) fast skeletal, BC071279; Chinese perch (Siniperca chuatsi) fast skeletal, AY454304; white croaker (Pennahia argentata) fast skeletal, AB039672; chum salmon (Oncorhynchus keta) fast skeletal, AB076182; amberjack (Seriola dumerili) fast skeletal, AB032020; carp (Cyprinus carpio) 30 fast skeletal, D89992; carp 20 fast skeletal, D89991; carp 10 fast skeletal, D89990; carp I-9 fast skeletal, AB182405 , hawkfish (Paracirrhites forsteri) fast skeletal, AJ243770; rockcod (Notothenia coriiceps) slow skeletal, AJ243769; walleye pollack (Theragra chalcogramma) fast skeletal, AB017819 and AB000214.
fast muscle fibers, was also monophyletic with the abovementioned ancestor type MYHs from other fish. Although the present tree had no outgroup, the phylogenetic relationships among torafugu MYHs were the same as those described in our previous report with scallop MYH as an outgroup (Ikeda et al., 2004).
4. Genomic structural analysis of torafugu MYH cluster Detailed comparison in the physical map of the human skeletal MYH cluster with that of torafugu revealed surprisingly that torafugu contained two syntenic regions and one of them, M454, contained only one MYH (Fig. 4). While one torafugu skeletal MYH was flanking at the end of M1876, M939 and M86 were connected by the BAC clone end sequences of B193A04 (70 kb). Further analysis on landmark BAC clone sequence of B218E09 revealed the existence of M6536 between M939 and M86. In addition, M2528-2 and M1034 were connected by the BAC clone end sequence of B230L18 (50 kb). The size of about 10 kb for the full-length torafugu MYH suggests that torafugu genome forms clusters of 3 – 4 MYHs in the
32
S. Watabe, D. Ikeda / Comparative Biochemistry and Physiology, Part D 1 (2006) 28 – 34
regions of M939-M6536-M86 and M2528-M1034. Detailed comparison of torafugu skeletal MYH cluster loci with the latest version of human genome (Built 34 Version 1) demonstrated a complex rearrangement in torafugu (Fig. 4). No human MYH is contained in the region corresponding to those of M939-M6536-M86, M2528M1034 and M743 (Fig. 4A – C), thus at least two clusters are likely specific to torafugu. The syntenic region of the torafugu MYH cluster of M2528-M1034 is also found in zebrafish contig (Ensembl #ctg24830), and thus this MYH cluster seems common to teleosts. Together, the present genomic structural analysis revealed that skeletal MYHs are not clustered in a single locus, but rather spread to at
A
Human
least four loci, with two of these locating to the mammalian syntenic regions. It is interesting that M1034 and M2528-2 MYHs forming one gene cluster are found within the same branch in the phylogenetic tree (see Fig. 3), whereas M743, M1876 and M454 MYHs, which do not form any gene cluster, are separated in the tree. Although M939 and M6536 MYHs form another gene cluster, these MYHs are not monophylic in the tree, suggesting the possible existence of different evolutional systems in torafugu genome at least for MYHs. Aforementioned results suggest that M1876 MYH corresponds to human MYHs and that a whole genome or a segmental duplication event had occurred before the
C
Fugu
Human
M939-M6536-M86
Fugu M743-M3457
16q22.1
RDH8 (19p13.2-p13.3) LOC123904
PSKH1 FF1C (1q32.1)
19p13.3
19p13.3 MBTPS1
BRD4
FLJ10815
FLJ22408
LOC54550
Notch3
COL5A3 (19p13.2)
MYH 16q22.1
19p13.1-2 STXBP2
NIP30 (16q13)
HYDIN
PCP2
LOC37474
CYP
XAB2
KIAA1864
MYH
KIAA1543 EDG4 (19p12) PBX4 (19p12)
B
Human
D
Fugu M2528-M1034
Human
Fugu
19q13
M1876
3p21.3 GSK3B
PH-4 6 genes
MYH
ERF CIC
SLC26A6
17p12 TSGA2 (21q22.3) ELAC2 1p36.1-36.3
LOC166348 (3q21.2)
MYH
3 genes MAP2K4
RERE LOC199873 ENO1
5 genes PPIL1 (6p21.1) SCL6A11 (3p25.3)
MDS006
SCO1
M454 SRRM2 (16p13.3)
MYH3 MYH2 MYH1
MYH
MYH4 MYH8 MYH13
RPS27AP1 GAS7 RCV1
Fig. 4. Physical map of torafugu skeletal MYH clusters and human syntenic regions containing MYHs. (A, B and C) No MYH is present in the human syntenic regions. (D) Only a single MYH is contained in the torafugu syntenic region of the human skeletal MYH cluster. Data cited from Ikeda et al. (2004) with modifications made by recent unpublished results. Black boxes represent MYHs. Other genes are shown with names used in the human content (NCBI Map Viewer, Statistics: Built 34 Version1, http://www.ncbi.nlm.nih.gov/mapview).
S. Watabe, D. Ikeda / Comparative Biochemistry and Physiology, Part D 1 (2006) 28 – 34
formation of M939-M6536-M86 and M2528-M1034 MYHs clusters. We are currently carrying out further analysis using the genome database of another pufferfish Tetraodon nigroviridis (Jaillon et al., 2004) and the results obtained will be reported elsewhere. Using the torafugu genome database, it was strongly suggested that there had been a whole genome duplication event early during the evolution of ray-finned fishes (Vandepoele et al., 2004). The spread skeletal MYH loci in torafugu are likely to be formed by a whole genome duplication event and it is assumed that tandem gene duplication, like human MYH cluster, has occurred at some loci (Ikeda et al., 2004). Such implication for gene duplication in torafugu genome has been also demonstrated with tropomyosin (TPM) genes (Ikeda et al., 2003; Toramoto et al., 2004). TPMs are abundant and ubiquitous proteins, with distinct isoforms in muscle including skeletal, cardiac and smooth ones and various non-muscle cells of all eukaryotes (LeesMiller and Helfman, 1991; Perry, 2001). Four TPMs are known in vertebrates and a number of alternatively spliced tissue-specific isoforms have been reported. The TPM4 generates only one isoform, whereas alternatively spliced exons account for the creation of at least 25 reported isoforms by other three genes amongst which, in mammals and birds, TPM1 is the most complex, producing several muscle and non-muscle isoforms. Besides the presence of singles genes each for TPM2 and TPM3 in torafugu, we found that TPM1 and TPM4 contained a set of two potentially duplicated genes, suggesting that torafugu TPM1 and TPM4 resulted from a gene duplication in the fish evolutionary lineage (Ikeda et al., 2003; Toramoto et al., 2004, Meyer and Van de Peer, 2005).
5. Conclusion Comprehensive investigation for fast skeletal MYHs based on the torafugu genome database and subsequent construction on their physical map revealed that torafugu contains at least 8 putative skeletal MYHs. Genomic structural analysis revealed that skeletal MYHs are not clustered in a single locus, but rather spread to at least four loci, with two of these locating to the mammalian syntenic regions. Such arrangement of torafugu MYHs are in a marked contrast to mammalian fast skeletal MYHs that are clustered in a single locus. These data confirm that an ancient segmental duplication or whole-genome duplication occurred in fish lineage as in many other reported torafugu genes.
Acknowledgements This study was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Science, Sports, and Technology of Japan. We would like to
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express our sincere thanks to Yvonne J.K. Edwards and Greg Elgar, Comparative Genomics Group, MRC UK HGMP Resource Centre, Hinxton, Cambridge CB10 1SB, UK, for analysis on torafugu MYHs. References Aparicio, S., Chapman, J., Stupka, E., Putnam, N., Chia, J.M., Dehal, P., Christoffels, A., Rash, S., Hoon, S., Smit, A., Gelpke, M.D., Roach, J., Oh, T., Ho, I.Y., Wong, M., Detter, C., Verhoef, F., Predki, P., Tay, A., Lucas, S., Richardson, P., Smith, S.F., Clark, M.S., Edwards, Y.J., Doggett, N., Zharkikh, A., Tavtigian, S.V., Pruss, D., Barnstead, M., Evans, C., Baden, H., Powell, J., Glusman, G., Rowen, L., Hood, L., Tan, Y.H., Elgar, G., Hawkins, T., Venkatesh, B., Rokhsar, D., Brenner, S., 2002. Whole-genome shotgun assembly and analysis of the genome of Fugu rubripes. Science 297, 1301 – 1310. Berg, J.S., Powell, B.C., Cheney, R.E., 2001. A millennial myosin census. Mol. Biol. Cell 12, 780 – 794. Brenner, S., Elgar, G., Sandford, R., Macrae, A., Aparicio, S., 1993. Characterization of the pufferfish (Fugu) genome as a compact model vertebrate genome. Nature 366, 265 – 268. Clark, M.S., Smith, S.F., Elgar, G., 2001. Use of the Japanese pufferfish (Fugu rubripes) in comparative genomics. Mar. Biotechnol. 3, S130 – S140. Desjardins, P.R., Murkman, J.M., Shrager, J.B., Allmond, L.A., Stedman, H.H., 2002. Evolutionary implications of three novel members of the human sarcomeric myosin heavy chain gene family. Mol. Biol. Evol. 19, 375 – 393. Elgar, G., Sandford, R., Aparicio, S., Macrae, A., Venkatesh, B., Brenner, S., 1996. Small is beautiful: comparative genomics with the puffer fish (Fugu rubripes). Trends Genet. 12, 145 – 150. Fry, F.E., Hart, J.S., 1948. Cruising speed of goldfish in relation to water temperature. J. Fish. Res. Board Can. 7, 169 – 175. George, E.L., Ober, M.B., Emerson Jr., C.P., 1989. Functional domains of the Drosophila melanogaster muscle myosin heavy-chain gene are encoded by alternatively spliced exons. Mol. Cell. Biol. 9, 2957 – 2974. Gerlach, G.F., Turay, L., Malik, K.T.A., Lida, J., Scutt, A., Goldspink, G., 1990. Mechanisms of temperature acclimation in the carp: a molecular biology approach. Am. J. Physiol. 259, R237 – R244. Goodson, H.V., Warrick, H.M., Spudich, J.A., 1999. Specialized conservation of surface loops of myosin: evidence that loops are involved in determining functional characteristics. J. Mol. Biol. 287, 173 – 185. Guo, X.F., Nakaya, M., Watabe, S., 1994. Myosin subfragment-1 isoforms having different heavy chain structures from fast skeletal muscle of thermally acclimated carp. J. Biochem. (Tokyo) 116, 728 – 735. Harrington, W.F., Rodgers, M.E., 1984. Myosin. Ann. Rev. Biochem. 53, 35 – 73. Hirayama, Y., Watabe, S., 1997. Structural differences in the crossbridge head of temperature-associated myosin subfragment-1 isoforms from carp fast skeletal muscle. Eur. J. Biochem. 246, 380 – 387. Ikeda, D., Toramoto, T., Ochiai, Y., Suetake, H., Suzuki, Y., Minoshima, S., Shimizu, N., Watabe, S., 2003. Identification of novel tropomyosin 1 genes of pufferfish (Fugu rubripes) on genomic sequences and tissue distribution of their transcripts. Mol. Biol. Rep. 38, 83 – 90. Ikeda, D., Clark, M.S., Liang, C.S., Snell, P., Edwards, Y.J.K., Elgar, G., Watabe, S., 2004. Genomic structural analysis of the pufferfish (Takifugu rubripes) skeletal myosin heavy chain genes. Mar. Biotechnol. 6, S462 – S467. Imai, J., Hirayama, Y., Kikuchi, K., Kakinuma, M., Watabe, S., 1997. cDNA cloning of myosin heavy chain isoforms from carp fast skeletal muscle and their gene expression associated with temperature acclimation. J. Exp. Biol. 200, 27 – 34. Iwami, Y., Ojima, T., Inoue, A., Nishita, K., 2002. Primary structure of myosin heavy chain from fast skeletal muscle of chum salmon Oncorhynchus keta. Comp. Biochem. Physiol. B 133, 257 – 267.
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Jaenicke, T., Diederich, K.W., Haas, W., Schleich, J., Lichter, P., Pfordt, M., Bach, A., Vosberg, H.P., 1990. The complete sequence of the human beta-myosin heavy chain gene and a comparative analysis of its product. Genomics 8, 194 – 206. Jaillon, O., Aury, J.M., Brunet, F., Petit, J.L., Stange-Thomann, N., Mauceli, E., Bouneau, L., Fischer, C., Ozouf-Costaz, C., Bernot, A., Nicaud, S., Jaffe, D., Fisher, S., Lutfalla, G., Dossat, C., Segurens, B., Dasilva, C., Salanoubat, M., Levy, M., Boudet, N., Castellano, S., Anthouard, V., Jubin, C., Castelli, V., Katinka, M., Vacherie, B., Biemont, C., Skalli, Z., Cattolico, L., Poulain, J., De Berardinis, V., Cruaud, C., Duprat, S., Brottier, P., Coutanceau, J.P., Gouzy, J., Parra, G., Lardier, G., Chapple, C., McKernan, K.J., McEwan, P., Bosak, S., Kellis, M., Volff, J.N., Guigo, R., Zody, M.C., Mesirov, J., LindbladToh, K., Birren, B., Nusbaum, C., Kahn, D., Robinson-Rechavi, M., Laudet, V., Schachter, V., Quetier, F., Saurin, W., Scarpelli, C., Wincker, P., Lander, E.S., Weissenbach, J., Roest Crollius, H., 2004. Genome duplication in the teleost fish Tetraodon nigroviridis reveals the early vertebrate proto-karyotype. Nature 431, 946 – 957. Karn, J., Brenner, S., Barnett, L., 1983. Protein structural domains in the Caenorhabditis elegans unc-54 myosin heavy chain gene are not separated by introns. Proc. Natl. Acad. Sci. U. S. A. 80, 4253 – 4257. Kikuchi, K., Muramatsu, M., Hirayama, Y., Watabe, S., 1999. Characterization of the carp myosin heavy chain multigene family. Gene 228, 189 – 196. Lees-Miller, J.P., Helfman, D.M., 1991. The molecular basis for tropomyosin isoform diversity. BioEssays 13, 429 – 437. Lyons, G.E., Ontell, M., Cox, R., Sassoon, D., Buckingham, M., 1990. The expression of myosin genes in developing skeletal muscle in the mouse embryo. J. Cell Biol. 111, 1465 – 1476. McGuigan, K., Phillips, P.C., Postlethwait, J.H., 2004. Evolution of sarcomeric myosin heavy chain genes: evidence from fish. Mol. Biol. Evol. 21, 1042 – 1056. Meyer, A., Van de Peer, Y., 2005. From 2R to 3R: evidence for a fishspecific genome duplication (FSGD). BioEssays 27, 937 – 945. Molina, M.I., Kropp, K.E., Gulick, J., Robbins, J., 1987. The sequence of an embryonic myosin heavy chain gene and isolation of its corresponding cDNA. J. Biol. Chem. 262, 6478 – 6488. Muramatsu-Uno, M., Kikuchi, K., Suetake, H., Ikeda, D., Watabe, S., 2005. The complete genomic sequence of the carp fast skeletal myosin heavy chain gene. Gene 349, 143 – 151. Nikovits Jr., W., Wang, G.F., Feldman, J.L., Miller, J.B., Wade, R., Nelson, L., Stockdale, F.E., 1996. Isolation and characterization of an avian slow myosin heavy chain gene expressed during embryonic skeletal muscle fiber formation. J. Biol. Chem. 271, 17047 – 17056. Nyitray, L., Jancso, A., Ochiai, Y., Graf, L., Szent-Gyo¨rgyi, A.G., 1994. Scallop striated and smooth muscle myosin heavy-chain isoforms are produced by alternative RNA splicing from a single gene. Proc. Natl. Acad. Sci. U. S. A. 91, 12686 – 12690.
Perry, S.V., 2001. Vertebrate tropomyosin: distribution, properties and function. J. Muscle Res. Cell Motil. 22, 5 – 49. Sellers, J.R., Goodson, H.V., 1995. Myosin. Protein Profile 2, 1323 – 1339. Shrager, J.B., Desjardins, P.R., Burkman, J.M., Konig, S.K., Stewart, S.K., Su, L., Shah, M.C., Bricklin, E., Tewari, M., Hoffman, R., Rickels, M.R., Jullian, E.H., Rubinstein, N.A., Stedman, H.H., 2000. Human skeletal myosin heavy chain genes are tightly linked in the order embryonic-IIa-IId/x-ILb-perinatal-extraocular. J. Muscle Res. Cell Motil. 21, 345 – 355. Strehler, E.E., Strehler-Page, M.A., Perriard, J.C., Periasamy, M., NadalGinard, B., 1986. Complete nucleotide and encoded amino acid sequence of a mammalian myosin heavy chain gene. Evidence against intron-dependent evolution of the rod. J. Mol. Biol. 190, 291 – 317. Togashi, M., Kakinuma, M., Hirayama, Y., Fukushima, H., Watabe, S., Ojima, T., Nishita, K., 2000. cDNA cloning of myosin rod and the complete amino acid sequence of myosin heavy chain of walleye pollack fast skeletal myosin. Fish. Sci. 66, 349 – 357. Toramoto, T., Ikeda, D., Ochiai, Y., Minoshima, M., Shimizu, N., Watabe, S., 2004. Multiple genes organaization of pufferfish Fugu rubripes tropomyosin isoforms and tissue distribution of their transcripts. Gene 331, 41 – 51. Vandepoele, K., De Vos, W., Taylor, J.S., Meyer, A., Van de Peer, Y., 2004. Major events in the genome evolution of vertebrates: paranome age and size differ considerably between ray-finned fishes and land vertebrates. Proc. Natl. Acad. Sci. U. S. A. 101, 1638 – 1643. Venkatesh, B., Gilligan, P., Brenner, S., 2000. Fugu: a compact vertebrate reference genome. FEBS Lett. 476, 3 – 7. Watabe, S., 2002. Temperature plasticity of contractile proteins in fish muscle. J. Exp. Biol. 205, 2231 – 2236. Watabe, S., Imai, J., Nakaya, M., Hirayama, Y., Okamoto, Y., Masaki, H., Uozumi, T., Hirono, I., Aoki, T., 1995. Temperature acclimation induces light meromyosin isoforms with different primary structures in carp fast skeletal muscle. Biochem. Biophys. Res. Commun. 208, 118 – 125. Watabe, S., Hirayama, Y., Nakaya, M., Kakinuma, M., Kikuchi, K., Guo, X.F., Kanoh, S., Chaen, S., Ooi, T., 1998. Carp expresses fast skeletal myosin isoforms with altered motor functions and structural stabilities to compensate for changes in environmental temperature. J. Therm. Biol. 22, 375 – 390. Weiss, A., McDonough, D., Wertman, B., Acakpo-Satchivi, L., Montgomery, K., Kucherlapati, R., Leinwand, L., Krauter, K., 1999. Organization of human and mouse skeletal myosin heavy chain gene clusters is highly conserved. Proc. Natl. Acad. Sci. U. S. A. 96, 2958 – 2963. Yoon, S.H., Kakinuma, M., Hirayama, Y., Yamamoto, T., Watabe, S., 2000. cDNA cloning and characterization of the complete primary structure for myosin heavy chain of white croaker fast skeletal muscle. Fish. Sci. 66, 1158 – 1162.
Review
Diversity of the pufferfish Takifugu rubripes fast skeletal myosin heavy chain genesB Shugo Watabe *, Daisuke Ikeda Laboratory of Aquatic Molecular Biology and Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo 113-8657, Japan Received 16 April 2005; received in revised form 23 October 2005; accepted 6 December 2005 Available online 2 February 2006
Abstract Myosin is a highly conserved, ubiquitous actin-based molecular motor that is distributed as diverse as from prokaryotes to mammalian tissues. Among various types in the myosin family proteins, class II, also called sarcomeric, myosin is a classical, conventional molecule that has been extensively studies on its functional and structural properties. It consists of two heavy chains (MYH) of about 200 kDa and four light chains of about 20 kDa. The exon – intron organization was determined for the major subunit of MYH, which contains ATP-hydrolysis and actin-binding sites, from torafugu (tiger pufferfish) Takifugu rubripes fast skeletal muscles. Comprehensive investigation for fast skeletal MYHs based on the fugu (torafugu) genome database and subsequent construction of their physical map revealed that torafugu contains at least 8 putative skeletal MYHs. Furthermore, genomic structural analysis revealed that skeletal MYHs are not clustered in a single locus, but rather spread to at least four loci, with two of them locating at the mammalian syntenic regions. Such arrangement of torafugu MYHs are in a marked contrast to mammalian fast skeletal MYHs that are clustered in a single locus. These data suggest that an ancient segmental duplication or whole-genome duplication occurred in fish lineage as in many other reported torafugu genes. D 2005 Elsevier Inc. All rights reserved. Keywords: Exon – intron organization; Fast skeletal muscle; Gene duplication; Myosin heavy chain gene; Physical map; Takifugu rubripes; Torafugu
Contents 1. Introduction. . . . . . . . . . . . . . . . . . . . . . . 2. Myosin heavy chain isoforms and muscle development 3. Myosin heavy chain genes . . . . . . . . . . . . . . . 4. Genomic structural analysis of torafugu MYH cluster . 5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . lineage . . . . . . . . . . . . . . . . . . . .
i This paper was presented at the TODAI International Symposium on Functional Genomics of Pufferfish Recent Advances and Perspective, The University of Tokyo, Yayoi Auditorium, Tokyo, Japan, 3rd – 6th Nov 2004. Abbreviations: I type, carp intermediate-type myosin heavy chain isoform; LMM, light meromyosin; MYH, myosin heavy chain; S1, myosin subfragment-1; S2, myosin subfragment-2. * Corresponding author. Tel.: +81 3 5841 7520; fax: +81 3 5841 8166. E-mail address: [email protected] (S. Watabe).
1744-117X/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cbd.2005.12.001
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28 29 30 31 33 33 33
1. Introduction The pufferfish Takifugu rubripes is called torafugu in Japanese, where ‘‘tora’’ means tiger, after its striped skin pattern. The muscle part is edible in Japan, which is one of the most delicious seafood dishes. Myosin accounts for about 50% in total muscle proteins and thus it is reasonably
S. Watabe, D. Ikeda / Comparative Biochemistry and Physiology, Part D 1 (2006) 28 – 34
predicted that myosin properties affect the quality of muscle in animals consumed as protein resources. For example, these determine postmortem changes and shelf life of fish. Besides such industrial demands, the expression of skeletal myosin isoforms is closely associated with muscle development and recruitment of muscle fibers and possibly nervous fibers as well. Thus it is important to characterize skeletal myosin isoforms both in the viewpoints of fundamental and practical purposes. Recent progress in molecular biological techniques has enabled cloning genes encoding myosin heavy chains (MYH) and light chains from fish. Subsequently the myosin molecule is now more deeply understood at the functional and structural levels. Comparing genome sequences and structures between vertebrates of different lineages is critical in understanding functions, mechanisms involved in transcriptional regulation, and molecular evolution of genes. The divergence of mammals from fish is far older, thus it is a good challenge to disclose the physical map of fish skeletal MYHs. However, in contrast with the mammalian skeletal MYH organization, little has been known about fish even for the total number of the genes (Kikuchi et al., 1999). Interestingly, retaining a gene repertoire similar to human, the torafugu genome size is about 400 Mb, i.e. 7.5 times smaller than that of human (Brenner et al., 1993). The torafugu genome is thus ideal for comparative genomics due to consequent small sizes of introns and intergenic regions (Elgar et al., 1996; Venkatesh et al., 2000) and thus the whole genome sequencing project has been carried out (Clark et al., 2001; Aparicio et al., 2002). Using the draft sequence assembly data with merits from its compactness, we have recently analyzed torafugu MYHs through in silico data mining (Ikeda et al., 2004).
2. Myosin heavy chain isoforms and muscle development lineage Myosin is highly conserved, ubiquitous actin-based motor protein that drives a wide range of motile processes in eukaryotic cells. It consists of various types divided into 9 –11 classes. Conventional type class II includes the extensively studied group of sarcomeric myosins (Berg et al., 2001). It is also the most abundant protein in the contractile apparatus as described above, essential for the contractile process. Sarcomeric myosin consists of two MYHs of about 200 kDa and four light chains of about 20 kDa (Harrington and Rodgers, 1984) (Fig. 1). An Nterminal half of each heavy chain folds into globular head, called subfragment-1 (S1), with two light chains. In contrast, C-terminal halves of two heavy chains, called rod, fold into a-helical coiled-coil structure and have ability to form thick filaments. Myosin is organized in situ into thick filaments in which the heads of the myosin molecules protrude from the thick filaments surface and form cross-bridges with actin-containing thin filaments. Each cross-bridge is thought to be composed of two
29
Essential light chain Regulatory light chain
HMM
LMM
S1
Rod S2 hinge
Fig. 1. The structure of sarcomeric myosin molecule. Abbreviations used are: HMM, heavy meromyosin; LMM, light meromyosin; S1, subfragment1; S2, subfragment-2.
myosin heads, or S1 units, and each of these heads contains a site for ATP hydrolysis and a site for interaction with actin. S1 contains functionally important regions called loop 1 and loop 2. Loop 1 is located near the nucleotide-binding pocket and likely to tune the rate constant for ADP release, whereas loop 2 is one of the actin-binding sites (Goodson et al., 1999). In mammalian skeletal muscles, members of MYH multigene family are expressed in a complex, sequential fashion during development (Sellers and Goodson, 1995). Embryonic, neonatal and adult isoforms are predominantly expressed during respective developmental stages. Furthermore, different isoforms are expressed in fast and slow skeletal muscle fibres and also in cardiac muscles besides skeletal muscles, eight sarcomeric MYHs including six skeletal and two cardiac, in mammals. Two developmental isoforms, embryonic (MYH3) and perinatal (MYH8) MYHs, belong to the fast skeletal type and are expressed during pre- and postnatal development of skeletal muscle, respectively (Lyons et al., 1990). Three adult skeletal muscle isoforms, types IIa (MYH2), IIb (MYH4) and IId/x (MYH1), are expressed primarily in fast fibres. Extraocular MYH (MYH13) is expressed in extrinsic eye muscle, whereas two cardiac types, h (also known as type I, MYH7) and a (MYH6), are expressed predominantly in heart and the h type is additionally expressed in slow fibres. Totally six genes encoding the fast skeletal MYH isoforms are found in tightly linked clusters on chromosomes 17 and 11 in human and mouse, respectively, and their genome organization is highly conserved in spite of their divergence approximately 75 – 110 million years ago. The physical order of these genes from the 5V end is embryonic, IIa, IId/x, IIb, perinatal and extraocular, but, unlike many clustered gene families, does not necessarily reflect the known temporal expression patterns (Weiss et al., 1999; Shrager et al., 2000). However, this evidence suggests that the physical organization of the clustered MYHs is significant for the regulation of their expression patterns. Although differential expression of muscle structural proteins including the class II MYH is a major contributing factor in the diversity of skeletal muscle
30
S. Watabe, D. Ikeda / Comparative Biochemistry and Physiology, Part D 1 (2006) 28 – 34
named 10 -C and 30 -C types, since they were predominantly contained in carp acclimated to their respective habitat temperatures (Imai et al., 1997). Another one defined as the intermediate type (I type) has an intermediate feature for both DNA nucleotide and deduced amino acid sequences between those of the 10 -C and 30 -C types. Most striking differences between the three carp MYH isoforms were found in the sequences in the two S1 loop regions (Hirayama and Watabe, 1997; Watabe, 2002). A considerable number of sarcomeric MYHs have been determined in both vertebrates and invertebrates. The complete sequences have been reported in vertebrates for several sarcomeric MYHs from rat (Rattus norvegicus) (Strehler et al., 1986) and chicken (Gallus gallus) embryonic (Molina et al., 1987), human (Homo sapiens) h cardiac (Jaenicke et al., 1990) and quail (Coturnix japonica) slow muscles (Nikovits et al., 1996). Among the invertebrates, the full-length MYHs have been reported for nematode (Caenorhabditis elegans) body wall (Karn et al., 1983), fruit fly (Drosophila melanogaster) skeletal (George et al., 1989) and scallop (Argopecten irradians) striated and smooth (Nyitray et al., 1994) muscles. The organization of vertebrate MYHs differs from that of invertebrates, although MYHs from both vertebrate and invertebrate have consensus DNA nucleotide and deduced amino acid sequences in the exon regions (Fig. 2). Interestingly, D. melanogaster skeletal (George et al., 1989) and scallop striated and smooth (Nyitray et al.,
fibers, this article is focused mostly on fast skeletal MYHs including those of embryonic type.
3. Myosin heavy chain genes Carp Cyprinus carpio has been used as a model fish for physiological and biochemical studies. Myosin is not exceptional and it has been revealed that its fast skeletal muscle contains sarcomeric myosin structurally similar to those of mammals (Watabe et al., 1998). Previously, Gerlach et al. (1990) and Kikuchi et al. (1999) have reported MYH gene multiplicity for carp. Kikuchi et al. (1999) also demonstrated 29 non-overlapping k clones which encoded skeletal or cardiac muscle type. Carp are known to tolerate broad seasonal temperatures and express temperature-related MYH isoforms in adult fish at both the protein and mRNA levels. Different MYH isoforms have different actin-activated Mg2+-ATPase activities (Guo et al., 1994). An isoform from low temperature exhibits a high ATPase activity, and vice versa for a high temperature isoform. It is well known that temperature acclimation of eurythermal fish such as goldfish Carassius auratus improve their swimming ability at their respective temperatures (Fry and Hart, 1948). We have isolated three types of cDNA clones encoding fast skeletal MYHs from thermally acclimated carp (Watabe et al., 1995; Imai et al., 1997). Two of the three types were Rat emb Exon1
Exon41
Chicken emb Exon1
Exon40
Carp Exon1
Exon41
Torafugu Exon3
Exon41
Nematode
5kbp Exon1
Exon9
Fruit fly Exon1
ab Exon3
a bcd Exon7
abc Exon9
ea b c d Exon11
ab Exon15
Exon19
Fig. 2. The structure of carp and torafugu MYHs in comparison with those of rat, chicken, nematode and fruit fly MYHs. Exons are represented with black boxes. Alternative exons in the fruit fly gene are alphabetically represented. Carp and torafugu represent adult fast skeletal MYHs of MYH I from carp (Cyprinus carpio) (Muramatsu-Uno et al., 2005) and M743 MYH from torafugu (Takifugu rubripes) (unpublished data), respectively. Rat emb and chicken emb represent rat (Rattus norvegicus) (Strehler et al., 1986) and chicken (Gallus gallus) (Molina et al., 1987) embryonic MYHs, respectively. Nematode and fruit fly represent the body wall muscle MYH from Caenorhabditis elegans (Karn et al., 1983) and flight muscle MYH from Drosophila melanogaster (George et al., 1989), respectively.
S. Watabe, D. Ikeda / Comparative Biochemistry and Physiology, Part D 1 (2006) 28 – 34
1994) muscle genes express different MYH mRNAs by alternative splicing from a single gene. Recent progress of the complete genome studies revealed the full sequences of various types of myosin heavy chain genes not only for human (Desjardins et al., 2002) but also torafugu (Ikeda et al., 2004; McGuigan et al., 2004) and zebrafish (Danio rerio) (McGuigan et al., 2004). Very recently, the full sequence of MYH isolated from a genomic library has been also determined for fast skeletal muscle myosin from carp (Muramatsu-Uno et al., 2005). The full length of carp MYH I, which encodes the intermediate-type MYH, was sequenced and totally 41 exons were distributed over 11 kb (Fig. 2). The total length of carp MYH is about half that of mammalian counterparts, mostly duet to the differences in the size of introns. The torafugu fast skeletal MYH shows the same exon –intron organization as and is smaller than the carp MYH I (Ikeda et al., 2004; McGuigan et al., 2004; Muramatsu-Uno et al., 2005). We searched the torafugu genome database using carp MYHI as a probe and found 16 regions which contained the complete or partial sequences of the skeletal MYHs. Although torafugu skeletal MYHs distributed to 38 CDS, only 4 out of 16 regions contained the full-length skeletal MYHs. The sequences of 12 genes were incomplete either due to the presence of gap or location flanking the mayffolds. To avoid overestimation of the gene number of torafugu skeletal MYH, we divided MYHs at the CDS level and counted them in every CDS, concluding that torafugu contains at least 8 skeletal MYHs. To investigate the relationship among fish MYHs including those of torafugu together with human MYHs, a phylogenetic tree was constructed by the neighbor-joining method (Fig. 3). The data of torafugu MYHs are based on the deduced amino acid sequences encoded by coding sequences (CDS) 1 – 8 which are common to MYHs of M454, M743, M939, M1034, M1876, M2528-2 and M6536. Unfortunately, M86 and M2528-1 MYHs lacked CDSs 1 –5 and 1– 14, thus these MYHs could not be used for the present analysis. While torafugu M743 and M2528-2 MYHs were expressed in embryos and adult slow muscle fibers, respectively, MYHs of M454, M1876, M1034, M6536 and M939 were expressed in adult fast skeletal muscle fibers (unpublished data). These seven skeletal muscle type MYH genes including that of an embryonic type were compared in the phylogenetic tree with those from other fish, where the full-length open reading frames are available. Interestingly, torafugu embryonic type M743 MYH formed a cluster with adult fast muscle type MYHs from white croaker (Pennahia argentata) (Yoon et al., 2000), walleye pollack (Theragra chalcogramma) (Togashi et al., 2000), and chum salmon (Oncorhynchus keta) (Iwami et al., 2002), together with embryonic type MYH of zebrafish (McGuigan et al., 2004). These results suggest that MYHs from white croaker, walleye pollack, and chum salmon are possibly those of an ancestor type of fast skeletal MYH. Torafugu M454 MYH, which is expressed in adult
31
Fig. 3. Phylogenetic tree constructed by the neighbor-joining method using the deduced amino acid sequences encoded by CDS 1 – 8 for carp and torafugu MYHs and fast skeletal MYHs from various teleosts. The amino acid sequences of MYHs were deduced from cDNA nucleotide sequences with accession numbers in the DDBJ/EMBL/GenBank databases for zebrafish (Danio rerio) fast skeletal, BC071279; Chinese perch (Siniperca chuatsi) fast skeletal, AY454304; white croaker (Pennahia argentata) fast skeletal, AB039672; chum salmon (Oncorhynchus keta) fast skeletal, AB076182; amberjack (Seriola dumerili) fast skeletal, AB032020; carp (Cyprinus carpio) 30 fast skeletal, D89992; carp 20 fast skeletal, D89991; carp 10 fast skeletal, D89990; carp I-9 fast skeletal, AB182405 , hawkfish (Paracirrhites forsteri) fast skeletal, AJ243770; rockcod (Notothenia coriiceps) slow skeletal, AJ243769; walleye pollack (Theragra chalcogramma) fast skeletal, AB017819 and AB000214.
fast muscle fibers, was also monophyletic with the abovementioned ancestor type MYHs from other fish. Although the present tree had no outgroup, the phylogenetic relationships among torafugu MYHs were the same as those described in our previous report with scallop MYH as an outgroup (Ikeda et al., 2004).
4. Genomic structural analysis of torafugu MYH cluster Detailed comparison in the physical map of the human skeletal MYH cluster with that of torafugu revealed surprisingly that torafugu contained two syntenic regions and one of them, M454, contained only one MYH (Fig. 4). While one torafugu skeletal MYH was flanking at the end of M1876, M939 and M86 were connected by the BAC clone end sequences of B193A04 (70 kb). Further analysis on landmark BAC clone sequence of B218E09 revealed the existence of M6536 between M939 and M86. In addition, M2528-2 and M1034 were connected by the BAC clone end sequence of B230L18 (50 kb). The size of about 10 kb for the full-length torafugu MYH suggests that torafugu genome forms clusters of 3 – 4 MYHs in the
32
S. Watabe, D. Ikeda / Comparative Biochemistry and Physiology, Part D 1 (2006) 28 – 34
regions of M939-M6536-M86 and M2528-M1034. Detailed comparison of torafugu skeletal MYH cluster loci with the latest version of human genome (Built 34 Version 1) demonstrated a complex rearrangement in torafugu (Fig. 4). No human MYH is contained in the region corresponding to those of M939-M6536-M86, M2528M1034 and M743 (Fig. 4A – C), thus at least two clusters are likely specific to torafugu. The syntenic region of the torafugu MYH cluster of M2528-M1034 is also found in zebrafish contig (Ensembl #ctg24830), and thus this MYH cluster seems common to teleosts. Together, the present genomic structural analysis revealed that skeletal MYHs are not clustered in a single locus, but rather spread to at
A
Human
least four loci, with two of these locating to the mammalian syntenic regions. It is interesting that M1034 and M2528-2 MYHs forming one gene cluster are found within the same branch in the phylogenetic tree (see Fig. 3), whereas M743, M1876 and M454 MYHs, which do not form any gene cluster, are separated in the tree. Although M939 and M6536 MYHs form another gene cluster, these MYHs are not monophylic in the tree, suggesting the possible existence of different evolutional systems in torafugu genome at least for MYHs. Aforementioned results suggest that M1876 MYH corresponds to human MYHs and that a whole genome or a segmental duplication event had occurred before the
C
Fugu
Human
M939-M6536-M86
Fugu M743-M3457
16q22.1
RDH8 (19p13.2-p13.3) LOC123904
PSKH1 FF1C (1q32.1)
19p13.3
19p13.3 MBTPS1
BRD4
FLJ10815
FLJ22408
LOC54550
Notch3
COL5A3 (19p13.2)
MYH 16q22.1
19p13.1-2 STXBP2
NIP30 (16q13)
HYDIN
PCP2
LOC37474
CYP
XAB2
KIAA1864
MYH
KIAA1543 EDG4 (19p12) PBX4 (19p12)
B
Human
D
Fugu M2528-M1034
Human
Fugu
19q13
M1876
3p21.3 GSK3B
PH-4 6 genes
MYH
ERF CIC
SLC26A6
17p12 TSGA2 (21q22.3) ELAC2 1p36.1-36.3
LOC166348 (3q21.2)
MYH
3 genes MAP2K4
RERE LOC199873 ENO1
5 genes PPIL1 (6p21.1) SCL6A11 (3p25.3)
MDS006
SCO1
M454 SRRM2 (16p13.3)
MYH3 MYH2 MYH1
MYH
MYH4 MYH8 MYH13
RPS27AP1 GAS7 RCV1
Fig. 4. Physical map of torafugu skeletal MYH clusters and human syntenic regions containing MYHs. (A, B and C) No MYH is present in the human syntenic regions. (D) Only a single MYH is contained in the torafugu syntenic region of the human skeletal MYH cluster. Data cited from Ikeda et al. (2004) with modifications made by recent unpublished results. Black boxes represent MYHs. Other genes are shown with names used in the human content (NCBI Map Viewer, Statistics: Built 34 Version1, http://www.ncbi.nlm.nih.gov/mapview).
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formation of M939-M6536-M86 and M2528-M1034 MYHs clusters. We are currently carrying out further analysis using the genome database of another pufferfish Tetraodon nigroviridis (Jaillon et al., 2004) and the results obtained will be reported elsewhere. Using the torafugu genome database, it was strongly suggested that there had been a whole genome duplication event early during the evolution of ray-finned fishes (Vandepoele et al., 2004). The spread skeletal MYH loci in torafugu are likely to be formed by a whole genome duplication event and it is assumed that tandem gene duplication, like human MYH cluster, has occurred at some loci (Ikeda et al., 2004). Such implication for gene duplication in torafugu genome has been also demonstrated with tropomyosin (TPM) genes (Ikeda et al., 2003; Toramoto et al., 2004). TPMs are abundant and ubiquitous proteins, with distinct isoforms in muscle including skeletal, cardiac and smooth ones and various non-muscle cells of all eukaryotes (LeesMiller and Helfman, 1991; Perry, 2001). Four TPMs are known in vertebrates and a number of alternatively spliced tissue-specific isoforms have been reported. The TPM4 generates only one isoform, whereas alternatively spliced exons account for the creation of at least 25 reported isoforms by other three genes amongst which, in mammals and birds, TPM1 is the most complex, producing several muscle and non-muscle isoforms. Besides the presence of singles genes each for TPM2 and TPM3 in torafugu, we found that TPM1 and TPM4 contained a set of two potentially duplicated genes, suggesting that torafugu TPM1 and TPM4 resulted from a gene duplication in the fish evolutionary lineage (Ikeda et al., 2003; Toramoto et al., 2004, Meyer and Van de Peer, 2005).
5. Conclusion Comprehensive investigation for fast skeletal MYHs based on the torafugu genome database and subsequent construction on their physical map revealed that torafugu contains at least 8 putative skeletal MYHs. Genomic structural analysis revealed that skeletal MYHs are not clustered in a single locus, but rather spread to at least four loci, with two of these locating to the mammalian syntenic regions. Such arrangement of torafugu MYHs are in a marked contrast to mammalian fast skeletal MYHs that are clustered in a single locus. These data confirm that an ancient segmental duplication or whole-genome duplication occurred in fish lineage as in many other reported torafugu genes.
Acknowledgements This study was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Science, Sports, and Technology of Japan. We would like to
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