Note that as the presence of other genes betweenMYH1andGAS7remains a possibility in the sea lamprey genome, the intergenic region is shown by a dashed line. from Japanese lampreyMYHs had no activity. These results suggest that the muscle-specific regulatory mechanisms are partially conserved between teleosts and tetrapods but not between cyclostomes and tetrapods, despite the conserved synteny. == Introduction == Myosin is a ubiquitous actin-based motor protein that drives a wide range of motile processes in eukaryotic cells. Muscle tissues contain the most classic sarcomeric myosin, called myosin II, which consists of 2 heavy chains (MYHs) and 4 light chains (reviewed in1). MYH contains 2 loop structures, loops 1 and 2, which are located Tiplaxtinin (PAI-039) in its N-terminal half, called the motor domain or subfragment-1 (S1), at the ATP- and actin-binding sites, respectively. MYH transduces chemical energy produced by splitting ATP to mechanical energy through the C-terminal half, called a rod, which has an -helical coiled-coil structure. Various types of sarcomeric MYH genes (MYHs) have been found in mammalian muscles. For example, 2 fast skeletalMYHs, called embryonicMYH3and perinatalMYH8, are expressed during pre- and postnatal development, respectively [2]. ThreeMYHs, types IIa (MYH2), IIb (MYH4) and IId/x (MYH1), are expressed in adult fast muscle. Extraocular MYH (MYH13) is expressed in extrinsic eye muscle. These 6 fast skeletalMYHs are clustered on a single chromosome, and this chromosomal organization is highly conserved among diverse mammalian species [3,4], suggesting their functional importance for temporal and spatial expression during development. However, the regulatory mechanisms involved in the gene expression and functional differences of encoded myosin molecules remain mostly unknown. More diverse sarcomericMYHs have been found in the fast muscle of teleosts, such as the common carpCyprinus carpio[5-8], zebrafishDanio rerio[9-12], medakaOryzias latipes[13-15] and torafuguTakifugu rubripes[16-19], at the genomic and Tiplaxtinin (PAI-039) transcriptional levels. In addition, genomic structural and syntenic analyses have revealed that at least 13 torafugu fast skeletalMYHs are distributed in 5 genomic regions, 3 of which contain multipleMYHs forming clusters A, B and C [18] (Figure S1). These clusters are also found in the green spotted pufferfishTetraodon nigroviridis, zebrafish and medaka [12,15,18], implying that such genomic arrangement Tiplaxtinin (PAI-039) is conserved in teleosts. Interestingly, human BMP7 fast skeletalMYHs form a single cluster, but their syntenic regions in torafugu, green spotted pufferfish and medaka are duplicated, although each locus contains a singleMYH[18]. Based on phylogenetic, genomic structural and syntenic analyses, teleostMYHs in clusters A, B and C were defined as those of fast skeletal types A, B and C, respectively; non-clustered teleostMYHs and those in the human cluster were defined to be of the fast D type [18] (Figure S1). Moreover, mammals, including mouse [3], dog [20] and the amphibianXenopus tropicalis[18] (tetrapod) have a single syntenic region similar to that of humans but not similar to that of teleosts. However, the evolutionary relationship of fast skeletalMYHs among vertebrates and the functional significance of their cluster formation have remained unknown. Lampreys and hagfishes are two groups of extant jawless vertebrates (agnathans). These vertebrates belong to a monophyletic group of cyclostomes and are thought to have diversified from a common ancestor shared with gnathostomes (jawed vertebrates) 535-462 million years ago [21]. Due to their unique phylogenetic history among vertebrates, evolutionary and developmental studies have been carried out extensively on lampreys (reviewed in21,22). Two fast skeletalMYHs (LjMyHC1andLjMyHC2) and 2 non-muscleMYHs (LjMyHC3andLjMyHC4) have been identified from an embryonic cDNA library of Japanese lampreyLethenteron camtschaticum(renamed fromLethenteron japonicumin the NCBI Taxonomy database) [23]. Although phylogenetic and gene expression analyses on Japanese lampreyMYHs were partially carried out [23], information about their gene structure and location in the genome that would be helpful in increasing our knowledge about the evolutionary relationship of vertebrateMYHs is not available. In the present study, to elucidate the evolutionary relationship among vertebrate fast skeletalMYHs, we compared those from teleosts and tetrapods with their lamprey counterparts. We.