Cloning and developmental expression of kinesin superfamily7 (kif7) in the brackish medaka (Oryzias melastigma), a close relative of the Japanese medaka (Oryzias latipes)
Introduction
Kinesins are molecular motor proteins that move unidirectionally along the surface of microtubules in a highly coordinated manner through ATP hydrolysis (Brendza et al., 2000, Endow, 2003, Hirokawa, 1996, Hirokawa, 1998). Kinesin heavy chains can be broadly divided into three domains: a globular motor domain, an α-helical stalk domain, and a globular tail region (Cole and Scholey, 1995, de Cuevas et al., 1992, Hirokawa et al., 1989). Each kinesin motor contains an about 340-amino acid sequence at the head region that forms the motor domain, which shares 35–45% identities between family members (Goldstein, 2001, Hirokawa, 1998, Vale, 2003). The motor domain possesses both the microtubule and nucleotide binding sites, and is sufficient to mediate the ATP-dependent movement along microtubules (Yang et al., 1990). The stalk and tail domains, which are divergent, even within a family, are the region responsible for the specific interaction with other subunits of the holoenzyme or with cargo molecules such as proteins, lipids or nucleic acids (Diefenbach et al., 1998, Hirokawa et al., 1989).
In zebrafish (Danio rerio), kinesin superfamily7 (kif7) is reported to be the vertebrate homologue of costal2 (cos2), which is a member of the hedgehog (hh) signaling system (Tay et al., 2005). As in Drosophila, zebrafish cos2 negatively regulates the sonic hedgehog (shh) signal transduction as the loss of cos2 by morpholino oligoneuclotide injection induces ectopic activation of the shh signaling in the myotome and the developing neural tube in the zebrafish embryo (Tay et al., 2005).
As it has been documented that exposure to environmental pollutants, such as mercury (Atchison and Hare, 1994, Castoldi et al., 2003, Johansson et al., 2007), cadmium (Aguilar and Kostrzewa, 2004, Chow and Cheng, 2003), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Dong et al., 2004, Henry et al., 1997, Teraoka et al., 2002) and atrazine (Wiegand et al., 2000, Ishaque et al., 2004), could interfere the development and functioning of the nervous system and the developing brain is much more susceptible to neurotoxicants than the brain in an adult, there is a need for having model organisms for better monitoring and evaluating the effects of environmental pollutants and neurotoxicants. Although several freshwater fish species including zebrafish (D. rerio), Japanese medaka (Oryzias latipes) and Japanese pufferfish (Takifugu rubripres) have been well developed with their entire genome completely or almost completely sequenced for molecular toxicological and developmental studies, no such well-developed estuarine or marine model organism is available. In this study, Oryzias melastigma, a brackish medaka and a close relative of the Japanese medaka (O. latipes), were selected as the model organism (Nelson, 1994). O. melastigma is well-adapted to high environmental salinity. It has the advantages of being small in size (3–4 cm in length) with easy maintenance and having a short generation time of 2–3 months. Males and females can be morphologically distinguished by a clearly dimorphic dorsal fin. Taking its advantage of being a close relative of the Japanese medaka, O. melastigma can serve as a good seawater and freshwater model organism for molecular toxicological and developmental studies by utilizing the resources developed from the Japanese medaka. As medaka is phylogenetically divergent from zebrafish and both separated from their last common ancestor by about 110 million years (Myr) (Furutani-Seiki and Wittbrodt, 2004, Nelson, 1994, Wittbrodt et al., 2002), the genetic and biochemical information from these distantly related teleosts is a useful tool for comparative studies on development and evolution as well.
In order to characterize the O. melastigma kif7 (omkif7), we have cloned the omkif7 by the way of primer design based on database search on the Japanese medaka database. We have characterized the expression profile of omkif7 in embryo and adult tissue through RT-PCR analysis in comparison with zebrafish kif7, and further examined the expression pattern of omkif7 in embryo by in situ hybridization.
Section snippets
Fish maintenance
Adult O. melastigma was imported from a Taiwan fish farm and zebrafish was purchased from a local retailer. O. mleastigma was maintained in aerated seawater at 26–27 °C and zebrafish was kept in aerated fresh water at 28.5 °C. Both fishes were subjected to a photoperiod of 14 h light and 10 h dark.
Database search
Database search was performed on the database of Medaka Genome Project developed by the NIG DNA Sequencing Center (http://dolphin.lab.nig.ac.jp/medaka/) using zebrafish kif7.
Cloning of omkif7
To clone the omkif7, the gene
Sequence analysis of omkif7
The cloned gene has an open reading frame (ORF) of 3762 bp and was deduced to encode a polypeptide of 1254 amino acids. In order to characterize which kinesin superfamily the cloned medaka gene belongs to, a phylogenetic analysis of the catalytic core, which is a well-conserved domain among kinesins, of the deduced amino acid sequence of the cloned gene with other 124 kinesins in human, mouse, rat, Xenopus, Drosophila, Caenorhabditis elegans and Saccharomyces cerevisiae that were reported in
Discussion
The omkif7 was cloned by Japanese medaka database search using the sequence of zebrafish kif7. During the medaka database search, it was noted that many scaffolds with high significance to the kinesin motor region were obtained. This was probably due to the existence of large amount of kinesin required for performing diverse cellular functions. In mice, a total of 45 Kifs have been identified. (Lawrence et al., 2004, Miki et al., 2005). As it was generally believed that genome duplication
Conclusion
The omkif7 has been identified and characterized with respect to their expression profile in embryonic stages and adult organs. Sequence analysis has revealed that omkif7 encodes a protein which shares a high identity and similarity with zebrafish kif7. All the putative ATP- and microtubule-binding motifs are also well conserved in the omkif7. As the gene was cloned based on primers designed according to the Japanese medaka, this demonstrates that brackish medaka O. melastigma could serve as a
Acknowledgement
The work described in this paper was fully supported by a grant from the CityU (Project No. 7002010).
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Present address: Institute of Toxicology and Genetics, Forschungszentrum Karlsruhe, P.O. 3640, D-76021 Karlsruhe, Germany.