Cloning of the murine unconventional myosin gene Myo9b and identification of alternative splicing
Introduction
Myosins are molecular motors that convert chemical energy, in the form of ATP, to mechanical force exerted against actin. Since the description of the first class of myosins in 1987, 14 additional classes, based on phylogenetic comparisons of the motor domains and features of the tail, have been identified (Sellers et al., 1996, Wang et al., 1998). These classes have been numbered chronologically. Unconventional myosins (i.e. anything not resembling the two-headed, filament forming conventional myosins of class-II) are thought to play important roles in cell migration, endocytosis and intracellular transport (Mermall et al., 1998, Mooseker and Cheney, 1995, Titus, 1997). The particular importance of myosins in a number of sensory systems, including hearing, balance and vision, has been well documented (Hasson and Mooseker, 1997). In fact, of the four mapped myosins associated with diseases, three have been implicated in human and/or murine deafness; myosin VI in the Snell's waltzer mouse mutation (Avraham et al., 1995), myosin VIIa in human Usher syndrome and mouse shaker-1 (Gibson et al., 1995, Weil et al., 1995), and MYO15 in human nonsyndromic deafness DFNB3 and mouse shaker-2 (Probst et al., 1998, Wang et al., 1998).
Human MYOXIb belongs to the same class as the fifth unconventional myosin identified from rat, myr5 (Reinhard et al., 1995, Wirth et al., 1996). Phylogenetically, myr5 is the most distant relative of conventional myosin (myosin-II). A closely related gene myr7 has recently been cloned from rat and human (Chieregatti et al., 1998). The MYR5/MYOIXb protein contains several functional domains not found previously in myosins, including a novel N-terminal domain of 140 amino acids and a 126-amino-acid insertion in the putative-actin binding domain, which has been proposed to interact with F-actin in a standard, ATP-sensitive fashion (Reinhard et al., 1995). In addition, the tail domain contains a putative GTPase-activating protein (GAP) domain of the rho/rac family of ras-like G-proteins (Reinhard et al., 1995, Wirth et al., 1996). It has been proposed that myr5 could be involved in the regulation of the organization of the actin filaments along which it might produce force and movement (Reinhard et al., 1995). Northern blot analysis of both myr5 and MYOIXb has shown that these myosins are widely expressed but show considerable variability in expression level, being especially concentrated in peripheral blood leukocytes (Reinhard et al., 1995, Wirth et al., 1996). Using anti-myr5 antibodies in immunofluorescence, Wells et al. (1997) showed Myo9b, along with several other members of the myosin superfamily, to be expressed at higher levels in murine myoblasts than in mature myotubes.
To date, myosin IXb cDNAs have only been cloned from human (Bement et al., 1994, Wirth et al., 1996) and rat (Reinhard et al., 1995). Here, we report a full-length cDNA for the mouse. Several alternative splice forms of murine Myo9b are described. In addition, an unusual polymorphism consisting of an in-frame imperfect duplication within the coding region of the gene has been identified.
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cDNA cloning and sequencing
An EST clone (GenBank Accession No. AA249912) with 90% nucleotide identity to the published sequence of myr5 was used as a probe to screen approximately 300 000 plaque-forming units of a mouse spleen λZAP II cDNA library. Final washes were performed at 65°C in 1× SSC, 0.1% SDS for 30 min. Four positive plaques were picked and resuspended in SM buffer (100 mM NaCl, 10 mM MgSO4, 35 mM Tris, pH 7.5) and rescreened twice before further analysis. The cDNAs were excised in vivo in pBluescript phagemid
Results and discussion
Based on its location on mouse chromosome 8 (Ceci and Mills, 1997), Myo9b could be considered as a candidate for the mouse mutations myodystrophy or quinky. Mouse models of human disease are invaluable for gene identification studies as well as for investigating disease pathogenesis. Although the precise function of class IX myosins remains unclear, the association of unconventional myosins with deafness is recognized (Hasson et al., 1999). Interestingly the human homologue, myosin IXB, maps to
Conclusions
- 1.
We report the cloning of a cDNA encoding the murine unconventional myosin IXB, Myo9b. The isolation of this cDNA is an important step towards the analysis of the genomic structure.
- 2.
The murine Myo9b gene has a widespread expression pattern, including spleen, brain, muscle and inner ear.
- 3.
The murine Myo9b gene contains at least three alternatively spliced exons. Two of these are located in equivalent positions to alternative sequences reported for myr7/MyoIXA (Chieregatti et al., 1998). The third
Acknowledgements
The authors thank Laurence Hall (Wellcome Trust, Grant no. 044327/Z/95/Z) for DNA sequencing and the MRC HGMP-Resource Centre for providing the EUCIB DNA. This work was funded by grants from the Muscular Dystrophy Group of Great Britain (P.K.G. and J.E.H.) and the Medical Research Council (M.M.). A.-M.J. was funded by an MRC HGMP Ph.D. studentship.
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Present address: Institute of Genetics, Queen's Medical Centre, University of Nottingham, Nottingham NG7 2UH, UK.