A potentially exhaustive screening strategy reveals two novel divergent myosins in Dictyostelium.
In recent years, the myosin superfamily has kept expanding at an explosive rate, but the understanding of their complex functions has been lagging. Therefore, Dictyostelium discoideum, a genetically and biochemically tractable eukaryotic amoeba, appears as a powerful model organism to investigate the involvement of the actomyosin cytoskeleton in a variety of cellular tasks. Because of the relatively high degree of functional redundancy, such studies would be greatly facilitated by the prior knowledge of the whole myosin repertoire in this organism. Here, we present a strategy based on PCR amplification using degenerate primers and followed by negative hybridization screening which led to the potentially exhaustive identification of members of the myosin family in D. discoideum. Two novel myosins were identified and their genetic loci mapped by hybridization to an ordered YAC library. Preliminary inspection of myoK and myoM sequences revealed that, despite carrying most of the hallmarks of myosin motors, both molecules harbor features surprisingly divergent from most known myosins.
Department of Molecular Cell Research, Max-Planck-Institute for Medical Research, Heidelberg, Germany.
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Schwarz
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Geissler
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Soldati
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D002478:Cells, Cultured; D018244:Chromosomes, Artificial, Yeast; D003001:Cloning, Molecular; D004023:Dictyostelium / Q000737:chemistry / Q000235:genetics*; D015723:Gene Library; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D024461:Myosin Type I; D009218:Myosins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D021141:Nucleic Acid Amplification Techniques; D010802:Phylogeny; D015800:Protozoan Proteins; D012313:RNA / Q000032:analysis; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017421:Sequence Analysis / Q000379:methods; D017386:Sequence Homology, Amino Acid
C118057:MyoK protein, Dictyostelium discoideum; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C121714:myoM protein, Dictyostelium discoideum; D012313:RNA; D024461:Myosin Type I; D009218:Myosins