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Chemical-Genetic Inhibition of Sensitized Mutant Unconventional Myosins

  • Ryan L. Karcher
  • D. William ProvanceJr.
  • Peter G. Gillespie
  • John A. Mercer
Part of the Methods in Molecular Biology™ book series (MIMB, volume 392)

Abstract

The construction of a sensitized mutant unconventional myosin is an excellent method for determining the function of the individual myosin against a background of related myosins with partially overlapping functions. In this chapter, we outline the steps involved in sensitizing myosin by mutation and screening them against panels of nucleotide analogs, including transfection, microinjection, and actin co-sedimentation in vitro. We also describe conditions and considerations involved in designing functional experiments after a mutant and cognate analog have been identified. The powerful strategy of chemical genetics, when correctly applied to unconventional myosins, enables both the specific and selectable inhibition of the target motor with outstanding internal controls.

Key Words

ATP hydrolysis chemical genetics myosin-Vb myosin-1c unconventional myosins 

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Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Ryan L. Karcher
    • 1
  • D. William ProvanceJr.
    • 1
  • Peter G. Gillespie
    • 2
  • John A. Mercer
    • 1
  1. 1.McLaughlin Research InstituteGreat Falls
  2. 2.Oregon Hearing Research Center and Vollum InstituteOregon Health and Science UniversityPortland

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