Forward and reverse genetic analysis of microtubule motors in Chlamydomonas

The ability to integrate biochemical, cell biological, and genetic approach es makes Chlamydomonas reinhardtii the premier model organism for studies o f the eukaryotic flagellum and its associated molecular motors. Hundreds of motility mutations have been identified in Chlamydomonas, including many t hat affect dyneins and kinesins. These mutations have yielded much informat ion on the structure and function of the motors as well as the roles of ind ividual subunits within the motors. The development of insertional mutagene sis has opened the door to powerful new approaches for genetic analysis in Chlamydomonas. Insertional mutants are created by transforming cells with D NA-containing selectable markers. The DNA is randomly integrated throughout the genome and usually deletes part of the chromosome at the site of inser tion, thereby creating mutations that are marked by the integrated DNA. The se mutations can be used for forward genetic approaches where one character izes a mutant phenotype and then clones the relevant gene using the integra ted DNA as a tab. The insertional mutants also may be used in a reverse gen etic approach in which mutants lacking a gene of interest ave identified by DNA hybridization. We describe methods to generate and characterize insert ional mutants, using mutations that affect the outer dynein arm as examples . (C) 2000 Academic Press.