PURIFICATION OF BASAL BODIES AND BASAL BODY COMPLEXES FROM CHLAMYDOMONAS-REINHARDTII

The microtubule organizing center (MTOC) in Chlamydomonas is defined as the pair of basal bodies, the rootlet microtubules, the distal and proximal striated fibers, and the nucleobasal body connectors (NBBCs). In Chlamydomonas, three different protocols can be used to obtain isolated basal body complexes. All of the isolated complexes include basal bodies and proximal fibers, but they differ in whether they contain other structures. Removal of the cell wall is a required step in preparing basal bodies. Autolysin is an enzyme produced by vegetative and gametic cells for removing the cell wall. Vegetative autolysin is used in the shedding of the mother cell wall following cell division. Gametic autolysin is used in the shedding of the cell wall prior to cytoplasmic fusion of mating cells. The steps required for preparation of autolysins have been mentioned in this chapter, along with the solutions and isolation of basal body complexes and of flagellum-basal body complexes. The isolated complexes can be treated with pressure and then centrifuged to separate basal bodies and proximal fibers from the flagella and other fiber systems. The presence of ethylene-diaminetetraacetic acid (EDTA) in this protocol is important to retaining the flagella and basal bodies together in the early steps. Isolation of nucleobasal body-flagellum complexes is a method for isolating a more complex structure that contains nuclei. This protocol relies on the use of autolysin or on cell wall-defective strains. The chapter also discusses components of basal body complexes. The isolation of mutations that affect the MTOC may be accomplished by classic means. Potential mutant phenotypes may include flagellar assembly defects and conditional lethality. The advent of insertional mutagenesis makes it possible to screen for mutations that are tagged with transforming DNA, which should facilitate the cloning of the locus of interest. © 1995, Academic Press Inc.