Chapter 81 Strategies for the Isolation of Ciliary Motility and Assembly Mutants in Tetrahymena

Abstract

Publisher Summary A screen for the isolation of mutations affecting ciliary regeneration in Tetrahyrnena thermophila has been deveoped. The development of transformation and gene replacement techniques in Tetrahymena should lead to increased interest in genetic analyses of ciliary function and assembly. Cells homozygous for the chp (cell cycle, heat shock, and phosphorylation defect) mutation fail to initiate ciliogenesis after deciliation and incubation at the restrictive temperature. Researchers showed that chp mutants are defective in the heat shock response. Cell maintenance medium includes 2% proteose peptone, 0.1% yeast extract, 0.2% glucose. All ingredients, bring to volume with water, aliquot approximately 6 ml into capped culture tubes, and autoclave are dissolved. Cells can be stored for 2-3 weeks between transfers. The chapter provides a clear description of the procedures and equipment for performing genetic analyses on Tetrahymena . Although the micronucleus is diploid, three ways are commonly used to bring recessive micronuclear mutations to expression in the macronucleus in Tetrahymena after mutagenesis and a single round of mating. One method is to induce cytogamy in mating pairs of Tetrahymena . To induce cytogamy, mating pairs of heterokaryons are subjected to an osmotic shock during mating. This prevents nuclear exchange and causes the production of whole-genome homozygotes in some fraction of the pairs.