Isolation of homozygous mutants after induced self-fertilization in Tetrahymena.

Abstract

(see ref. 5 for a review). The micronucleus, a diploid nucleus with about the same genome as the macronucleus (6), is capable of mitotic and meiotic division, is generally inert in transcription, but is active in conjugation; new macronuclei are developed following meiosis and subsequent exchange and fertilization of the micronuclei of a mating pair.The ability to isolate induced mutations is an obvious necessity in any modem experimental genetic system. Unfortunately, in Tetrahymena the ability to generate cells suitable for mutant selection is complicated by the separation of somatic and germinal functions into different nuclei. To be expressed, a mutation must reside in the macronucleus; to be heritable through conjugation, it must be in the micronucleus. Since progeny macronuclei are developmental derivatives of micronuclei following conjugation, schemes for mutagenesis must insert a mating between induction of mutations and selection for mutant phenotypes. In order to generate homozygotes for the expression of recessive mutations, the mating should be some form of self mating.The phenomenon of genomic exclusion has held great promise for mutagenesis since Allen (7,8) demonstrated that the first round of mating results in an induced self-fertilization; Fig. 1 outlines the events associated with the entire process. The sequence is initiated by mating with a strain containing a defective micronucleus; C*, a vegetative derivative of inbred strain C (7) is the best known of these, but other strains can induce the phenomenon. Although round one mating produces cells with homozygous micronuclei derived from the non-C* conjugant, the developmental process seemingly aborts before the production of a new macronucleus; newly formed macronuclei only occur as a consequence of a second round of mating. In order to recover homozygotes in mutagenesis, round one pairs must be isolated, cloned, and mated, to insure a remating of the same conjugants in round two (10).A related paper (9) 25 ,g/ml of cycloheximide (cy) and 15 ,ug/ml of 6-methylpurine (6-mp), respectively (17).As previously described (9), functional heterokaryons for each of the two dominant markers were created by constructing the resistant heterozygotes, subcloning after vegetative growth, and retaining subclones that expressed sensitivity. Homozygous heterokaryons were created by passing heterozygous functional heterokaryons through the first round of mating in genomic exclusion, and identifying by a suitable testcross the clones which had become homozygous for the desired alleles in the 3243

LF4/MOK and a CDK-related kinase regulate the number and length of cilia inTetrahymena

“…The conjugating cells were spread as a thin layer onto a 10-cm wide Petri dish containing a layer of Tris-Agar (10 mM Tris-HCl pH 7.4, 1.5% agar) and positioned at the bottom-most slot of PDS1000/He. To generate homokaryons, we either used the standard procedure based on two rounds of genomic exclusion or the short circuit genomic exclusion (SCGE) [121] with either B*VI or B*VII [83]. Specifically, the homokaryons for SUP1, CDKR1-KO, LF4A-KO_CDKR1-KO, ovGFP-LF4A and ovGFP-LF4A_CDKR1-KO were made using the SCGE.…”

Tetrahymena in the Laboratory: Strain Resources, Methods for Culture, Maintenance, and Storage

“…Since both partners now contain identical functional micronuclei, the second round of mating proceeds normally, giving rise to progeny that are whole genome homozygotes. Star strains are a valuable genetic tool, useful for an array of genetic applications, including the construction of homozygous strains (Allen, 1967b), the creation of functional heterokaryons including gene knock-out (KO) strains (Dave et al, 2009; Hai et al, 2000), conjugation rescue (Satir et al, 1986), short circuit genomic exclusion (Bruns et al, 1976) , and uniparental cytogamy (Cole and Bruns, 1992). …”

A Genetic Screen for Vegetative Gene Expression in the Micronucleus of Tetrahymena thermophila

“…like normal conjugation, involves cross-fertilization and nuclear reorganization to produce sexually immature exconjugants. However, during round one with strain C*(III), about 4% of the conjugants (0.4% with mutagenized cells) have been found to go on to complete the formation of a new mac in the variant pathway called "short-circuit genomic exclusion" [6] (see Fig. 2).…”