Genomic Exclusion: A Rapid Means for Inducing Homozygous Diploid Lines in
            <i>Tetrahymena pyriformis</i>
            , Syngen 1

Allen, Sally Lyman

doi:10.1126/science.155.3762.575

Cited by 114 publications

(58 citation statements)

References 13 publications

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“…Round I genomic exclusion (1,8,21) is a special type of abortive mating between WT and star (*) strains which have defective, hypodiploid MICs. Star strains can form pairs with WT but are not able to produce pronuclei during nuclear exchange and fertilization stages of conjugation (18,50,78).…”

Section: Methodsmentioning

confidence: 99%

“…To clarify this issue, a WT MIC was reintroduced into the S134A rescued strain to create a "rejuvenated" strain. This is possible in Tetrahymena through round I genomic exclusion (1,8,21), a special type of abortive mating between WT and star (*) strains which have defective, hypodiploid MICs (see Materials and Methods for details). Since the S134A rescued cells stopped mating at meiosis II and did not produce pronuclei, they behaved like star cells, suggesting that they had defective MICs.…”

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confidence: 99%

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Phosphorylation of the SQ H2A.X Motif Is Required for Proper Meiosis and Mitosis in Tetrahymena thermophila

Song

Gjoneska

Ren

et al. 2007

Molecular and Cellular Biology

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Phosphorylation of the C terminus SQ motif that defines H2A.X variants is required for efficient DNA double-strand break (DSB) repair in diverse organisms but has not been studied in ciliated protozoa. Tetrahymena H2A.X is one of two similarly expressed major H2As, thereby differing both from mammals, where H2A.X is a quantitatively minor component, and from Saccharomyces cerevisiae where it is the only type of major H2A. Tetrahymena H2A.X is phosphorylated in the SQ motif in both the mitotic micronucleus and the amitotic macronucleus in response to DSBs induced by chemical agents and in the micronucleus during prophase of meiosis, which occurs in the absence of a synaptonemal complex. H2A.X is phosphorylated when programmed DNA rearrangements occur in developing macronuclei, as for immunoglobulin gene rearrangements in mammals, but not during the DNA fragmentation that accompanies breakdown of the parental macronucleus during conjugation, correcting the previous interpretation that this process is apoptosis-like. Using strains containing a mutated (S134A) SQ motif, we demonstrate that phosphorylation of this motif is important for Tetrahymena cells to recover from exogenous DNA damage and is required for normal micronuclear meiosis and mitosis and, to a lesser extent, for normal amitotic macronuclear division; its absence, while not lethal, leads to the accumulation of DSBs in both micro-and macronuclei. These results demonstrate multiple roles of H2A.X phosphorylation in maintaining genomic integrity in different phases of the Tetrahymena life cycle.

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

Phosphorylation of the SQ H2A.X Motif Is Required for Proper Meiosis and Mitosis in Tetrahymena thermophila

Song

Gjoneska

Ren

et al. 2007

Molecular and Cellular Biology

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“…Temperature-sensitive mutants blocked in cytokinesis have been collected (Frankel et al, 1976), and a general procedure for the selection of temperature-sensitive mutants has recently been outlined for Tetrahymena (Bruns & Sanford, 1978). The potential utility of Tetrahymena as a cell type for genetic analysis has increased dramatically with the exploitation of phenotypic assortment (Sonneborn, 1974) to produce functional heterokaryons (Bruns & Brussard, 1974a), genomic exclusion (Allen, 1967) to yield homozygotes in a matter of hours , and the microtitre technology for mass testing of clones (Orias & Bruns, 1976). The availability of a procedure for selection of mating mutants should be of value in coupling genetic and biochemical analyses to the sequence of events surrounding the development of the sexual pathway.…”

Section: Discussionmentioning

confidence: 99%

Mass Selection of Conditional Mating Mutants of Tetrahymena thermophila

Wolfe

1979

Journal of General Microbiology

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1The mating reaction in Tetrahymena thermophila includes a starvation period and two distinct cell interactions, co-stimulation and cell pairing, before the cells are cytoplasmically joined as conjugants. A selection procedure for harvesting mutants unable to mate at a restrictive temperature has been developed. A conjugant pair consisting of one cycloheximide-resistant cell and one wild-type cell (cycloheximide-sensitive) was itself sensitive to the drug. By adding cycloheximide and nutrient medium to a cross made at the restrictive temperature, only cycloheximide-resistant cells that did not successfully conjugate could survive and grow. Repetition of the selection procedure enriched for cells unable to conjugate at the restrictive temperature. The selected cells were able to grow at 38 "C and could conjugate at 28 "C. This procedure may be narrowed to select specifically for cell interaction mutants.

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“…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.…”

mentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

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

Bruns¹,

Brussard²,

Kavka³

1976

Proc. Natl. Acad. Sci. U.S.A.

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(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

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Genomic Exclusion: A Rapid Means for Inducing Homozygous Diploid Lines in Tetrahymena pyriformis , Syngen 1

Cited by 114 publications

References 13 publications

Phosphorylation of the SQ H2A.X Motif Is Required for Proper Meiosis and Mitosis in Tetrahymena thermophila

Phosphorylation of the SQ H2A.X Motif Is Required for Proper Meiosis and Mitosis in Tetrahymena thermophila

Mass Selection of Conditional Mating Mutants of Tetrahymena thermophila

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

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