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Selection system for temperature sensitive mutants in Paramecium aurelia

University of British Columbia

Four temperature-sensitive DNA deficient mutants of Paramecium aurelia have been isolated through a new application of UV light photolysis of 5-bromouracil substituted DNA, following nitrosoguanidine mutagenesis! Bromouracil was introduced by bacterial vector to achieve specific incorporation into DNA. Non mutant cells presumably incorporated 5-bromouracil at the restrictive temperature, mutants did not. The selection procedure took a month to complete, requiring two successive sexual generations. There were two lethal periods for wild type cells in the screening system; the first occurs during the irradiation of BU substituted macronuclei by UV light, the second, following autogamy (a result of homozygosis of UV light damage to the bromouracil labelled micronuclei). From 1300 isolated survivors from the screening procedure, 29 lines were classed as temperature-sensitive. Approximately 14 percent of these were ts DNA deficient (the ratio of restrictive to permissive temperature incorporation was 10 percent or less). Protein synthesis, and food-vacuole formation at the restrictive temperature deviated no more than 50 percent from the normal level. . The selection procedure was shown to be efficient in retrieving ts DNA deficient mutants. A genetically marked ts DNA mutant (isolated through the selection system earlier) was recovered at an enrichment of 7500 fold over the wild type (mean seven runs). The methods of production of bromouracil substituted bacteria have been standardized, and the durations of various parameters of the selection system have been set. For example, the length of UV light exposure following bromouracil substitution, and the length of restrictive temperature pretreatment (Equilibration time before bromouracil treatment) have been optimized. The relative effects of parts of the screening procedure are presented along with discussion of the results, and suggestions for future applications of the isolated Paramecium lines.

Text

2010-01-19

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http://hdl.handle.net/2429/18683

Genetics

University of British Columbia

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