The isolation and phenotypic characterization of Paramecium tetraurelia temperature-sensitive cell cycle mutants

Conditional cell cycle mutants which block cell division under restrictive conditions, increase their cell mass as a result of continued growth. Here, we develop a protocol to screen for large Paramecium temperature-sensitive cell cycle mutants by sizeselecting centrifugal elutriation. By using the ccl mutant as a standard for Paramecium temperature-sensitive cell cycle mutants, we determine that 14 hours of heat treatment produces sufficient size difference for the elutriator to discriminate cell cycle mutants from wild-type cells. In addition, this duration of heat treatment does not reduce the viability nor delays recovery rates significantly in cell cycle mutants. We demonstrate that multiple elutriations are able to retrieve ccl cells at cell cycle mutant frequencies of 0.02%. Two mutants, cc5 and cc8 have been isolated from this screen. Temperature-shift experiments reveal that these two mutants possess similar temperature-sensitive periods to ccl. The cc5 and cc8 mutants are allelic to each other but extragenic to ccl. The presence of a "pseudo" class in the F2 progeny of the cc5 X ccl cross suggests an interaction between the cc5 and ccl gene products.Science, Faculty ofZoology, Department ofGraduat