[“Beyonder Vs Molecule Man

For UVC irradiation, cells were reinnoculated into prewarmed and conditioned media for a recovery period of minutes, and then ltered and irradiated as above.Mockirradiated cells were ltered and then placed back into medium.Cell cycle progression was followed by septation indices or septa in synchronous cultures.Septation data were derived from the average of counts of at least live cells, and was determined by phasecontrast microscopy every or minutes during the course of the experiments.UVC survival assays were performed in cultures grown in dened medium, plated at various densities on rich medium. This plasmid was cotransformed together with a LEU marked plasmid. Transformants were selected by leucine prototrophy.These were then replicaplated to score for loss of the ura marker and then conrmed for the appropriate integration event.Several isolates were obtained, and all were wild type for cell length and cell cycle progression.DNA damage checkpoint mutants are hypersensitive to DNA damaging agents.Therefore, we rst assessed the UVC and ionising irradiation sensitivities of these strains, using wildtype and chkdeleted strains as controls.We conclude that neither of these strains is signicantly hypersensitive to DNA damaging agents and that this observation suggests these strains are therefore checkpoint procient.We next assessed the checkpoint kinetics in these strains by two assays.In the case of wildtype cells, irradiation induced a transient cell cycle arrest and hence the septation index fell.For the cdcw and cdcw cdcD strains, the results clearly showed wildtype arrest kinetics, and therefore both strains are checkpoint procient as was predicted by their lack of radiation hypersensitivity.We then assessed the checkpoint status in cultures synchronised in G by centrifugal elutriation. For these experiments only wildtype and cdcw cdcD cells were analysed, as although a uniformly sized population of cdcw cells could be obtained by this protocol, this population could not be sufficiently synchronised for cell cycle progression due to their heterogeneity of size at division.This was expressed from a single integrated copy of the nmt promoter.In the presence of thiamine, expression from the promoter is lower, but still complements cdcD.Therefore, these conditions were used to assay the response to UVC irradiation.This strain showed wildtype levels of UVC sensitivity, and wildtype checkpoint kinetics in cells that were synchronised in G by elutriation. Cells were synchronised by elutriation, allowed to recover for minutes, and then irradiated. The indicated strains were transformed with an nmt expression plasmid alone. Cells were grown in the presence of thiamine, washed times, and reinnoculated into medium lacking thiamine to cellsml and grown for hours at C.In tetrad dissections, cells deleted for both wee and cdc showed poor viability, were frequently diploid, and were hence too genetically unstable for timecourse experiments.However, two other strains utilising temperature sensitive alleles were analysed: cdcD wee, and cdc weeD each grown J.Survival curves showed that cdcD wee cells were substantially more UVC sensitive than controls. However, the double mutant showed a high frequency of diploid colonies following irradiation, which is consistent with mitotic abnormalities due to chromosome segregation defects resulting from checkpoint failure.We assayed the checkpoint status of both the cdcweestrains and found that they failed to arrest cell cycle progression following irradiation.

Leave a Reply