30][“Molecule H”

The severity of these phenotypes was similar to those observed for chkD cells, whereas no evidence for checkpoint failure was seen in controls. We were unable to conrm these data in synchronised cultures, as the cell size, particularly for the wee cdcD strain, was too variable to select a synchronous population of cells by centrifugal elutriation, and their genetic background precludes classical temperaturedependent block and release experiments.We also tested the response of both weecdc strains to chk overexpression.This may reect residual activity in the temperature sensitive enzyme.These cells were difficult to transform with either plasmid, and in the case of nmt: chk, do not grow well even in the presence of thiamine.With the wide length distribution and altered size control in this strain, we cannot be sure of the specicity of this response.Wildtype cultures grown at C behaved essentially identically to those grown at C. The percentage aberrant mitoses were counted from samples of cells.Cells were grown at C in the presence of thiamine, washed times, and reinnoculated into medium lacking thiamine and grown for hours at C.Neither modication was evident in cells synchronised in G by elutriation or mutation in cdc. However, with the variation in plasmid copy number that occurs in ssion yeast, we were unable to verify the specicity of this effect.Wildtype and chkD cells were irradiated as in and samples taken at the indicated times following radiation, and total protein and RNA extracts made.The wee mRNA was detected with a probe spanning the wee ORF.Protein extracts and western blotting was performed as in. In ssion yeast, cdcF mutants, which cannot be inhibited by this means, are nonresponsive to checkpoint signals. In this study we have addressed how ssion yeast cells control Y phosphorylation after having suffered DNA damage.Cdc is retained in the cytoplasm in an inactive state and is unable to dephosphorylate and activate cdc.Although this is also formally possible for the weecdc strains, it is hard to envisage how this could be the case for two lossoffunction mutations.Activation of spcpstyp advances mitosis under conditions of cellular stress, also by undened mechanisms, and this response is also blocked by sump overexpression. However, the protocol used in these experiments may not trigger a strong checkpoint delay that would be evident through cell elongation at the end of the experiment and it is unclear from this study as to the total dose of ionising radiation delivered during the experiment.These ndings will therefore impact on the eld in general as ssion yeast genetics is used to complement biochemical studies in higher eukaryotes.Wee becomes an essential gene when the checkpoint is inactivated, even without extrinsic DNA damage. Mikp appears to contribute to G DNA damage checkpoint maintenance, but our results here demonstrate that mikp alone is insufficient to maintain Y phosphorylation following DNA damage.Further a checkpoint arrest induced by overexpression of regions of the replication initiation protein J.Therefore, checkpoint control in ssion yeast and budding yeasts may not be as different as we have previously thought.Science. Genetics. Science. Science. Science.

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