| Research Abstract |
In order to generate conditional lethal caml mutants, mutations have been introduced by site-directed mutagenesis into the Ca^<2+>-binding loop(s)in the caml gene. These mutants are cam1-T64, cam1F102, cam1-T137, cam1-(T64, F102)and caml-(F102, 7 Three of these mutants, cam1-T64, cam1-F102 and cam1-T137, were phenotypically indistinguishable from the wild type in their growth rate and efficiencies in mating and sporulation. The mutant allele cam1-(T64, F102), was shown to be lethal, whereas the other mutation, designated cam1-(F102, T137), resulted in a temperature sensitive(ts)phenotype. This ts mutant(cam1^<ts>), which is unable to complete the cell cycle at the restrictive temperature(37ﾟC), does not accumulate with a uniform terminal morphology ; 50% of the cells arrest with single nuclei, 35% of the cells with a defect in cell separation or an asymmetrically situated septum in the absence of any nuclear division, and 15% of the cells with 2 nuclei per cell or separated chromosomes
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. These observations lead to the suggestion that CaM might be involved in various events of the cell cycle in S. pombe.
On the other hand, diploid strains homozygous for cam1^<ts> are blocked in meiosis even at the permissive temperature, indicating that CaM is a multi-functional protein which regulates not only sporulation but also meiosis in the fission yeast.
The distribution of CaM in S. pombe was investigated by fluorescence microscopy using anti-CaM. The cam1^+ gene product is distributed in the cytoplasm through the cell cycle, however, the localization of CaM during cytokinesis shows a close correlation with the known pattern of cell wall deposition as well as that of F-actin. The distinct localization patterns of CaM apparently disappear in a temperature sensitive cell division mutant, cdc3, which accumulates swollen elongated cells with 2-4 nuclei per cell and contains disorganized cell plate material, at the restrictive temperature. These results strongly suggest that one of the functions of CaM in cell cycle control in S. pombe may be during cytokinesis, i. e. septum formation and cell separation. Less
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