Cope, James Warren; (1996) Cytological, biochemical and genetical investigation of actin and tubulin in fission yeasts. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

A combined cytological, biochemical and genetical approach was applied to the distribution and composition of the microtubule and actin components of the fission yeast. Indirect immunofluorescence techniques were used to study the depolymerisation / polymerisation of microtubules in Schizosaccharomyces pombe and Schizosaccharornyces japonicus var. versatilis cells treated for 40 min at 1°C, subsequently re-warmed to 25°C. Microtubules depolymerised totally after 40 min at 1°C, but reappeared in 2 min at 25°C and at 30 min were indistinguishable from control cells. The effect of this treatment on actin distribution was examined. Actin was delocalised in cold treated cells, with some cells showing a ring of actin around the nucleus. Reformation of the normal actin array occurred even in cells in which microtubule polymerisation was blocked with 100μg ml-1 thiabendazole. Therefore polymerised microtubules are not required for the normal distribution of actin to be maintained. One and two-dimensional gel electrophoresis and Western blotting using a monoclonal anti-actin antibody were carried out on block-released cdc25.22 cells to determine whether actins' state changed throughout the cell cycle. Actin levels varied through the cell cycle. Attempts to determine whether any of the four species detected by two-dimensional gel electrophoresis were the result of post-translational modification gave equivocal results. Classical genetic techniques were utilised to identify suppressors of the cold-sensitive β-tubulin mutant nda3-KM311 and thereby identify possible interacting proteins. Two approaches were employed: (i) 552 phenotypically revertant colonies were isolated after EMS-mutagenesis of nda3-KM311; these were further tested for temperature sensitivity at 36°C; (ii) three phenotypically revertant colonies were isolated by spontaneous mutation of nda3-KM311. Ten revertants from the chemically induced group (four temperature sensitive and six not) and the three spontaneous revertants were crossed to wild type cells and the resulting asci dissected, replica plated and scored for growth at 20°C. The cold sensitive phenotype did not segregate out, suggesting that all of the mutations were intragenic. This was confirmed by free spore analysis.

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