Résumé des résultats (Abstract)
(Anglais)
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As one of the Swiss groups participating in the European functional analysis network (EUROFAN 1) for novel yeast genes, we also performed a basic functional analysis for 6 open reading frames. Besides the standard program, growth and sporulation assays with gene deletions, construction and tests of gene deletion cassettes and generation of cognate clones we also analysed carboxy-terminal and amino-terminal fusions with the green fluorescent protein (GFP). The double modules GFP-KanMX and GFP-HIS3MX used for these fusions were constructed and tested during this EUROFAN program. All GFP fusions led to observable green fluorescence but only in one third of 24 cases investigated at clearly identifyable subcellular locations. One GFP gene fusion located to the spindle pole body. The deletion of that gene caused a slow growth phenotype with 50% of the cells carrying two or more nuclei. This gene was named CNM67 due to the chaotic nuclear migration of the deletion and the predicted molecular weight of the gene product. Electron microscopic investigations (in collaboration with J. Kilmartin, Cambridge) of embedded thin sections showed a loss of the outer plaque of the spindle pole body in the CNM67 deletion strain. This part of the spindle pole body is the organization center of cytoplasmic microtubules. We could show that in the absence of the outer plaque at least a few cytoplasmic microtubules are attached to the half bridge of the spindle pole body throughout the cell cycle. This is most likely a rescue pathway used by the cell to assure at least partially functional nuclear migration in the absence of the outer plaque. Further analysis of this novel yeast protein is presently performed in EUROFAN 2.
A major investment and effort was the establishment of in vivo video fluorescence microscopy for the dynamic analysis of GFP marked organelles and also proteins. Meanwhile we succeeded in following in vivo nuclear migration, spindle pole body separation and microtuble dynamics by time lapse video microscopy using strains carrying a histone-GFP, a CNM67 GFP and an alpha tubulin fusion, respectively.
Publications (in addition to those mentioned in report 95-0191-1)
Wach, A., Brachat, A., Rebischung, C., Steiner, S., Pokorni, K. te Heesen, S., Philippsen, P. (1998) PCR-based gene targeting in S. cerevisiae. In Methods in Microbiology (A.J. Brown and Tuite, M.F., eds.), Vol 26, pp. 67-81. Academic Press, San Diego.
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