Abstract
(Englisch)
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In 1995 a consortium of 125 European laboratories (11 from Switzerland) was founded aiming at the functional analysis of 1'000 novel S. cerevisiae genes. Funds were made available by the EU and for the Swiss participants by the BBW in January 1996. Our group in Basel wrote the technical outline of this basic analysis program including a detailed methods section, took responsibility for coordination of and help in systematic analysis of 180 novel genes from chromosome XIV, and worked on the improvement of methods for gene analyses. 62 of the 180 novel genes were assigned to Swiss participants: M. Aebi, Zürich, A. Conzelmann, Fribourg, M. Hall, Basel, W. Heyer, Bern, P. Linder, Geneva, H. Riezman, Basel, C. Sengstag, Zürich, F. Thoma, Zürich, A. Wach, Basel and our lab. The work load for fundamental analysis of 21 of these 62 genes was shared by three groups (M. Hall, P. Philippsen, A. Wach). Gene deletions were constructed and their phenotype analysed, gene deletion cassettes were cloned and tested, cognate gene clones were generated and used in complementation assays. In addition, the open reading frame of the green fluorescent protein (GFP) was fused to the last sense codon of all 21 yeast genes and for 10 genes to the first codon. Main results are as follows: Two gene deletions led to cell death, three to slow growth and two to lack of sporulation. Two proteins, when overexpressed, led to slow growth. In vivo fluorescence microscopy (see report 05.0191-12) revealed the subcellular localization of five GFP fusion proteins, two to the cytoplasm (tubular structures), one to the nucleus, one to the nucleolus and one to the spindle pole body. The latter gene was further extensively studied (see report 05.0191-12). All basic gene analysis data will be made publically available by December 31, 1998 via the EUROFAN 1 web page at MIPS: http://web.archive.org/web/20001203133800/http://websvr.mips.biochem.mpg.de/proj/eurofan/index.html. A major part of our efforts focussed on establishment of various PCR-based gene targeting methods. The performance of our heterologous selection markers KanMX and HIS3MX for gene deletions and gene truncations was tested with respect to targeting fidelity, targeting efficiency and targeting affinity. Target guide sequences of 40 bp flanking these markers are sufficient to achieve over 95% correct gene targeting as tested at 20 different gene loci. Single base pair differences between target guide and genomic target sequence reduced gene deletion frequencies by a factor of five, two or three base pair differences by a factor of 10 to 20. Correctly targeted deletions were achieved for up to 15 kb. In a competitive situation, two potential genomic target sites, one with two 40 bp the other with two 400 bp homology regions to the target guide sequences, still 5% of the deletions were targeted to the short region of homology. This allows to generate homozygous deletions in diploid strain by two consecutive PCR-based targeting steps. In addition to these systematic experiments double modules (GFP-KanMX, GFP-HIS3MX, HA-KanMX and other epitopes) for gene tagging were constructed and tested (see also report 05.0191-12). One functional node of Eurofan 1 was devoted to relations between the S. cerevisiae genome and other fungal genomes. We contributed to this node with an analysis of the ends of 100 plasmid clones carrying 3.5 to 5.5 kb genomic DNA of the filamentous fungus Ashbya gossypii . This fungus is a cotton pathogen and is also commercially used for vitamin B2 production. Our initial sequence analysis of 100 DNA clones and heterologous complementation experiments had already shown a surprisingly close relationship between this exclusively filamentously growing fungus and the unicellular fungus S. cerevisiae. The sequence and bioinformatic analysis, with help of Qiagen and the Martinsried Institute for protein sequences, respectively, for this second set of Ashbya clones revealed for 90% of the Ashbya sequences significant homology to S. cerevisiae genes. More importantly, a conservation of gene order plus transcription orientation (synteny) was found in one third of the plasmids. This result extends our earlier observations of a four gene synteny at the THR4 locus and of two and sometimes three genes syntenies at other loci. This finding is very important for future whole genome analysis of fungi once similarities in gene sequence and gene organization with respect to the known S. cerevisiae genome have been established. This approach has meanwhile been used by two EUROFAN 1 labs (A. Hinnen, Jena and B. Dujon, Paris). For the pathogenic yeast Candida albicans sequence similarities but low level of synteny were found, for the yeast Kluyveromyces lactis a high level of sequence similarity and synteny was observed.
Wach, A., Brachat, A., Alberti-Segui, C., Rebischung, C. and Philippsen P. (1997) Heterologous HIS3 marker and GFP reporter Modules for PCR-targeting in Saccharomyces cerevisiae. Yeast 13:1065-1075. Longtine, M., McKenzie, A., Demarini, D., Shah, N., Wach, A., Brachat, A., Philippsen, P., Pringel P. (1998) Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14:953-961 Bähler, J., Wu, J., Longtine, S., Shah, N., McKenzie, A., Steever, A., Wach, A, Philippsen, P and Pringle, J. (1998) Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe Yeast 14:943-951. Brachat, A., Kilmartin, J. Wach, A. Philippsen P (1998) Saccharomyces cerevisiae cells with defective spindle pole body outerplaques accomplish nuclear migration via half-bridge-organized microtubules. Mol. Biol. Cell 9:977-991. Brachat, A., Rebischung, C., Demchyshyn, V., Liebundguth, N., Lemire S., Howald, I., Düsterhöft, A., Möstle, D., Pöhlmann, R., Hall, M., Wach, A. and Philippsen P. (1998) Analysis of 21 novel yeast genes (submitted).
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