Résumé des résultats (Abstract)
(Anglais)
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An international consortium of 14 yeast groups aims at generating deletion mutants for all 6200 protein-coding genes of S. cerevisiae and labelling each deletion with two unique sequences of twenty bp each (bar-coded deletions). With the help of high density DNA chips, this collection can be used for simultaneous testing of all deletions in order to identify in a single experiment those deletions with improved or impaired growth at specific conditions (e.g. unusual nutrients, presence of stimulating or inhibiting drugs, stress factors, etc). The logistics of this project is as follows: The group of R. Davis (Stanford University) produces by PCR sets of 96 deletion fragments. Each fragment carries the KanMX4 selection marker (constructed in Basel) flanked on each side by unique sequence-codes of 20 bp plus 45-mers homologous to the 5' and 3' side of the target genes, respectively. These sets are distributed to four groups in the US (J. Boeke, Baltimore; S. Friend, Seattle; M. Johnston, St. Louis; M. Snyder, New Haven) one group in Canada (H. Bussey, Montreal) and nine groups in Europe (B. Andre, F. Foury, F. Messenguy and G. Volckaert, Belgium; H. Hegemann, Germany; S. Kelly, UK; J. Revuelta, Spain; G. Valle, Italy; P. Philippsen, Switzerland). In addition, Stanford supplies these groups with primers for verification of correct gene targeting and, as a back-up, with all oligonucleotides used for the PCR reactions.
A reference diploid strain is transformed with the PCR-made deletion fragments. The genetic background of this strain is identical with the genome which was sequenced between 1998 and 1996 by an international consortium of laboratories in Europe, USA, Canada and Japan. Deletions are verified by PCR. Two independent heterozygous deletants are subjected to sporulation in order to isolate haploid deletion strains. At this step essential genes (about 15%) are recognized by the failure of haploid deletants to grow. For each non-essential gene one viable MATa haploid deletion and one viable MATa haploid deletion are isolated from two independent transformants and used for generation of homozygous deletant strains. This strategy minimizes the risk of expression of unwanted phenotypic changes in the homozygous deletants due to uncontrolled mutations introduced during the transformation procedure. The collection of homozygous deletions will mainly be used for high throughput screening and as a reliable source of gene deletion constructed using the same quality criteria.
During the first round of transformations 80-95% of successful deletions were obtained for each 96-pack. They were processed as described above. The unsuccessful transformations were redistributed. Replicas of strains produced by the European groups are processed at the EUROFAN strain collection at Frankfurt and are also sent to Stanford and Research Genetics (the collection centers in the US). Vice versa the replicas of deletion strains produced in the US are sent to the EUROFAN strain collection. By the end of 1999 the first round of deletions and strain constructions was finished for 90% of all 96-packs. A manuscript outlining the deletion procedure and describing verification data and use of DNA chips for identification of slow growers has been published (Winzeler, A., Science 1999 Aug., Vol. 285:901-906).
Our group in Basel represents the European view points in the meetings in the US, acts as coordination center for the European participants and is responsible to generate the strains for 400 of the 2600 gene deletions which were allocated to EUROFAN, the European Functional Analysis Network for studying novel S. cerevisiae genes. By the end of 1999 we had finished 80% of our workload.
Our group is also partner in the cell architecture sub-project of EUROFAN and we are investigating in novel methods in the field of fluorescence video microscopy. During 1999 we established a high sensitivity video camera system which allowed us to follow nuclear migrations for many generations in S. cerevisiae wild type and spindle pole body mutant strains. A manuscript describing that work has been published (D. Hoepfner, et al., Molecular Biology of the Cell 2000 Apr., Vol. 11:1197-1211). The nuclei were labelled with a fusion of histone H4 and the green fluorescent protein, a nuclear fluorescent marker constructed in our lab during the first phase of EUROFAN. The video microscope set-up was used in 1999 to study nuclear migration in several cell architecture mutants of S. cerevisiae generated by the cell architecture team of EUROFAN.
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