Obiettivo The main objective of the 'Pilot Study' was to define strategies in order to discover the physiological role and the molecular mechanisms governed by the novel genes discovered by systematic sequencing of the budding yeast. Several complementary approaches have been developped. These approaches were based on techniques of molecular and physiological genetics as well as protein and RNA analyses. They have been applied to the search of functions of novel genes located on chromosome III.The main achievement is that, by applying a large-scale systematic approach, one can prove that the majority of Open Reading Frames of previously unknown function do correspond to true genes, since their inactivation leads to experimentally detectable physiological or biochemical changes. Therefore, the feasibility of this multifactorial strategy has been demonstrated and its extension to thousands of novel genes should be encouraged.MAJOR SCIENTIFIC BREAKTHROUGHS:The major breakthroughs consisted: (i) in the construction of a library of 81 individual gene deletions obtained by oligonucleotide targeted mutagenesis. All deletions have been verified by southern blotting and tetrad analyses. Numerous mistakes (mainly frame-shift errors) in the published sequence of chromosome III have been corrected; (ii) in the establishment of conditions for phenotypic tests in microtiter plates using a battery of diagnostic metabolic inhibitors and various environmental stress conditions different from the routine laboratory tests. Several essential genes, those involved in mitochondrial activities, sporulation, cell and nuclear division, cytoskeleton architecture, membrane permeability, ionic and thermotolerance were uncovered. It has been shouwn by tetrad analyses and by complementation with the wild type genes, that the phenotypes discovered are due to inactivations of cognate genes. The unravelling of interactions between different genes has begun by isolating functional multi-copy suppressors and synthetic co-lethal mutations, both within genes of chromosome III and between the genes from this chromosome and others. It should be stressed that for numerous genes whose physiological role has been discovered, there exist structural homologues of completely unknown function in higher organisms, including humans. Thus, the study of yeast mutants opens new perspectives for the understanding of plant, animal and human genes; (iii) in the commencement of `proteome' oriented research which is the logical counterpart of the `genome' oriented research, by establishing the reference wild-type 2-dimensional protein map and by identifying several hundreds of gene products. It is of special interest that many secondary changes (either decrease or increase in protein abundancy) concerning proteins which are not coded by the deleted genes were uncovered indicating that this approach should be fruitful for the dissection of Cellular Integrated Networks; (iv) in the application to the study of gene expression of two complementary approaches based, on the one hand, upon the estimation of RNA-transcript abundancy under various growth conditions and, on the other hand, upon the regulation by the general energy central system of fused reporter genes. Campo scientifico natural sciencesbiological sciencesbiochemistrybiomoleculesproteinsproteomicsnatural sciencesbiological sciencesgeneticsmutationnatural sciencesbiological sciencesgeneticsRNAnatural sciencesbiological sciencesgeneticschromosomesnatural sciencesbiological sciencesgeneticsgenomes Programma(i) FP3-BIOTECH 1 - Specific research and technological development programme (EEC) in the field of biotechnology, 1990-1994 Argomento(i) 1.2 - Gene structure Invito a presentare proposte Data not available Meccanismo di finanziamento CSC - Cost-sharing contracts Coordinatore CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE Contributo UE Nessun dato Indirizzo Avenue de la Terrasse, BFtiment 26 91198 GIF SUR YVETTE Francia Mostra sulla mappa Costo totale Nessun dato Partecipanti (5) Classifica in ordine alfabetico Classifica per Contributo UE Espandi tutto Riduci tutto Centre National de la Recherche Scientifique (CNRS) Francia Contributo UE Nessun dato Indirizzo Avenue des Facultés 33405 Talence Mostra sulla mappa Costo totale Nessun dato Heinrich-Heine-Universität Düsseldorf Germania Contributo UE Nessun dato Indirizzo Universitätstraße 1 40225 Düsseldorf Mostra sulla mappa Costo totale Nessun dato National and Kapodistrian University of Athens Grecia Contributo UE Nessun dato Indirizzo Panepistimiopolis 10679 Athens Mostra sulla mappa Costo totale Nessun dato UNIVERSITE DE PARIS-SUD XI Francia Contributo UE Nessun dato Indirizzo Rue Georges Clemenceau 15, BFtiment 400 91405 GOMETZ LA VILLE Mostra sulla mappa Costo totale Nessun dato Århus Universitet Danimarca Contributo UE Nessun dato Indirizzo 240,Wilhelm Meyers Alle 8000 Århus C Mostra sulla mappa Costo totale Nessun dato
