EUROFAN: European network for functional analysis of yeast genes discovered by systematic DNA sequencing

• Forschungsstelle: EU FRP
• Projektnummer: 95.0191-9
• Projekttitel Englisch: EUROFAN: European network for functional analysis of yeast genes discovered by systematic DNA sequencing

Schlüsselwörter (Englisch)

Gene disruptions; endocytosis; synthetic lethality

Alternative Projektnummern (Englisch)

EU project number: BIO4-CT95-0080

Forschungsprogramme (Englisch)

EU-programme: 4. Frame Research Programme - 4.1 Biotechnology

Kurzbeschreibung (Englisch)

See abstract

Abstract (Englisch)

Our laboratory has now completed the scientific part of the EUROFAN I project. We have finished the six pack of gene disruptions and vector constructions. Of our six genes, one was essential and one showed a defect in sporulation. This work took substantially longer than planned due to unexpected difficulties in cloning certain DNA fragments. All of the strains have been sent to EUROSCARF to be put into the collection of gene disruptants.
Our lab was also responsible to set up two assays for the EUROFAN II and this work has been completed successfully. We have created a strain that permits us to test for sythetic lethality with the vma2::LEU2 mutation. This strain is MATa ura3 leu2 his3 ade2 ade3 lys2 bar1 vma2::LEU2 with a plasmid pVMA2::URA3::ADE3::CEN-ARS. The efficiency of disruption and detection of endocytosis mutants has been evaluated using known end mutants and is satisfactory. We have screened seven disruptants to begin the screening protocol and have already identified one ORF, YOL018c (later named TLG2) that shows synthetic lethality . The endocytosis phenotype of this mutant has been evaluated. The internalization step of endocytosis is normal, but delivery of endocytic markers to the vacuole is delayed two fold. Using a newly developed method to examine the endocytic pathway at the ultrastructural level we have shown that the delay in transport leads to the accumulation of small vesicles with trapped endocytic markers. This mutant shows no detectable defects in the secretory pathway, but does secrete low levels of the vacuolar enzyme, carboxypeptidase Y. Localization of the Tlg2 protein using a GFP fusion construct showed that it is found in small membraneous compartments in the periphery of the cell, consistent with a localization in early endosomes. Endocytosis of positively charged nanogold showed an accumulation of label in what appeared to be primary endocytic vesicles and a reduction in amount in early endosomes. This work has been published.
Due to the tardy availability of gene disruption cassettes we have developed an alternative protocol to identify endocytosis defective cells among the disruption mutants generated by EUROFAN I. This involves the analysis of the accumulation of a small fluorescent endocytic marker, lucifer yellow CH, in the yeast vacuole. This method turns out to be more rapid than the synthetic lethal approach and will be used to screen the bank of disruption mutants.

Datenbankreferenzen (Englisch)

Swiss Database: Euro-DB of the
State Secretariat for Education and Research
Hallwylstrasse 4
CH-3003 Berne, Switzerland
Tel. [phone]
Swiss Project-Number: 95.0191-9