1. Yeast Two-Hybrid system for working with proteins affecting yeast growth Maarten de Smit m.smit@chem.leidenuniv.nl

2. Y2H: Interaction activates reporter GAL4-DBD-bait (hybrid 1) GAL4-AD-prey (hybrid 2; single or library) bait-prey interaction: GAL4 activates reporter GAL1 UASGAL1 TATAReporter GAL4 DNA RNA selectable or detectable protein DBD AD

3. Screening a library of prey cDNAs Y2H selective agar plate: activated reporter = growth Colonies contain DNA encoding interacting proteins Sequencing!

4. Transformation or mating bait plasmid prey library 1 transformant needed > 5.000.000 transformants needed bait plasmid 5.000.000 prey-transformants purchased mating > 5.000.000 diploid clones needed

5. A bad pilot... st1 bait plasmid prey library Matchmaker™ Two-Hybrid System 3 (Clontech) Bait: SV40 small-t antigen fragment 1-82 ("st1") st1-fusion causes transformation problems? Yeast with bait + prey vector:1·10 4 cfu/µg Empty yeast + prey vector:2·10 5 cfu/µg

6. Sick yeast: plasmid loss during growth prey library prey library 10% 90% 10% 90% st1-fusion makes yeast sick: 90% loss of bait plasmid st1 bait plasmid T d = 200 min T d = 100 min

7. Wanted: controllable promoter for bait bait plasmid prey library Bait promoter off: yeast happy efficient transformation Bait promoter on: bait expressed two-hybrid selection YTH selective plate off on

8. Copper-controlled promoters Copper induction: CUP1-CYC1 fusion promoter Copper repression: CTR3-CYC1 fusion promoter 0.2220100 CuSO 4 (µM) 0.2220100 CuSO 4 (µM) reporter fusion: GAL4BD - p53

9. Copper-controlled plasmid loss Bait fusion-protein down = plasmid loss down 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 CUP1-CYC1 st1 CTR3-CYC1 st1 ADH1 st1 ADH1 empty 0.2 2 20 100 CuSO 4 (µM) fraction of cells with plasmid bait fusion protein plasmid loss measured after overnight growth in selective medium (becomes worse after growth in rich medium before transformation!)

10. Copper severely reduces colony formation - from freshly transformed cells - under two-hybrid selective conditions So we use copper repression, not induction - copper added before library transformation - no copper in selective plates Copper kills selection... Cu bait plasmid prey library YTH selective plate Cu offonoff

11. Preliminary results of pilot screen (small-t 1-82): screened ~15% of library (human normal keratinocytes) on ~50 plates (15 cm  ) picked 21 candidates 13 grow on "medium stringency" selective plates 2 grow on "high stringency" selective plates Y2H with bait protein affecting yeast growth (plasmid retention) is now feasible Preliminary results

12. Thanks!