The Yeast Deletion Collection Constructed by the “Yeast Consortium” A resource for the whole community Total of about 16

1. The Yeast Deletion Collection Constructed by the “Yeast Consortium” A resource for the whole community Total of about 16,000 strains

2. The Consortium U.S./ Canada Jef Boeke, Johns Hopkins University Howard Bussey, McGill University Ron Davis, Stanford University Mark Johnston, Washington University at St. Louis Jasper Rine, University of California at Berkeley Rosetta Inpharmatics, Kirkland, Washington Jeffery Strathern & David Garfinkel, Frederick Cancer Research and Development Center Michael Snyder, Yale University EUROFAN: Bruno Andre, University Libre de Bruxelles Francoise Foury, Universite Catholique de Louvain Johannes Hegemann, Justus-Liebig-Universitaet Giessen Steve Kelly, University of Wales Aberystwyth Peter Philippsen, Biozentrum, Basel Bart Scherens & F. Messenguy, Institut de Recherches du CERIA Jose Revuelta, Universidad de Salamanca Giorgio Valle, University of Padova Guido Volckaert, Katholieke Universiteit Leuven

4. LiOAC transformation into strain: Diploid (70%) BY4743: MATa/a his3D1/his3D1 leu2D0 /leu2D0 lys2D0/LYS2 MET15/met15D0 ura3D0 /ura3D0 Haploids (30%) BY4741: MATahis3D1 leu2D0 met15D0 ura3D0 Or BY4742   MATa his3D1 leu2D0 lys2D0 ura3D0

5. Strains were verified in either haploids or diploids

6. Total of ~ 6200 deletions 383 genes could not be deleted

7. STRANGE THINGS ABOUT TRANSFORMATION From Peter Philippsen: 53/819 (6.5%) of diploids segregated unlinked lethals G418 G418 G418 + + or + + + let 2:0 Essential 2:2 Non-essential + (~5000) (~1000)

8. 96 well format = 76 plates (Omnitrays) Combine four plates = 384 format = 19 Omnitrays Four Sets 2 haploid (MATa ot MAT) 2 diploids (homozygous and heterozygous)

9. One good use is to transfer mutants to your genetic background KanMX4 200 bp 200 bp 200 bp

10. Screen the library to test for any phenotype BUT BEWARE Strange things about strains From Rosetta 22/290 strains that were expression profiled were aneuploid (7.5%)

11. Simple Genetic Screens Drug Resistance

12. Using the deletion set Replica Pinner ~5000 mutants (orf::G418) Pin the strains onto plates +/- drug

13. YPD YPD + Benomyl

14. Advantages: • Simple • Comprehensive (at least for non-essential genes) • Instant gene identification • Eliminates cloning by complementation • Eliminates confirming the cloned gene (right gene vs suppressor) • However, still requires proof (CAN YOU THINK OF AT LEAST TWO • METHODS FOR HOW YOU WOULD DO THIS?) • (WHAT A JUICY EXAM QUESTION !!!!!! )

15. Very Useful for screening for phenotypes But what if you wanted to do genetics with the entire collection ……such as make double mutants? For example, do any of the deletion mutants suppress yfg1? How would you make the double mutants? You could PCR amplify and transform into yfg1….(6000 transformations!!!) Too boring. You need….The Magic Marker

16. Must recall mating type determination in yeast DEFAULT sg (OFF) a1 asg (ON) MATa (ON) sg 1 2 asg (OFF) MAT (OFF) sg a1 1 2 asg (OFF) MATa/MAT

17. GENETICS USING THE DELETION SET MFA1 gene encodes the mating factor “a” therefore an asg expressed only MATa cells MAT PMFA1-HIS3 yfg1:URA3 his3 ura3 x MATa orf:G418 his3 ura3 his- ura- G418R his- ura+ G418S Select diploids SC-ura+G418 Cross to deletion collection in MATa

18. MATa PMFA1-HIS3 yfg1:URA3 orf::G418 MAT + + + Sporulate We are not about to dissect tetrads from 6000 crosses • Therefore we do “random spore analysis” • Select haploids on SC-his • Must be MATa and have the PMFA1-HIS3 • ½ the spores are URA+, ½ are G418R • Therefore ¼ are URA+ and G418R (double mutant)

19. Synthetic lethality Two genes exhibit a synthetic lethal interaction when mutations in either gene by itself result in viable cells, but the double mutant is dead. xy x y Mutant x is viable Mutant y is viable Double mutant xy is inviable

20. From Boone, Bussey and Andrews, 2007

21. Systematic Genetic Array Analysis xy x y Mutant x is viable Mutant y is viable Double mutant xy is inviable yfg1::NAT Orf::G418

22. SGA From Boone, Bussey and Andrews, 2007

23. (double mutant selection) 768 format Haploid selection plates

24. A Typical Screen Yields ~ 200 hits Secondary Screen Serial 10 fold dilutions of the same spores #1 #2 #3 #4 #1 #2 #3 #4 Haploid selection Double Mutant

25. Confirm the interactions • Tetrads (PD, NPD, T for NAT and G418) • NPD’s are 2:2 for viability • or • 2. Random spore analysis

26. G418 + + NAT Synthetic lethality Random Spore Analysis Select haploid spores, replica plate to: no drug, single drug, double drug measure ratio of markers Diploid Spores + + G418 + + NAT G418 NAT

27. Haploid selection plate Replica plate to: No synthetic lethality Synthetic lethality auxotrophs

28. The spindle checkpoint Bub1 Bub3 Mps1 Bub2 Byr4 Tem1, MEN Mad1 Mad2 Mad3 Cdc14 Cdc20 Swi5 Cdh1 Pds1 Sic1 Clb2 Esp1 G1 METAPHASE ANAPHASE

32. BioMatrix Robot with 192 plates Plates are bar coded and loaded Pinning at 1536 density = 5 plates/genome Can do 38 different SGA crosses per run with BioMatrix 5000/38= 132 runs to do 5000 mutants (the genome) The goal is to have a complete SGA analysis of the genome (5,000 x 5000)

33. A competing technology is called DSLAM (Diploid-based Synthetic Lethal Analysis by Microarrays) DSLAM uses the unique 20-mer “barcodes” of each mutant called the uptag and dntag. Both are flanked by universal primers

34. From Boone, Bussey and Andrews, 2007

35. From Boone, Bussey and Andrews, 2007