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

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

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

Strains were verified in either haploids or diploids

Total of ~ 6200 deletions 383 genes could not be deleted

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

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)

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

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%)

Simple Genetic Screens Drug Resistance

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

YPD YPD + Benomyl

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 !!!!!! )

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

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

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+ G418S his- ura- G418R Cross to deletion collection in MATa Select diploids SC-ura+G418

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)

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

From Boone, Bussey and Andrews, 2007

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

SGA From Boone, Bussey and Andrews, 2007

(double mutant selection) 768 format Haploid selection plates

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

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

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

No synthetic lethality Haploid selection plate Replica plate to: ORF No drug G418 NAT G418+NAT YOR381W 50 22 26 11 YJL204C 80 42 38 24 YJR018W 61 29 20 YJR050W 110 58 59 YML013C-A 90 49 8 YDR452W 82 1 YDL198C 67 34 No synthetic lethality Synthetic lethality auxotrophs

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

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)

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

From Boone, Bussey and Andrews, 2007

From Boone, Bussey and Andrews, 2007