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: MATa his3D1 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
2:2
Non-essential
2:0
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
sg
(ON)
1 2
asg
(OFF)
MAT
sg
(OFF) 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+ G418S
his- ura- G418R
Cross to deletion collection in MATa
Select diploids SC-ura+G418

18. MATa
PMFA1-HIS3
yfg1:URA3
orf::G418
MAT + + +
Sporulate
We are not about to dissect tetrads from 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 Analysisxy 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. 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 G
NAT
G418 NAT

27. 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

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

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