Transcript Lecture 2 - University of Virginia

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
+
+
let
2:2
Non-essential
+
(~5000)
or
+
+
2:0
Essential
(~1000)
96 well format = 76 plates (Omnitrays)
Combine four plates = 384 format = 19 Omnitrays
Four Sets 2 haploid (MATa ot MATa) 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
~5000 mutants (orf::G418)
Replica Pinner
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
a1
MATa
a1
a2
asg
(OFF)
asg
(ON)
asg
(ON)
asg
(OFF)
asg
(OFF)
asg
(OFF)
MATa
a1
a1
a2
MATa/MATa
GENETICS USING THE DELETION SET
MFA1 gene encodes the mating factor “a”
therefore an asg expressed only MATa cells
MATa PMFA1-HIS3 yfg1:URA3 his3 ura3 x MATa orf:G418 his3 ura3
his- ura- G418R
his- ura+ G418S
Cross to deletion collection in MATa
Select diploids SC-ura+G418
MATa
MATa
PMFA1-HIS3
+
yfg1:URA3
orf::G418
+
+
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.
x
Mutant x
is viable
y
Mutant y is
viable
xy
Double mutant
xy is inviable
From Boone, Bussey and Andrews, 2007
Systematic Genetic Array Analysis
x
y
Mutant x
is viable
Mutant y is
viable
yfg1::NAT
Orf::G418
xy
Double mutant
xy is inviable
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
Haploid selection
#4
#1
#2
#3
Double Mutant
#4
Confirm the interactions
1. 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
+ G418
+
NAT
Spores
+
+
G418
+
+
NAT
G418 NAT
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
0
YJR050W
110
58
59
0
YML013C-A
90
24
49
8
YDR452W
82
1
38
1
No synthetic
lethality
Synthetic
lethality
auxotrophs
YDL198C
67
0
34
0
The spindle checkpoint
Bub1 Bub3 Mps1
Bub2 Byr4
Mad1 Mad2 Mad3
Tem1,
Cdc20
Cdc14
MEN
Swi5
Cdh1
Pds1
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