The Yeast Deletion Collection

Constructed by the “Yeast Consortium”

A resource for the whole community

Total of about 16,000 strains

  U.S./ CanadaJef Boeke, Johns Hopkins UniversityHoward Bussey, McGill UniversityRon Davis, Stanford UniversityMark Johnston, Washington University at St. LouisJasper Rine, University of California at BerkeleyRosetta Inpharmatics, Kirkland, WashingtonJeffery Strathern & David Garfinkel, Frederick Cancer Research and Development Center Michael Snyder, Yale University EUROFAN:Bruno Andre, University Libre de BruxellesFrancoise Foury, Universite Catholique de LouvainJohannes Hegemann, Justus-Liebig-Universitaet GiessenSteve Kelly, University of Wales AberystwythPeter Philippsen, Biozentrum, BaselBart Scherens & F. Messenguy, Institut de Recherches du CERIAJose Revuelta, Universidad de SalamancaGiorgio Valle, University of PadovaGuido Volckaert, Katholieke Universiteit Leuven

The Consortium

Diploid (70%)

BY4743: MATa/ his31/his31 leu20 /leu20 lys20/LYS2 MET15/met150 ura30 /ura30

Haploids (30%)

BY4741: MATa his31 leu20 met150 ura30

Or

BY4742   MAT his31 leu20 lys20 ura30

LiOAC transformation into strain:

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

+

+

2:2Non-essential

or +

+

2:0Essential

let

+ (~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

BUT BEWARE

Strange things about strains

From Rosetta

22/290 strains that were expression profiled were aneuploid (7.5%)

Screen the library to test for any phenotype

Simple Genetic Screens

Drug Resistance

~5000 mutants (orf::G418) Replica Pinner

Using the deletion set

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

MATa

MAT

MATa/MAT

a1

asg

sg

asg

1 2

sg (OFF)

(ON)

DEFAULT

a1

1 2 asg

sg

(ON)

(OFF)

(OFF)

(OFF)

MFA1 gene encodes the mating factor “a”

therefore an asg expressed only MATa cells

GENETICS USING THE DELETION SET

MAT PMFA1-HIS3 yfg1:URA3 his3 ura3 x MATa orf:G418 his3 ura3

Cross to deletion collection in MATa

his- ura+ G418S his- ura- G418R

Select diploids SC-ura+G418

MATa

MAT

PMFA1-HIS3

+

yfg1:URA3

+

orf::G418

+

We are not about to dissect tetrads from 6000 crosses

Sporulate

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 lethalityTwo genes exhibit a synthetic lethal interaction when mutations in either gene by itself result in viable cells, but the double mutant is dead.

Mutant x is viable

Mutant y is viable

Double mutant xy is inviable

x y xy

From Boone, Bussey and Andrews, 2007

Systematic Genetic Array Analysis

yfg1::NAT Orf::G418

Mutant x is viable

Mutant y is viable

Double mutant xy is inviable

x y xy

From Boone, Bussey and Andrews, 2007

SGA

Haploid selection plates

(double mutant selection)

768 format

A Typical Screen Yields ~ 200 hits

Haploid selection Double Mutant

Serial 10 fold dilutions of the same spores

Secondary Screen

#1 #1#2 #2#3 #4 #3 #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

+NAT

G418 +

+ +G418 + + NATG418 NAT

Diploid Spores

Synthetic lethality

Haploid selection plate

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

YDL198C 67 0 34 0

No synthetic lethality

Synthetic lethality

auxotrophs

Replica plate to:

Bub1 Bub3 Mps1

Mad1 Mad2 Mad3

Cdc20

Pds1

Bub2 Byr4

Tem1,

Cdc14

Cdh1

Clb2

METAPHASE ANAPHASE G1Esp1

MEN

Sic1

Swi5

The spindle checkpoint

BioMatrix Robot with 192 plates

Plates are bar codedand loaded

Pinning at 1536 density= 5 plates/genome

Can do 38 different SGA crosses per run with BioMatrix5000/38= 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 unique20-mer “barcodes” of each mutant called the uptag and dntag. Both are flanked by universalprimers

From Boone, Bussey and Andrews, 2007

From Boone, Bussey and Andrews, 2007