Yeast as a model organism

• Model eukaryote– Experimental genetics– Gene function – Orthologs, family members– Pathway function - “Biological synteny”

• Testbed for genomic technologies– Genome sequenced (4/96)

relatively less complex– Ability to assess biological relevance of the

data

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Genomics technology developmentYeast as a testbed

• Gene expression patterns– DNA microarrays, SAGE

• Genomic DNA scans– Mapping complex traits (SNPs)

• Phenotype screening– Genome-wide knockouts

• Genetic interaction networks– Synthetic lethals

• Protein interaction networks– Two-hybrid, mass spectrometry

Affymetrix whole genome yeast array

• Each gene is probed by multiple oligonucleotide probes (>19).

• A control probe is synthesized adjacent to each actual probe

• ~120,000 different oligonucleotide sequences for the entire genome.

• Entire yeast genome is on 5 arrays (~ 65,000 25 mers on each).

2 kbGene 1 Gene 2

25mers 25mers

Lisa Wodicka, Dave Lockhart, Affymetrix

Mapping complex traits

• Construct a SNP genetic map

• Perform cross

• Analyze rare segregants

• Identify regions inherited solely from one parent

YJM789• Isolated from the lung of an AIDS

patient.

• Able to grow at 42 °C, form pseudohyphae and undergo colony-morphology switching.

• Hypersensitive to cycloheximide.

• Polymorphic

– one difference every 150 bases relative to sequenced strain

Laboratory strain

YJM789 parent

Allelic variation between two strains can be detected using arrays.

Laboratory strain (non-pathogenic)

YJM789(virulent wild isolate)

2 kbGene 1

25mers

Mismatch control probe (position 13 of 25)

2 kbGene 1

25mers

* ** *

missing signals = markers

Polymorphisms

Yeast Array

Since probe locations are known, a genetic map can be constructed:Since probe locations are known, a genetic map can be constructed:interesting loci (virulence) can be mapped and positionally cloned for study.interesting loci (virulence) can be mapped and positionally cloned for study.

Allelic variation in YJM789

• 3808 markers detected by automated analysis of scanned images.– Largest gap = 56 kb– Average frequency = 3000 bases (1.0 cM)

• More markers identified in one hybridization than in the past 40 years of yeast genetics.

Verification of markers by tetrad analysis

Expect 90 cross-overs per genome.Expect 90 cross-overs per genome. Expect clear recombination breakpointsExpect clear recombination breakpoints Expect most markers to segregate 2:2.Expect most markers to segregate 2:2.

Segregation of markers in one tetrad (one chromosome)

96 crossovers 96 crossovers (90 expected).(90 expected).

96% of markers 96% of markers segregate 2:2.segregate 2:2.

Clear breakpointsClear breakpointsobserved.observed.

Markers segregate as expectedMarkers segregate as expected

spore 1 spore 2

spore 4spore 3

Laboratory strain (S96)genotype: MATa, lys5, LYS2, ho, CYH

Wild Isolate (YJM789)genotype MAT LYS5, lys2, ho::hisG, cyh

Diploid

Haploid Haploid116

1

16

1

16 16

161

...

...

...

...

... ... ...

(mat lys2, LYS5, ho, cyh)

Inheritance of markers in 10 lys2 segregants

Results of mapping five phenotypic loci in 10 segregants.

• Five regions identified that were inherited solely from one parent.

• Four encompassed known locations of MAT, LYS5, LYS2, and HO.

• Minimum intervals ranged from 12 to 90 kb.

Cycloheximide sensitivity = pdr5

• Cycloheximide sensitivity maps to remaining 56 kb interval on Chromosome XV adjacent to pdr5.

• PDR5 is deleted in YJM789.

• Wildtype strain, deleted for pdr5 is unable to complement YJM789.

Mapping Complex Traits: Feasibility Summary

• Identified 3808 genetic markers.

• Demonstrated that traits can be mapped using these markers.

• Next step: Map virulence loci.

Virulence in YJM789

• Virulence is a multigenic trait with 5 loci contributing.

– Only 5 of 200 segregants from crosses between YJM789 and laboratory strain are virulent.

• Genes cannot be cloned by complementation.

• Hybridization with arrays is an appropriate way to map all contributing loci simultaneously.

Assigning Function through Mutational Analysis

• Inactivate gene product (delete gene).

• Grow mutant strain under different selective or stress conditions.

• Identify mutants with growth defects.

• Function of gene product may be revealed.– UV sensitivity = DNA repair protein– Adenine auxotrophy = Adenine biosynthesis

Construction of yeast deletion strains

KanR

plasmid

Deletion Cassette

Chromosomal Gene

Amplify selectable marker gene using primers with yeast gene

homology at 5’ ends

Replace yeast gene by homologous recom-

bination

yeast sequence

International Deletion Consortium Members

Mike Snyder, Jasper Rine, Mark Johnston, Jef Boeke, Mike Snyder, Jasper Rine, Mark Johnston, Jef Boeke, Howard Bussey, Rosetta, Acacia, Peter Philippsen, Howard Bussey, Rosetta, Acacia, Peter Philippsen, Hans Hegemann, Francoise Foury, Guido Volckaert, Hans Hegemann, Francoise Foury, Guido Volckaert, Bruno Andre, Giogio Valle, Jose Revuelta, Steve Bruno Andre, Giogio Valle, Jose Revuelta, Steve Kelly, Bart ScherensKelly, Bart Scherens

24,000 strains in 3 years

Serial analysis of deletion strains

Apply Selection

Identify deletion strains with growth defects

1

2

3

6,000

Molecular tags as strain-identifiers

Unique 20-mersUnique 20-mers Good hybridization propertiesGood hybridization properties Similar melting temperaturesSimilar melting temperatures More than 5 base differences between eachMore than 5 base differences between each

1.1 x 10 12 possible 20mers 12,000 best

Shoemaker et al., 1996. Nature Genetics, 14:450-456

Can be introduced during strain constructionCan be introduced during strain construction Two different tags (UPTAG and DOWNTAG) per strainTwo different tags (UPTAG and DOWNTAG) per strain

KAN RTAG

TAG

TAGTAG

TAG

TAG

Detecting molecular tags in yeast pools

PCR-amplify tags from pooled genomic DNA using fluorescently-labeled primers

Hybridize labeled tags to

oligonucleotide array

containing tag complements

Each tag has unique location

Tags can be used to perform negative selections on pools

Growth in minimal media identifies all known auxotrophic strainsWinzeler et., 1999 Science 285:901-906

Genomic profiling of drug sensitivitiesvia “induced haploinsufficiency”

Decreased gene dosage from two copies to one copy in heterozygous strains results in increased sensitivity, or

drug- induced haploinsufficiency

Strains that are heterozygous for drug target are haploinsufficient

in the presence of drug:

0123456

0 2 4 6 8 10 12

alg7/ALG7ALG7/ALG7

O.D.

600

time (hrs)

1. a) 0µg/ml tunicamycin

0123456

0 2 4 6 8 1012time (hrs)

b) 0.5µg/ml tunicamycin

0123456

0 2 4 6 8 1012time (hrs)

c) 2µg/ml tunicamycin

Giaever et al., 1999. Nature Genetics, 21:278-283

Tunicamycin sensitivity

Analysis of pools of heterozygous (and homozygous) strains reveals primary and secondary drug targets

G. Giaever, unpublished results

Examples- global screens

Synthetic lethals

Synthetic dosage lethals

Heterozygous diploids Haploinsufficiency modifiers Increased drug sensitivity- (target ID)

Direct phenotype screening

Method for genomic synthetic lethal (SL) screenMethod for genomic synthetic lethal (SL) screen

Tong et al., 2001 Science,Vol. 294, 2364-2368--- (Boone Lab)

YF mutation,plasmid,reporter,……

each deletion strain in quadruplicate

Final double mutant selection

MAT a deletion set

no growth potential SL interaction

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