yeast as a model organism model eukaryote –experimental genetics –gene function – orthologs,...
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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
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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
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Mapping complex traits
• Construct a SNP genetic map
• Perform cross
• Analyze rare segregants
• Identify regions inherited solely from one parent
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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
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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.
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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.
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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.
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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
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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)
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Inheritance of markers in 10 lys2 segregants
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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.
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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.
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Mapping Complex Traits: Feasibility Summary
• Identified 3808 genetic markers.
• Demonstrated that traits can be mapped using these markers.
• Next step: Map virulence loci.
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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.
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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
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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
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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
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Serial analysis of deletion strains
Apply Selection
Identify deletion strains with growth defects
1
2
3
6,000
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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
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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
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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
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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
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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
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Tunicamycin sensitivity
Analysis of pools of heterozygous (and homozygous) strains reveals primary and secondary drug targets
G. Giaever, unpublished results
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Examples- global screens
Synthetic lethals
Synthetic dosage lethals
Heterozygous diploids Haploinsufficiency modifiers Increased drug sensitivity- (target ID)
Direct phenotype screening
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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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