Transcript MEDG505.Yeast.testbed.05

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
Common Ancestor
Years x10
6
~800
~350
~80
Human
Mouse
Fly Worm
Yeast
Mam malian Model
- transgenic m ice
- disease phenotype
Multicellular Biology
- genetic analysis
- organismal biology
Unicellular Biology
- Gene function
classical genetics
recombinant genetics
~30,000 genes
~15,000 genes
~6,000 genes
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Genomics technology development
Yeast 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
2 kb
Gene 1
25mers
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Gene 2
25mers
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).
Lisa Wodicka, Dave Lockhart, Affyme
Mapping complex traits
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Construct a SNP genetic map
Perform cross
Analyze rare segregants
Identify regions inherited solely
from one parent
YJM789
Laboratory strain
YJM789 parent
• Isolated from the lung of an AIDS
patient.
• Able to grow at 42 °C, form
pseudohyphae and undergo colonymorphology switching.
• Hypersensitive to cycloheximide.
• Polymorphic
– one difference every 150 bases
relative to sequenced strain
Allelic variation between two strains can be detected using arrays.
Laboratory strain
(non-pathogenic)
2 kb
Gene 1
YJM789
(virulent wild
isolate)
Polymorphisms
*
25mers
*
2 kb
Gene 1
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25mers
Mismatch control probe
(position 13 of 25)
Yeast Array
missing signals = markers
Since probe locations are known, a genetic map can be constructed:
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 clear recombination breakpoints
 Expect most markers to segregate 2:2.
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Markers segregate as expected
96 crossovers
(90 expected).
96% of markers
segregate 2:2.
Clear breakpoints
observed.
Segregation of markers in one tetrad (one chromosome)
Haploid
1
Haploid
Diploid
16
...
1
1
16
...
1
16 16
...
Laboratory strain (S96)
genotype: MATa, lys5,
LYS2, ho, CYH
Wild Isolate (YJM789)
genotype MAT LYS5,
ho::hisG, cyh
16
...
spore 1
...
spore 2
...
spore 3
(mat  lys2, LYS5, ho, cyh)
...
spore 4
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
yeast sequence
KanR
Amplify selectable marker gene
using primers with yeast gene
homology at 5’ ends
plasmid
Deletion Cassette
Chromosomal Gene
Replace yeast gene by
homologous recombination
International Deletion Consortium Members
Mike Snyder, Jasper Rine, Mark Johnston, Jef Boeke,
Howard Bussey, Rosetta, Acacia, Peter Philippsen,
Hans Hegemann, Francoise Foury, Guido Volckaert,
Bruno Andre, Giogio Valle, Jose Revuelta, Steve
Kelly, Bart Scherens
24,000 strains in 3 years
Serial analysis of deletion strains
Apply Selection
1
2
3
6,000
Identify deletion strains
with growth defects
Molecular tags as strain-identifiers
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Unique 20-mers
Good hybridization properties
Similar melting temperatures
More than 5 base differences between each
1.1 x 10 12 possible 20mers
12,000 best
Can be introduced during strain construction
 Two different tags (UPTAG and DOWNTAG) per strain
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Shoemaker et al., 1996. Nature Genetics, 14:450-456
Detecting molecular
tags in yeast pools
T G
A
KA
R
N
T G
A
T G
A
T G
A
T G
A
T G
A
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 strains
Winzeler et., 1999 Science 285:901-906
Genomic profiling of drug sensitivities
via “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:
1. a) 0µg/ml tunicamycin b) 0.5µg/ml tunicamycinc) 2µg/ml tunicamycin
6
5
4
3
2
1
0
6
5
4
3
2
1
0
6
5
600
O. 4
3
D.
2
1
0
0 2 4 6 8 10 12
time (hrs)
alg7/ALG7
ALG7/ALG7
0 2 4 6 8 10 12
time (hrs)
0 2 4 6 8 10 12
time (hrs)
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) screen
MAT a deletion set
YF mutation,
plasmid,reporter,……
each deletion strain in
quadruplicate
Final double mutant selection
no growth
potential SL interaction
Tong et al., 2001 Science,Vol. 294,
2364-2368--- (Boone Lab)
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