Transcript Dr. Hieter`s Lecture

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
Mammalian Model
- transgenic mice
- 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
QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture.
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
•
•
•
•
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
Assigning function by analyzing
gene expression
•
•
•
•
•
Isolate mRNA
Label mRNA
Hybridize to array
Detect hybridization
Measure the abundance of every mRNA
– Test different growth conditions
– Test different genetic backgrounds
Measuring gene expression using the Affymetrix array
Whole genome mRNA
expression pattern for
yeast grown in rich media
Lisa Wodicka,
Dave Lockhart
Affymetrix
Transcriptional analysis of the yeast cell
cycle
M
• Events of cell growth, DNA
replication, cell division and
chromosome segregation are tightly
controlled.
G1
• Cultures can be synchronized with
respect to the cell cycle.
• Cyclic regulation of transcription
expected.
G2
S
Cell cycle transcriptional regulation of two genes
CLN1
late G1 phase
S phase
G1 phase
S phase
YML027W
Normalized Transcript Level
Genes induced in S and M phase
2.5
2.0
1.5
1.0
0.5
0
0
20
G1
40
S
60
80 100 120
Time (minutes)
G2 M G1
S
140 160
G2 M
Cell-cycle transcription in yeast
• 425 open reading frames were identified
that displayed cell-cycle dependent
fluctuations in transcript levels.
• 40% were of unknown function.
• 30% are located next to other cell-cycle
transcribed genes (possible enhancer effect).
• Correlation with known and unknown
promoter elements.
Mapping complex traits
•
•
•
•
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
*
*
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.

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




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

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 tunicamycin c) 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
Saccharomyces cerevisiae Genome Deletion Project
•Collaboration of eight North American and eight European labs
to generate a complete set of yeast nonessential deletion mutants
•~4,700 nonessential genes deleted with kanMX = fifty 96 well plates
•~6,000 heterozygous diploids also available
96 well plate
frozen glycerol stock
pin 96 strains
onto G418 plates
condense 4
plates onto 1
Examples- global screens
Synthetic lethals
Synthetic dosage lethals
Heterozygous diploids
Haploinsufficiency modifiers
Increased drug sensitivity- (target ID)
Direct phenotype screening
Yeast as a tool to discover drugs
and their mechanism of action
Identification of natural compounds that
inhibit invasion
• Metastasis responsible for 90% of cancer deaths
• Metastasis requires invasion of adjacent tissue
and blood vessels by tumour cells
Lianne McHardy, Cal Roskelley, Shoki Dedhar,
Ali Karsan,David Williams, Raymond Andersen,
Michel Roberge
N
N
H
NH2
Motuporamine C
N
N
H
DihydroMotuporamine C
Xestospongia exigua
from outer reef off
Motupore Island,
Papua New Guinea
NH2
How to identify the mechanism of action
of motuporamines?
- Invasion is a complex process, incompletely understood
- Structure of motuporamines gives no clue to function
-Motuporamines are not good candidates for biochemical
approaches
Drug-Induced Haploinsufficiency
Drug
Y/Y
Y
Y
Alive
Y
Y
Alive
Drug
y∆/Y
Y
Alive
Y
Dead
Drug-induced haploinsufficiency
Proof of principle study:
Giaever et al. Genomic profiling of drug sensitivities via
induced haploinsufficiency. Nat Genet 21, 278-83. (1999)
Can these techniques really identify the target
or targetted pathways of a drug with an
unknown mechanism?
Can they predict the target in human cells?
Steps of drug-induced haploinsufficiency
screen
1- selection of a drug-induced phenotype
2- systematic high-throughput drug-induced phenotypic
screen of yeast heterozygous deletion diploid set
3- quantitative ranking of drug sensitivity PRIORITIZATION
4- confirmation of drug mode of action in yeast
5- assessment of cognate mode of action in the
mammalian system
dhMotC affects yeast growth
liquid culture
Screen with or without 60 µM dhMotC
and identification of strains showing increased sensitivity
8 strains
in duplicate
QuickTime™ and a
Photo - JPEG decompressor
are needed to see this picture.
Treatment:
DMSO
dhMotC
Heterozygous deletion strains sensitive to dhMotC
ORF
YCL034W
YNL314W
YML099CARG81
YBR078W
YNL267W*
NAME
Biological Process
LSB5
actin filament organization
DAL82
allantoin catabolism and transcription initiation from
arginine metabolism
ECM33
cell wall organization and biogenesis
PIK1 *
cytokensis, post Golgi transport and signal transduction YLR286C
CTS1
cytokinesis, completion of separation
YDL192W
ARF1
ER to golgi transport and intra-golgi transport
YBR290W
BSD2
heavy metal ion transport and protein-vacuolar targeting
YLR025W
SNF7
late endosome to vacuole transport
YHR147CMRPL6
protein biosynthesis
YOL040C*
RPS15 *
protein biosynthesis
YAL005C SSA1
protein folding and protein-nucleus import, translocation YIL047C
SYG1
signal transduction
YBR265W*
TSC10 *
sphingolipid biosynthesis
YMR296C*
LCB1 *
sphingolipid biosynthesis
YJR007W*
SUI2 *
translation initiation
YML092C*
PRE8 *
ubiquitin-dependent protein catabolism
YER140W
YER140W
unknown
YER188W
YER188W
unknown
Which are more relevant?
YGR205W
YGR205W
unknown
YLR294C
YLR294C
unknown
* essential genes
Ranking of strain sensitivity in liquid culture using low dhMotC concentration (20 µM)
Supersensitive strains (Integrated Growth Curve Difference >2)
Dihydrosphingosine rescues growth inhibition by dhMotC
Can these techniques really identify the mode of action of a drug?
YES
Can they predict the target/target pathway in human cells?
YES
Advantages
-systematic, unbiased and genome-wide
-adaptable to other phenotypes.
-pathway conservation = physiological phenotype
-development of chemical probes
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)
QuickTime™ and a TIFF (Uncomp resse d) de com press or are nee ded to s ee this picture.
QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture.
DONE
ressor are needed to see this picture.
QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture.
QuickTime™ and a TIFF (Uncompressed) dec ompressor are needed to see this picture.
QuickTi me™ a nd a TIFF (Uncompre ssed ) decomp resso r are need ed to se e th is p icture.
QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture.
CSG2 deletion rescues
growth inhibition by dhMotC
dhMotC reduces
cellular ceramide
levels
Ceramide partially rescues
dhMotC toxicity in
mammalian cells