Transcript Slide 1

Saccharomyces cerevisiae
a model system representing a simple
eukaryote
Hörður Guðmundsson og Guðrún P. Helgadóttir
Advantages of S. cerevisiae
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Nonpathogenic
Rapid growth (generation time ca. 80 min)
Dispersed cells
Ease of replica plating and mutant isolation
Can be grown on defined media giving the
investigator complete control over
environmental parameters
Well-defined genetic system
Highly versatile DNA transformation system
Therapeutic products from yeasts
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Prokaryotic products: Tetanus toxin fragment C; Streptokinase
Surface antigens of viruses: Hepatitis B; HIV; Foot and mouse
disease; Influenza; Polio; Polyoma; Epstein-Barr; Oncogenic
retroviruses
Malaria antigen
Animal products: Hirudin; porcine interferon; interleukin; trypsin
inhibitor
Human hormones: Insulin; parathyroid hormone; growth hormone,
chorionic gonadotropin
Human growth factors: IGF1; NGF; EGF; tissue factor; CSF; GMCSF; TNF
Human blood proteins: Hemoglobin; factors VIII and XIII; alpha-1antitrypsin; antithrombin III; serum albumin
Various human enzymes; CFTR; estrogen receptor; INF-alpha; INFbeta1
General advantages of S. cerevisiae
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Strains have both a stable haploid and
diploid state
Viable with a large number of markers
→recessive mutations are conveniently
manifested in haploid strains and
complementation tests can be carried
out with diploid strains
General advantages of S. cerevisiae
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The ease of gene disruptions and single step gene
replacements offers an outstanding advantage for
experimentation
Yeast genes can functionally be expressed when
fused to the green fluorescent protein (GFP) thus
allowing to localize gene products in the living cell by
fluorescence microscopy
The yeast system has also proven an invaluable tool
to clone and to maintain large segments of foreign
DNA in yeast artificial chromosomes (YACs) being
extremely useful for other genome projects and to
search for protein-protein interactions using the twohybrid approach
General advantages of S. cerevisiae
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Transformation can be carried out directly with
short single-stranded synthetic oligonucleotides,
permitting the convenient productions of
numerous altered forms of proteins
Extensively exploited in the analysis of gene
regulation, structure–function relationships of
proteins, chromosome structure, and other
general questions in cell biology
The yeast genome
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S. cerevisiae contains a haploid set of
16 well-characterized chromosomes,
ranging in size from 200 to 2,200 kb
Total sequence of chromosomal DNA is
12,8 Mb
6,183 ORFs over 100 amino acids long
Advantage of using S.cerevisiae in this study
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Parallell analysis of yeast strains with heterozygous deletions of
drug target genes can be used to monitor compound activities
in vivo
The cellular targets of clinically proven small molecules are used
to identify proteins that can be safely and effectively targeted in
humans
Many human disease-associated genes have highly conserved
yeast counterparts and therefore S. cerevisiae has been proven
to be a powerful tool for mechanistic studies of clinically
relevant compounds
In this study reported targets for many well-characterized
compounds were correctly identified
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Many potentially novel drug targets were also identified
Since this study was completed tagged heterozygous deletion
strains have been made available for virtually every gene in the
yeast genome
Greinin
Discovering Modes of Action for
Therapeutic Compounds Using a
Genome-Wide Screen of Yeast
Heterozygotes
Lum et al., Cell, 116, pp. 121–137
Yeast Genome Deletion
Project
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Generating a library of gene deletion strands
Using a PCR-based gene deletion strategy
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Homologus recombination
Four different collections generated:
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Haploids Mat a
Haploids Mat 
Homozygous diploids (for non-essential genes)
Heterozygous diploids (used in this experiment)
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Reducing the number of copies of a gene results in
sensitization to drugs targeting the protein product of
that gene
PCR-based gene deletion (in YGDP)
Replacement DNA (in YGDP)
Common
5´ barcode
(18bp)
(20 bp)
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KanR
3´ barcode Common
(20 bp)
(18 bp)
Two specific 20 bp barcode sequenses for
each insertion, you only need to sequence
the barcode to know which strain you have
Using primers in KanR and common it is
possible to PCR all the strands using the
same primers (vital to this exp.)
Competitive growth
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Reducing number of copies of a gene results in
sensitization to drugs targeting the protein product of
that gene
Constructed a heterozygous deletion pool containing
3503 deletion strands (about ½ the yeast genes)
Grown for 10 generations in normal medium then
grown for 10 generations in medium with drug
Isolation of genomic DNA
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DNA isolated
G-0 PCR with Cy3 markt primer
G-20 PCR with Cy5 markt primer
Hybridization
Identification
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Tags printed in
triplicate on the
microarray
Color intesity for G-0
and G-20 compared
Fitness profiles of 78
compounds
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Assess the cellular effects of
78 compounds
Three groups of
compounds:
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Group I: showed no drugspecific fitness changes (18)
Group II: a small number of
significant outliers (56)
Group III: showed wide
spread fitness changes (4)
Advantages of fitness assay
for analyzing drug activities
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Requires no prior knowledge of
compounds mode of action
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Allows truly novel drug activities to be
uncovered in a systematic and unbiased
fashion
Biological processes that are affected by
a given compound are identified in
addition to the precise protein targets
Limitations
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Compound of interest must be able to affect
the growth rate of the cell
The activity level of the targeted protein must
be influenced by the dosage level of the
corresponding gene
Compounds that exert their effects through
direct interaction with non-protein elements
in the cell do not appear suitable for this
approach
Thank you