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sequences. The more important a gene is for growth in
a given condition, the more rapidly the associated deletion
strain will diminish relative to the other strains in the
culture, reflected by the decreased intensity of the associ-
ated molecular barcodes on the 'tag' array. Thus, all genes
required for growth are identified and quantitatively ranked
in order of their relative importance for resistance to the
condition of interest. This powerful assay has been applied
in
comprehensive genome-wide datasets from these various
platforms. Some of the genomic technologies that were
first developed and tested in yeast include the first
comprehensive gene expression microarray (following
proof of concept [14] ) and the molecular barcode micro-
array (the 'TAG series') designed for fitness profiling
assays. In addition, yeast was used to test several gener-
ations of technology improvements of the microarray,
many testing the performance of ever-increasing oligo-
nucleotide densities. More recently, the first complete 4 bp
resolution tiling array for both DNA strands of the yeast
genome allowed the characterization of the complete
transcriptome, leading to one of the first observations of
pervasive transcription [15] . Other genomic technologies
developed and tested using S. cerevisiae include, for
example, platforms that measure protein modifications
[16] and protein activity [17] ,protein
10 000 individual studies, a large percentage of which
have been driven by the HIPHOP chemogenomic screening
platform, a variation on the fitness assay ( Box 8.1 ),
allowing the systematic interrogation of the relative
requirement of each gene for resistance to the small
molecule or drug tested (described in detail in the HIPHOP
section).
>
lipid interactions
[18] , genetic interactions (synthetic genetic array (SGA)
technology) [19,20] and chemical
e
Impact of Yeast in the Development
of Genomic Technologies
genetic interactions
[21] , among others. SGA technology, developed by
Charlie Boone and Brenda Andrews at the University of
Toronto, has attracted particular attention, allowing the
identification of all (non-essential) genes that genetically
interact with a chosen 'query' gene in a single experiment,
e
The impact of the YGP and YKO projects is clear from
the
3000 citations of the two publications describing the
YKO collection [10,11] .Yeastmovedontobecome
a highly respected innovator and test bed for virtually all
'omics'
>
technologies, generating a vast number of
BOX 8.1 Fitness-Based Chemogenomic Dosage Assays
Chemogenomic profiles are generated by screening a library of
yeast strains and measuring the sensitivity or resistance of each
individual strain to small molecule of interest In
l MoBY-ORF 2.0 collection; each gene carried on a single-
copy CEN plasmid expressed from its native promoter [25].
The non-competitive assays typically involve growing
strains on plates where the fitness phenotype is quantified by
colony size (e.g., [26] ). In these assays, small molecules are
added to solid media in an automation-friendly petri dish
(known as an 'omni tray') to serve as the test plate;
,
there are several full genome yeast collections available
comprised of strains that each carry a gene whose copy number
is either reduced or increased compared to a wild-type strain.
Here, we restrict our discussion to the yeast collections that
carry DNA barcodes (20 bp in length) and therefore allowing
parallel chemogenomic screens to be performed (as described
in Fig. 8.1 ). These include:
l
S. cerevisiae
600 such
assays have been performed to date. Individual strains are
arrayed onto the plates by 'pinning' from a 96- or 384-well
formatted deletion collection. Sensitivity or resistance to small
molecules is then measured by comparing the colony size of
each strain to the same strain grown on a mock-treatment
control plate, or to a control wild-type strain on the same plate,
Colony size has often been assessed by visual inspection,
although image analysis software can now be applied for better
quantification. The collection of resulting relative growth
measurements defines a chemogenomic profile specific to the
tested small molecules, with the number of significant differ-
ences observed dependent on the sensitivity and precision of
the growth metric used. The limitations of chemical screens
performed in plate assays are that they are time consuming,
costly in terms of the small molecule (requiring 10-100X the
quantity required for a pooled liquid assay) and the cost/plate is
2-5 USD.
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Yeast Knock Out (YKO) collection [10,11] , each strain
carries a start-to-stop deletion:
l
homozygous deletions strains; complete deletion of
a nonessential gene in a diploid strain background
haploid deletion strains; complete deletion of a nones-
sential gene in a haploid strain background
l
heterozygous deletion strains; single-copy deletion of an
essential or nonessential gene in a diploid strain
background
l
Decreased Abundance by mRNA Perturbation (DAmP) [22]
collection [23] ; each strain carrying a DAmP allele of an
essential gene expressing ~10% of
l
the wild-type gene
dosage from its native promoter
Molecular Barcoded Yeast Open Reading Frame (MoBY-
ORF) collection; each gene carried on a high-copy 2
l
m
plasmid expressed from its native promoter [24]
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