Addinall et al. 2008 – Supplementary Information.
1. Supplementary Methods
a. Growth media
b. Growth assays
c. Screening for cdc13-1 suppressors and synthetic interactors
d. Scoring of growth assays, photography and image analysis
e. Sample tracking
f. Hierarchical clustering
2. Supplementary Tables
3. Supplementary Figure Legends
4. Supplementary References
5. Supplementary Figures
SUPPLEMENTARY METHODS
Growth media
All strains created using synthetic genetic array (SGA) in this study were routinely
cultured in “DROP-2 + Canavanine + G418” media. This was adapted from Tong &
Boone (TONG and BOONE 2006) and was made as follows. 2 g Amino acid
supplement powder (-His, -Arg, -Leu, -Ura, -Ser) + 1.7 g yeast Nitrogen base + 1 g
monosodium glutamate + 20 g Bacto agar + 6 ml 1M NaOH, made up to 950 ml with
H2O and autoclaved; after cooling, 50 ml 40% dextrose + 0.5 ml 100 mg/ml
Canavanine + 1 ml 200 mg/ml G418 were added. The deletion library was routinely
cultured as spots on solid (2% Bacto agar) YEPD (yeast extract, peptone and
dextrose) containing 0.2 mg/ml G418. For all solid media, 6 ml of 1 M NaOH, per
litre, was added to prevent the otherwise low pH causing degradation of agar, which
sometimes lead to piercing of agar by pintools.
Growth assays
Growth assays were first performed manually (screens 1–2, Table S1), and
subsequently robotically (screens 3–7, Table S1). Manual spot-tests used sterile
wooden toothpicks to inoculate cdc13-1 strains from 768-colony SGA plates (384
duplicates) into liquid DROP-2 + G418 + Canavanine media in 96-well plates
(Greiner). After two days growth at 20°C, without shaking, aliquots of the resulting
yeast cultures were diluted 7 x in water, twice. The dilution series was then manually
plated onto solid YEPD + G418 media (Omnitrays, Greiner) using a sterile, manual, 2
mm diameter, 96-pin tool (SIGMA). Strains were incubated at 20, 27.3 and 36°C and
if growth was detected at 36°C (indicating contamination) and/or 27.3°C (indicating
suppression), the duplicate colony was tested by this same procedure.
For robotic spot tests (screens 3–7, Table S1), colonies were inoculated into 96-well
plates containing 200 µl DROP-2 + canavanine + G418 in each well, from solid agar
SGA plates, using a Biomatrix BM3-09 robot equipped with a 96 x 1 mm diameter
pin tool. These were grown to saturation (usually two days), without shaking, at 20°C.
The following 3 steps were then performed as part of an integrated robotic procedure
using a Beckman Biomek FX pipetting robot equipped with a magnetic, floating, 96pin, tool (V&P Scientific, Inc., San Diego, CA, USA). Yeast cultures were
resuspended, then diluted by dipping pins into the saturated culture and transferring to
a 96-well plate containing 200 µl H2O in each well; diluted cultures were then spotted
(after further mixing) onto solid DROP-2 + canavanine + G418. Each test plate was
spotted with either 96 or 384 strains. For 96-spot experiments (screens 3 and 4, Table
S1), pins were 3 mm in diameter and resuspension of yeast cultures was achieved by
repeated dipping of the pintool into cultures during the robotic procedure. For 384spot experiments (screens 5–7, Table S1; Figure 1), 2 mm diameter pins were used
and resuspension was achieved by shaking (Teleshake; 1000 rpm for 20s). To spot
384 strains, the contents of four 96-well plates were spotted onto each agar plate (see
Figures S1 and 1). Bar-coded plates were used to track individual strains through the
process. In both manual and robotic assays, single cultures were spotted onto multiple
agar plates and incubated under different conditions (e.g. 20°C, 27°C, 36°C).
Screening for cdc13-1 suppressors and synthetic interactors
Query strain DLY2304, containing the recessive cdc13-1 allele flanked by dominant
selectable markers URA3 and LEU2 (Materials and Methods and (DOWNEY et al.
2006)), was crossed to the collection of viable single gene deletion strains in 768colony per plate format (384 duplicates per plate). Spot test growth assays (see above)
for one replicate (Screen 1, Table S1) were incubated at 20°C, 27.3°C and 36°C, then
growth under each condition was scored by eye. Strains which demonstrated growth
at 27.3°C were identified as potential suppressors of the temperature sensitivity of
cdc13-1. Growth at 36°C was used as an indication of failure of the SGA process or
spontaneous reversion, since previous experience with the cdc13-1 mutation indicated
that genetic suppression would only rarely permit growth at such a high temperature
(ZUBKO et al. 2004; ZUBKO and LYDALL 2006). Since no gene deletion allowed
growth of cdc13-1 cells consistently at 36°C, this control was robust. For those strains
that appeared to grow at 36°C and/or 27.3°C, the second replicate was tested. This
whole process was repeated (Screen 2, Table S1), resulting in a minimum of 2 and a
maximum of 4 replicates corresponding to each viable gene deletion.
In order to produce a robust and exhaustive list of cdc13-1 interacting genes, the
screen was repeated – two independently isolated strains similar to DLY2304 but
containing the Schizosaccharomyces pombe His5 gene instead of the S. cerevisiae
HIS3 gene for haploid selection (DLY3241, DLY3242; Table S2) were each crossed
in duplicate, in 768-format (384 duplicates per plate), to a subset of viable single gene
deletion strains (Screens 3 and 4, Table S1). One replicate from each cross was tested
for suppression of temperature-sensitivity using robotic growth tests in 96-spot format
(Materials and Methods). Growth under each condition (20, 27, 28 and 36°C) was
scored by eye as above and strains which demonstrated growth at 27°C were
identified as potential suppressors (Figure 1).
At this stage a minimum of four and a maximum of six biological replicates had been
tested for each gene deletion (screens 1–4, Table S1). From these data 461 genes,
deletion of which allowed growth at the non-permissive temperature in 2 or more
replicates, were identified as possible cdc13-1 suppressors. The corresponding 461
gene deletion strains were mated to DLY2304 in 384-format (Screen 5, Table S1) and
the subsequent SGA strains tested for their ability to grow at the non-permissive
temperature relative to controls. Combining this replicate with the previous 4–6
replicates, gene deletions which allowed cdc13-1 mutants to grow at the nonpermissive temperature in the majority of replicates were designated as suppressors
(Table 1).
For strains scored as growing at 36°C in one or more replicates of the SGA libraries,
the corresponding single gene deletion strains were mated again to DLY2304 (Screen
6, Table S1) in 1536-format (384 quadruplicates per plate) and their phenotypes
tested. Another subset of strains were identified as either slow-growing or nongrowing, indicating potential synthetic interactions with the cdc13-1 mutation. For
these genes, the corresponding single gene deletion strains were mated to DLY2304
(Screen 7, Table S1) in 1536-format (384 quadruplicates per plate) and their growth at
27°C re-tested. For these follow-up SGA experiments, growth tests were spotted in
384-format and measured by image analysis and strain locations were tracked using
bar-coded plates and "plate", "row" and "column" identifiers (Materials and Methods;
Figure S1). The results of these were used to complete and refine the list of gene
deletions that suppress cdc13-1 temperature sensitivity (Table 1). They also allowed
the identification of genes which exhibited synthetic lethal and synthetic sick
interactions with cdc13-1 (Table 2). Where mating between the query and deletion
strain was shown to be successful (ie. growth occurred on diploid selection plates;
(TONG and BOONE 2006; TONG et al. 2001)) deletion of a gene was considered
synthetic lethal with cdc13-1 when at least 6 out of 8 biological replicates failed to
produce viable haploid progeny at the end of the SGA process (Figure S2, Table 2).
On those few occasions where 1 or 2 matings out of 8 produced viable cells, the
colonies showed very strong growth indicative of spontaneous reversion, second-site
suppression or failure of the SGA procedure. Gene deletions which, when combined
with the cdc13-1 mutation, exhibited consistently poor growth at the permissive
temperature for cdc13-1 were classed as synthetic sick with cdc13-1. This was judged
at the final stage of SGA experiments, by eye, from photographs of solid agar plates
(Figure S2).
A group of deletion strains which consistently performed poorly in SGA experiments
(TONG et al. 2001) were treated differently from other strains from screen 3 onwards
(Table S1). Some were absent from all subsequent experiments due to use of a
slightly different strain collection (Table S1). Others which were included in the
SGAv2 collection were removed from the list of synthetic lethal or synthetic sick hits
when scored as such, however they were permitted to be scored as suppressors or as
hits in the UP-DOWN assay. Other genes removed from our list of interactors were
those involved as selective markers during the SGA strain construction procedure or
which function in the same pathways as those genes. These included CAN1 (scored as
a suppressor) and HIS1, HIS2, HIS4, HIS6, HIS7, URA1, URA2, URA4, LEU1 (scored
as synthetic sick or synthetic lethal). In addition, because we routinely neutralize the
acidic pH of solid media by addition of NaOH prior to sterilization (see methods), we
excluded RIM101 and RIM20 (involved in the response to alkaline pH and both
scored as suppressors) from our list of interactors.
Finally, some genes classified as “dubious” open reading frames in the SGD database
were identified as hits in our screen. Most of these overlap neighbouring genes which
are the likely cause of their deletion phenotypes (Table S4). We have, however, left
YEL033W (MTC7) as a putative UDS hit since its deletion has previously been
demonstrated to cause shortened telomeres and YEL033W does not extensively
overlap with neighbouring genes.
Scoring of growth assays, photography and image analysis
Manual growth tests were scored by eye directly from the agar plates. Robotic growth
tests in 96-spots format were also scored directly from the agar plates by eye. Robotic
growth tests in 384-spot format were photographed on a Biomatrix BM3-09 robot
equipped with an integrated Canon Powershot A620 camera. Images were captured in
32 bit RGB resolution, and saved in .jpg format. They were subsequently converted to
8-bit greyscale .pgm images for analysis. Contrast between yeast spots and the
background was good, permitting the use of a simple thresholding algorithm for
segmentation. The threshold was selected using an iterative threshold selection
algorithm (PARKER 1997; SONKA et al. 1993). Spot positions were then demarcated
by applying a grid to the image. Individual rows and columns of the grid were
adjusted to minimize the sum of white pixels across each line profile. Due to
irregularities in the growth of spots – some failed to grow at all, whilst others
overgrew the boundaries set by the grid and sometimes merged visually with
neighbouring spots – this adaptive approach was not applicable to all plates. Plates
with non-uniform growth were thresholded and gridded using default values
calculated as the average of those from several hundred uniformly-growing plates.
The area and sum of grey values was calculated for each identified colony and output
alongside bar-code, "row", "column", date and time information for each spot, in a tab
delimited “imagelog” file (see below).
Sample tracking
Strains were tracked using bar-codes and the reporting functions of each robotic
setup. Inoculation was reported by the Biomatrix BM3-09 in the form of a text
"inoclog" file which links the bar-code of each 96-well plate to the bar-code of the
solid agar plate from which it was inoculated. Spotting was reported by the Beckman
Biomek FX in the form of multiple spreadsheet "spotlog" files. Spotlog files carry the
bar-code of the plate they are describing in their filename. The spreadsheet describes a
384-spot pattern divided into 4 x 96-spot quadrants and indicates the bar-code of
which 96-well plate was spotted into which quadrant. Photography was reported by
automatic appending of bar-code, time and date information to each image-file name,
which was combined with image analysis data in a tab-delimited "imagelog" file.
Original strain positions were described in a "master" spreadsheet file defining
"plate", "row" and "column" numbers for each strain.
Strain location and tracking information were linked to each other by uploading of
inoclog, spotlog, imagelog and master files into a "Robot Object Database" (ROD)
which also stored metadata regarding "media" (the contents of each bar-coded plate)
and "treatment" (how each plate was treated, for example "incubation at 20˚C").
For each complete replicate of a screen, a summary of the data was exported from
ROD in a tab-delimited file and examined along with data from other replicates.
Hierarchical clustering
Data from two telomere length studies (ASKREE et al. 2004; GATBONTON et al. 2006;
SHACHAR et al. 2008) were combined as follows. Genes which were found to affect
telomere length in both studies were given a score (negative for deletions which cause
shorter telomeres and positive for deletions which result in longer telomeres) of 2,
those which were identified in only one study were similarly given a score of 1. Gene
deletions which result in sensitivity to MMS (JELINSKY and SAMSON 1999) or UV
irradiation (BIRRELL et al. 2001) were given a score of -1 in the corresponding
category and gene deletions which result in sensitivity to ionizing radiation (BENNETT
et al. 2001) were scored as -1 if they confer a delayed recovery phenotype and -2
otherwise. Genes which are regulated by nonsense-mediated decay (HE et al. 2003)
were assigned a score of 1 and genes regulated by MMS were scored as either +1 (upregulated) or -1 (down-regulated). Gene deletions which affect the replication of a
positive-strand RNA virus were scored as +1 (replication of the virus is enhanced) or 1 (replication is inhibited). In all of the above cases, genes were given a score of 0 (no
result) or no score (not tested) or 0 if no distinction was made between these two
possibilities. Our own data was scored as follows. Strong suppressors (+2) and
suppressors (+1) were combined in one category, synthetic lethal (-2) and synthetic
sick (-1) were combined as “enhancers”, UP-DOWN sensitive (-1 to -3 depending on
severity of phenotype) and UP-DOWN resistant (+1) were also combined. Only genes
identified in our study that conferred a phenotype in at least two categories were
included in the clustering.
SUPPLEMENTARY TABLES
Table S1. Screen progression during this study.
Screen
Starter strain
Library
(library layout)
1
2
3
4
5
6
7
DLY2304
DLY2304
DLY3241 (F1A)
DLY3242 (C4B)
DLY2304
DLY2304
DLY2304
Euroscarf
Euroscarf
SGAv2
SGAv2
SGAv2-hits
SGAv2-cont
SGAv2-son
(21 plates, (WINZELER
et al. 1999))
(21 plates, (WINZELER
et al. 1999))
(14 plates, (TONG et al.
2001))
(14 plates, (TONG et al.
2001))
2 plates (genes scored as
hits in screens 1–4)
3 plates (genes scored as
contaminated in screens
3 and 4)
1 plate (genes scored as
slow or non-growers in
screens 1–4)
In each of screens 3–7, 76 “control” his3::KANMX strains were positioned around the edge of each plate (TONG et al. 2001).
Comments
Spots per plate for
matings (replicates)
SGA robot
Spotting method
Dilutions for spotting
Strains tested per plate
(relative throughput)
Liquid media
Spotting media
Scoring
768 (2)
768 (2)
768 (2)
768 (duplicates)
384 (1)
1536 (4)
1536 (4)
Virtek Versarray
Virtek Versarray
Virtek Versarray
Virtek Versarray
Biomatrix BM3-09
Biomatrix BM3-09
Biomatrix BM3-09
Manual
Manual
Biomek FX with 96-pin
tool
Biomek FX with 96-pin
tool
Biomek FX with 96pin tool x 4
Biomek FX with 96-pin
tool x 4
Biomek FX with 96-pin
tool x 4
3 x 7-fold
3 x 7-fold
pin tool dipped in culture
then in 200 µl H2O
pin tool dipped in culture
then in 200 µl H2O
pin tool dipped in
culture then in 200 µl
H2O
pin tool dipped in culture
then in 200 µl H2O
pin tool dipped in culture
then in 200 µl H2O
32 (1)
32 (1)
96 (~3)
96 (~3)
384 (~10)
384 (~10)
384 (~10)
DROP-2 + G418 +
Canavanine
DROP-2 + G418 +
Canavanine
DROP-2 + G418 +
Canavanine
DROP-2 + G418 +
Canavanine
DROP-2 + G418 +
Canavanine
DROP-2 + G418 +
Canavanine
DROP-2 + G418 +
Canavanine
YEPD + G418
YEPD + G418
DROP-2 + G418 +
Canavanine
DROP-2 + G418 +
Canavanine
DROP-2 + G418 +
Canavanine
DROP-2 + G418 +
Canavanine
DROP-2 + G418 +
Canavanine
By eye from plates
(RH)
By eye from plates
(MB)
By eye from plates (MZ)
By eye from plates (MZ)
Image analysis and
confirmed by eye from
photographs
Image analysis and
confirmed by eye from
photographs
Image analysis and
confirmed by eye from
photographs
Number colonies tested
Dates
1 (2)
1 (2)
1
1
4
4
4
6/2003
8/2003
9/06
11/06
4/2007
8/2007
9/2007
1 colony each from screens 3 and 4 were analysed in
the UP-DOWN assay and tested for growth at 28°C
Comments
4 colonies each from screens 6 and 7 were analysed in
the UP-DOWN assay
Table S2. Strains used in this study.
Strain
Genotype
Alternative Name
Reference
Source
BY4741
MATa ura3 leu2 his3 met15
BY4742
MAT ura3 leu2 his3 lys2
Y2454§
BY4742 mfa::MFA1pr-HIS3 can1
DLY1622
Y2454 cdc13-1 int
DLY2198
DLY1622 cdc13-1 int::URA3
DLY2304
DLY2198 LEU2::cdc13-1 int::URA3
DLY3241
MAT mfa::MFA1pr-spHIS5+ LEU2::cdc13-1 int::URA3 can1
F1A
This work.
DLY3242
MAT mfa::MFA1pr-spHIS5+ LEU2::cdc13-1 int::URA3 can1
C4B
This work.
Strain Collection
Construction
Reference
Source
SGA-v2
BY4741 orfX::KANMX
(TONG et al.
2001)
Charlie Boone
SGA-cdc13-1
DLY2304 x SGA
(DOWNEY et al.
2006)
SGA-v2-F1A
DLY3241 x SGA-V2
This work.
SGA-v2-C4B
DLY3242 x SGA-V2
This work.
(TONG et al.
2001)
(DOWNEY et al.
2006)
SGA-v2-hits
DLY2304 x SGA-V2 (461 possible suppressors)
This work.
SGA-v2-son
DLY2304 x SGA-V2 (288 slow or non-growers)
This work.
SGA-v2-cont
DLY2304 x SGA-V2 (976 contaminated in Screens 3 and 4)
This work.
Table S3. Statistical analysis of gene ontology terms applied to cdc13-1 interacting
genes.
Strong suppressors: Biological Process
GOBPID
Pvalue^
OddsRatio
ExpCount*
Count Size
Term
GO:0000077 5.39E-07
101.2533
0.0756
4
10
DNA damage checkpoint
GO:0000075 7.30E-06
23.8005
0.2875
5
38
cell cycle checkpoint
GO:0000184 7.68E-06
146.1538
0.0454
3
6
mRNA catabolic process, nonsense-mediated decay
Suppressors: Biological Process
GO:0009987 1.06E-10
3.4394
120.3922
158
2521
cellular process
GO:0006996 3.04E-07
2.3777
33.1902
61
695
organelle organization and biogenesis
GO:0043170 6.41E-06
1.9713
63.4673
92
1329
macromolecule metabolic process
GO:0000723 8.32E-06
3.2404
8.5005
23
178
telomere maintenance
140.2586
163
2937
intracellular part
Suppressors: Cellular Component
GO:0044424 9.38E-06
2.5707
Synthetic lethal or Synthetic sick: No over-represented terms in any category
UDS NOT suppressors: Biological Process
GO:0016571 4.05E-09
51.3013
0.2922
7
14
histone methylation
GO:0031324 8.20E-09
7.6991
2.7974
16
134
negative regulation of cellular metabolic process
GO:0000723 1.23E-08
6.5177
3.7160
18
178
telomere maintenance
GO:0006996 1.98E-08
3.8401
14.5093
36
695
organelle organization and biogenesis
GO:0065007 3.07E-08
3.8522
13.3194
34
638
biological regulation
GO:0008213 3.51E-08
32.6114
0.3757
7
18
protein amino acid alkylation
GO:0048519 5.67E-08
6.1788
3.6325
17
174
negative regulation of biological process
GO:0006325 5.65E-07
5.8240
3.2985
15
158
establishment and/or maintenance of chromatin
architecture
GO:0050794 6.41E-07
3.6730
10.1043
27
484
regulation of cellular process
GO:0006342 8.41E-07
9.2606
1.3987
10
67
chromatin silencing
GO:0000279 1.07E-06
5.5033
3.4655
15
166
M phase
GO:0043414 1.10E-06
17.0365
0.5845
7
28
biopolymer methylation
GO:0040029 1.28E-06
8.7904
1.4613
10
70
regulation of gene expression, epigenetic
GO:0009987 1.41E-06
7.3114
27.3294
42
1919
cellular process
GO:0031497 1.90E-06
8.3650
1.5240
10
73
chromatin assembly
GO:0022402 2.14E-06
4.4252
5.1983
18
249
cell cycle process
GO:0043283 2.91E-06
2.9246
20.7933
40
996
biopolymer metabolic process
GO:0006281 2.95E-06
6.3148
2.3799
12
114
DNA repair
GO:0006338 3.10E-06
6.9613
1.9832
11
95
chromatin remodeling
GO:0007001 4.61E-06
5.2294
3.4322
14
194
chromosome organization and biogenesis
GO:0006313 5.88E-06
65.7719
0.1461
4
7
transposition, DNA-mediated
GO:0000335 5.88E-06
65.7719
0.1461
4
7
negative regulation of DNA transposition
GO:0019222 7.37E-06
3.6186
7.4530
21
357
regulation of metabolic process
GO:0009719 7.86E-06
5.2388
3.0688
13
147
response to endogenous stimulus
GO:0006730 8.25E-06
11.8968
0.7724
7
37
one-carbon compound metabolic process
GO:0016481 8.34E-06
6.2038
2.1920
11
105
negative regulation of transcription
GO:0009090 8.77E-06
Inf
0.0626
3
3
homoserine biosynthetic process
GO:0051053 8.92E-06
24.9466
0.3131
5
15
negative regulation of DNA metabolic process
GO:0000075 9.94E-06
11.5099
0.7933
7
38
cell cycle checkpoint
3
3
telomerase activity
UDS NOT suppressors: Molecular Function
GO:0003720 8.77E-06
Inf
0.0626
UDS NOT suppressors: Cellular Component
GO:0044422 1.14E-09
4.0339
22.2338
48
1065
organelle part
GO:0043232 7.47E-09
4.4990
9.3945
29
450
intracellular non-membrane-bound organelle
GO:0043234 5.01E-07
3.5902
11.3152
29
542
protein complex
GO:0044428 1.75E-06
3.9126
7.4405
22
370
nuclear part
GO:0044427 7.03E-06
7.1003
1.7536
10
84
chromosomal part
GO:0005697 8.77E-06
Inf
0.0626
3
3
telomerase holoenzyme complex
RAD9-like / EXO1-like: Biological Process
GO:0000075 1.56E-08
54.5336
0.1884
6
38
cell cycle checkpoint
GO:0000077 8.90E-08
169.2
0.0495
4
10
DNA damage checkpoint
GO:0006259 6.72E-07
12.5555
1.5866
10
320
DNA metabolic process
GO:0006974 2.74E-06
16.0155
0.6991
7
141
response to DNA damage stimulus
GO:0051726 6.66E-06
17.6812
0.5106
6
103
regulation of cell cycle
^Statistical analysis performed using GOstats (FALCON and GENTLEMAN 2007) as
described in Materials and Methods. *truncated at 4 decimal places.
Table S4. Dubious open reading frames which were identified in this study.
Gene
SGD description (SGD 2008)
phenotype
comments
YNL171C
Dubious open reading frame unlikely to encode a
functional protein; based on available
experimental and comparative sequence data
UDS
YNL171C is adjacent to APC1 and
overlaps PSD1. Deletion of YNL171C
likely affects expression of the essential
APC1 gene to give an UDS phenotype
YNL235C
Dubious open reading frame unlikely to encode a
protein, based on available experimental and
comparative sequence data; partially overlaps the
verified ORF SIN4/YNL236W, a subunit of the
mediator complex
UDS
Deletion of SIN4 is synthetic with wildtype in SGA technique
YCL060C
Merged open reading frame, does not encode a
discrete protein; YCL060C was originally
annotated as an independent ORF, but as a result
of a sequence change, it was merged with an
adjacent ORF into a single reading frame,
designated YCL061C
UDS
Deletion of YCL061C (MRC1) gives UDS
phenotype
YBR174C
Dubious open reading frame unlikely to encode a
protein, based on available experimental and
comparative sequence data; partially overlaps the
verified ORF YBR175W; null mutant is viable
and sporulation defective
UDS
Deletion of YBR175W (SWD3) gives UDS
phenotype
YDR290W
Dubious open reading frame unlikely to encode a
protein, based on available experimental and
comparative sequence data; partially overlaps the
verified ORF RTT103
UDS
Deletion of RTT103 gives UDS phenotype
SUPPLEMENTARY FIGURE LEGENDS
Figure S1. Scheme for robotic growth tests of yeast strains in 384-spot format.
In this SGA procedure, 384 gene deletions per plate are crossed to a cdc13-1 query
strain in quadruplicate, resulting in 1536 double-mutant colonies per plate (left).
Hence colonies at positions 1,1; 1,2; 2,1 and 2,2 (coloured red) are four replicates of
the same gene deletion crossed to the query strain. One of these replicates is
inoculated into liquid growth media in 96-well plates using a 96-pin tool which
inoculates 96 out of 1536 colonies each time. In order to inoculate one replicate for
each of 384 gene deletions, four different “quadrants” (indicated as red, blue, green
and purple) are inoculated into four different 96-well plates containing growth media.
After growth at 20°C, cultures are diluted in water, then the four quadrants from one
repeat are spotted in 384-format onto a solid agar plate (right) in the same pattern as
the original SGA plate (as indicated by colours). The process can be repeated to test
up to four replicates.
Figure S2. Scoring of synthetic interactions from SGA experiments.
The same region of plates from two different stages of the SGA process are presented.
At the diploid selection stage (left), all spots are expected to grow unless the
particular gene deletion or query strain mutations have deleterious effects on mating.
At the haploid selection stage, however, growth is indicative of the success of the
SGA procedure. A gene deletion which consistently fails to produce progeny in SGA
(such as whi3∆ in this example) likely has a synthetic lethal interaction with the query
mutation, unless the strain has already been identified as one which generally gives
poor results in SGA (such as ctk1∆). The sin4∆ deletion consistently produced either
poor or no growth when combined with cdc13-1 and so was categorized as synthetic
sick with cdc13-1.
Figure S3. Interactions between groups of functionally related suppressor genes.
Groups of functionally related cdc13-1 suppressor genes are displayed using
OSPREY and numbered as described in Table 5. Interactions described in the BioGrid
database which link groups are displayed as lines connecting groups – the stroke of a
line being indicative of the number of separate interactions (as illustrated in the key).
Individual genes are represented as filled circles, colour-coded by OSPREY with each
colour (as per the key in Figure 2) representing a gene ontology term, up to a
maximum of four.
Figure S4. Interactions between Casein Kinase II, Tbf1 and Rap1.
Display of BioGrid interactions between Casein Kinase II (CK2) and RAP1 or TBF1.
Many of the genes which connect CK2 with either RAP1 or TBF1 in BioGrid are
known to be involved in gene silencing, transcription and/or chromatin remodeling,
affect telomere length or have been identified in our screen (see gene anotations in
figure). Individual genes are represented as filled circles, colour-coded by OSPREY
with each colour (as per Figure 2) representing a gene ontology term, up to a
maximum of four. Types of interactions are colour coded as per the key. Bold text in
the accompanying list highlights genes identified as cdc13-1 interactors in this study.
Figure S5. Interactions between groups of functionally related UDS genes.
Groups of functionally related UDS genes are displayed using OSPREY and
numbered as described in Table 5. Interactions described in the BioGrid database
which link groups are displayed as lines connecting groups – the stroke of a line being
indicative of the number of separate interactions (as illustrated in the key). Individual
genes are represented as filled circles, colour-coded by OSPREY with each colour (as
per the key in Figure 2) representing a gene ontology term, up to a maximum of four.
Note that vesicular traffic (Group 5) has been split into two (vacuole and endosome
function/peroxisome function), the mitochondrial group (Group 8) has been spilt into
three (genome integrity/electron transport/transport and integrity) and the ribosomal
group (Group 7) has been split into two (large subunit/other) for this figure.
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