Supporting Online Material


Abstract
Full Text
Systematic Identification of Pathways That Couple Cell Growth and Division in Yeast
Paul Jorgensen, Joy L. Nishikawa, Bobby-Joe Breitkreutz, Mike Tyers

Supplementary Material

Materials and Methods
Strains and cell size analysis

Deletion strains were obtained from H. Bussey (McGill University, Montreal), Research Genetics and EUROSCARF. As it was not present in available collections, a precise ORF deletion of SCH9 was constructed by PCR using a nourseothricin resistance (natR) marker cassette. Cultures were grown overnight in XY medium (2% peptone, 1% yeast extract, 0.01% adenine, 0.02% tryptophan) containing 2% glucose, diluted ~300-fold into fresh medium and grown for at least 5 hours (which corresponded to three population doublings for wild-type) at 30°C to a final density of 0.3-3x107 cells/mL, a cell density range in which wild-type size distributions do not vary. To obtain each size distribution, 100Greek Letter MuL of culture was diluted into 10mL of IsotonII, sonicated gently for 10s to disperse aggregated cells, and analyzed with a Coulter Channelizer Z2 (Beckman-Coulter). Cell size distributions were saved in a tabular form, as a function of cell counts in each of 256 size bins.

Hierarchical clustering
To eliminate background particles, size bins smaller than 10 fL (for haploid deletion strains) or 30fL (for heterozygote deletion strains) and larger than 128 fL (for both haploid and heterozygote deletion strains) were eliminated. Each size distribution was normalized as a percentage of total counts and then smoothed by averaging over a seven bin sliding window. Size distributions were clustered (S1) and bins with a number % greater than 0.485 (an arbitrary threshold) were colored with the AFM4.0 program (S2) such that color intensity was proportional to number % value.

Definition of Lge and Whi phenotypes
Daughter cell size was defined as the cell size at which the number % is half maximal on the left hand side of the cell size distribution; this value was 23fL for wild-type cells. Peak median size was defined as the middle of the peak component of a cell size distribution; this value was 38fL for wild-type cells. Daughter cell size and peak median size were determined by visual inspection for each of the 4,812 haploid deletion strains. Median cell size (from 10-128fL) was also calculated for each haploid deletion strain; this value was 41fL for wild-type cells. The smallest 5% of strains in the haploid deletion set had a daughter cell size of less than 18fL and/or a peak median size of less than 32fL. Each of these 238 strains was tested for viability on XY 2% glycerol medium; failure to grow was presumed to reflect mitochondrial defects or appearance of spontaneous rho-mutation and so these strains were excluded from further analysis. In addition, all strains deleted for a ribosomal subunit were excluded, except for determination of the baseline cell growth-cell size correlation (Fig. 2B). The remaining 103 candidate whi strains were sized a further three times from an independent set of deletion strains (EUROSCARF) to yield a final set of 61 highly reproducible whi strains. The 61 reproducible whi strains and the 135 strains that either were deleted for a structural component of the ribosome or did not grow on 2% glycerol media were categorized by function (S3) and are listed in Table S1 (below).The largest 5% of viable haploid deletion strains had a peak median cell size of >45fL. The size distributions of 249 candidate lge strains were measured again from an independent set of deletion strains (EUROSCARF) to yield a final set of 202 reproducible lge strains. The 202 reproducible lge strains were categorized by function (S3) and listed in Table S1 (below). The median cell size (30-128fL) was calculated for each of the 1,142 strains heterozygous for the deletion of an essential gene. The 53 largest and smallest heterozygous essential diploid strains was determined by median cell size; all were >7% larger or smaller than wild-type. These size distributions of these106 strains were measured again to yield 26 whi and 27 lge reproducibly haploinsufficient strains; these strains were categorized by function (S3) and listed in Table S2 (below).

Synthetic Genetic Array (SGA) analysis
The 249 lge strains were arrayed into a 768 pin format and manipulated with a CPCA robot (Virtek/ESI, Toronto). The array was mated to SGA MATUppercase Greek Letter Delta starting strains can1Uppercase Greek Letter Delta:: natR (YCB3047), swi4Uppercase Greek Letter Delta::natR (MTY2365), cln3Uppercase Greek Letter Delta::LEU2 (MTY2342), or bck2Uppercase Greek Letter Delta:: natR (MTY2341). The can1Uppercase Greek Letter Delta:: natR strain served as a wild-type control. After selection for heterozygous diploids, sporulation, and selection for double mutant MATa meiotic progeny, colony growth was scored (S4). SGA analysis was carried out with each starting strain at least twice to reduce the rate of false positives. lge strains that consistently exhibited reduced colony size in can1Uppercase Greek Letter Delta:: natR screens and in 14 other wild-type control screens (A. Tong and C. Boone, unpublished data) were eliminated from consideration. SGA detected 23 candidate genetic interactions, 12 of which were confirmed by direct tetrad analysis on XY 2% glucose medium (Fig. 2A). In addition, epistasis analysis between whi mutants identified 6 additional synthetic genetic interactions (Fig. 2A, pink nodes). Of these 18 genetic interactions, 10 resulted in synthetic lethality where few or no double mutant spore colonies were isolated (swi4Uppercase Greek Letter Deltavs. cdh1Uppercase Greek Letter Delta, cla4Uppercase Greek Letter Delta, ypl055cUppercase Greek Letter Delta, bem1Uppercase Greek Letter Delta, bem2Uppercase Greek Letter Delta, bem4Uppercase Greek Letter Delta; cln3Uppercase Greek Letter Deltavs. bck2Uppercase Greek Letter Delta; sch9Uppercase Greek Letter Deltavs. sfp1Uppercase Greek Letter Delta, gpa2Uppercase Greek Letter Delta; cdh1Uppercase Greek Letter Deltavs. whi5Uppercase Greek Letter Delta) and 8 resulted in synthetic, i.e. greater than additive, growth defects (swi4Uppercase Greek Letter Deltavs. sec66Uppercase Greek Letter Delta, rps21bUppercase Greek Letter Delta, cdc73Uppercase Greek Letter Delta; cln3Uppercase Greek Letter Deltavs. sst2Uppercase Greek Letter Delta; bck2Uppercase Greek Letter Deltavs. sst2Uppercase Greek Letter Delta, swi4Uppercase Greek Letter Delta; sfp1Uppercase Greek Letter Delta vs. cdh1Uppercase Greek Letter Delta, swi4Uppercase Greek Letter Delta

Determination of critical cell size
For isolation of early G1 daughter cells, cultures were grown overnight in 1L of XY 2% raffinose at 30°C to a density of ~ 3 x 107 cells/mL, then fractionated with a Beckman JE-5.0 elutriator as described previously (S5). Early fractions containing predominantly small unbudded cells were resuspended in XY 2% glucose and incubated at 30°C. Samples were harvested at regular intervals to monitor bud index, DNA content, and the abundance of CLN2, RNR1, and ACT1 transcripts as cells progressed through G1 phase.

Flow cytometry and budding index
Cultures were grown as for cell size analysis (above) and ~ 1 x 107 cells were fixed in 70% ethanol for FACS analysis; 1 mL of culture was fixed in 3.7% formaldehyde for budding index determination. To determine % budded, at least 300 cells were counted for each sample. For FACS analysis, fixed cells were sonicated, incubated with RNaseA, digested with proteinase K, then stained with 1 Greek Letter MuM Sytox Green (Molecular Probes). Samples were sonicated just prior to analysis. Flow cytometry analysis was carried out with a Becton Dickinson FACSCalibur and CellQuest Pro software (BD Biosciences). %1N DNA content was calculated by dividing number of events under the 1N DNA peak by the total number of events under the 1N and 2N DNA peaks; cells in S-phase were excluded from this analysis. Budding index and %1N DNA content were performed twice, the results averaged, and standard deviation calculated.

Determination of alpha-factor resistance
Cultures were grown as for cell size analysis (above). ~500 exponentially growing cells from each strain were plated on wells containing XY + 2% glucose and increasing concentrations (0, 0.1, 0.25, 0.5, 1.0, 2.5, 5 and 10 Greek Letter MuM) of alpha-factor peptide. Cells were grown at 30°C for 3 days and alpha-factor resistance scored. The assay was repeated at least twice and strains reproducibly resistant or sensitive to alpha-factor were classified as such.

Epistasis tests
12 of the whi mutants (cdh1Uppercase Greek Letter Delta, gpa2Uppercase Greek Letter Delta, kap122Uppercase Greek Letter Delta, prs3Uppercase Greek Letter Delta, ptk2Uppercase Greek Letter Delta, rpa14Uppercase Greek Letter Delta, sch9Uppercase Greek Letter Delta, sfp1Uppercase Greek Letter Delta, sky1Uppercase Greek Letter Delta, ssf1Uppercase Greek Letter Delta, whi5Uppercase Greek Letter Delta, whi6Uppercase Greek Letter Delta) from Table 1 were subjected to epistatic analysis with deletions of three known Start regulators (cln3Uppercase Greek Letter Delta, bck2Uppercase Greek Letter Delta, swi4Uppercase Greek Letter Delta) and the newly identified regulator (cdh1Uppercase Greek Letter Delta) (Fig. 3A). The identity of the 12 MATakanR marked deletion strains was first confirmed by sequencing of unique deletion barcodes flanking each deletion cassette(S6, S7) and then mated to the following congenic MATGreek Letter Alpha strains: wild-type (BY4742) carrying a pRS315 CEN LEU2> plasmid, cln3Uppercase Greek Letter Delta::LEU2 (MTY2125), cdh1Uppercase Greek Letter Delta:: natR (MTY2293), bck2Uppercase Greek Letter Delta:: natR (MTY2296), and swi4Uppercase Greek Letter Delta:: natR (MTY2365). Two sets of tetratype progeny were sized. In rare cases where the size distributions of isogenic meiotic progeny did not perfectly overlap, at least two more tetratype sets were analyzed to reach a consensus. As epistasis was only scored if single and double mutant distributions completely overlapped, so more subtle relationships were not noted. Two complex genetic relationships, where the double mutant distributions were neither additive nor epistatic were observed (details in Fig. 3A legend).

DNA microarray analysis
A sfp1::kanR-GAL1-SFP1 (MTY2129) strain was constructed by insertion of a kanR marked cassette bearing a GAL1 promoter just upstream of SFP1. In glucose or raffinose, this strain had a growth rate and cell size distribution that was indistinguishable from an sfp1Uppercase Greek Letter Delta strain. For time course experiments, congenic wild-type (BY4741, S288c MATa) and sfp1::kanR-GAL1-SFP1 (MTY2129) were grown to early log phase (OD600 ~0.2) at 30°C in 2.5L of XY 2% raffinose media and t = 0 samples (800mL) harvested. Galactose was then added to 2% and samples (200mL) harvested 10, 20, 30, 40, 50, 60, and 90min later. All cells were harvested by rapid centrifugation at room temperature then flash frozen in liquid nitrogen. For experiments with asynchronous cultures, congenic wild-type (BY4741, S288c MATa) and sfp1Uppercase Greek Letter Delta::URA3 (MTY2148) strains were grown to log phase (OD600 ~0.3) in 300mL of XY 2% glucose and samples harvested as above. For each sample, total RNA was prepared by glass bead lysis in phenol and polyA RNA isolated on poly-dT agarose columns (5). Cy3 or Cy5 conjugated cDNA was prepared for each poly-A sample by indirect labeling (see www.microarrays.org/pdfs/amino-allyl-protocol.pdf). Differentially labeled experimental and control cDNA preparations were pooled, heated to 100°C for 2min, and hybridized overnight at 45°C in 4x SSC 0.34% SDS to an oligonucleotide microarray. Arrays were washed 3x 10min in 2xSSC 0.2%SDS at 37°C, rinsed 3x in 0.1xSSC, and spun dry prior to scanning with a GenePix 4000B (Axon Instruments). Spots were found and quantitated with Quantarray 1.0 software (GSI Lumonics). As each oligonucleotide probe was spotted twice on the microarrays, twin spots that gave more than 2-fold differences in expression ratio were eliminated. In addition, low intensity spots at or near background levels were eliminated. Expression ratios were normalized and median centered by Quantarray Data Handler 3.0 (S2) and analyzed with AFM 4.0 (S2). Oligonucleoltide microarrays were constructed by spotting the Yeast 70mer Genome Set version 1.0 (Qiagen/Operon) on poly-L-lysine coated slides with a SDDC-2 microarrayer (Virtek). The complete data set used to create Fig. 4A is available at http://www.mshri.on.ca/tyers/.

Groups of genes induced at t = 30 or 40 min after GAL1-SFP1 induction were categorized by function (S3). In similar experiments with the Met4 transcription factor (met4::GAL1-MET4), a similar lag of ~ 30 min was observed between galactose addition and activation of known Met4 target genes (Jorgensen et al., in preparation), suggesting that the genes induced at t = 30 or 40 min (see Fig. 4A) are direct targets of Sfp1.

For promoter analysis, the MEME/MAST system was employed. Sequences from the �500 to 0 region upstream of the 94 genes induced within 40 min in Fig. 4A were entered into the MEME program to search for common promoter elements within the 94 different promoters. The previously identified RRPE (GAAAA(A/T)TTT) and PAC (GCGATGAGNT) elements were significantly enriched (PAC E-value: 1.8 x 10-98, RRPE E-value: 1.4 x 10-61) and were each found in the �500 to 0 region of >75% of the 94 genes (S8, S9). In similar fashion, input of the �500 to 0 region upstream of 64 ribsomal protein (RP) genes into the MEME program allowed for the identification of the RAP1 binding site (GTA(T/C)GGG, E-value: 7.3 x 10-59) (S9). The MEME program generated a position-specific probability matrix for each of the three elements, based on input sequences. This matrix was then queried versus the �950 to +50 promoter regions of all ORFs (~6000) in the yeast genome with the MAST program in order to identify promoters with the most significant matches to the RRPE, PAC, and RAP1 position-specific probability matrices. With a P-value threshold of 100, 472/6000 (7.9%) promoters possess RRPE element, of which 60 are in the 116 genes (51.7%) induced early in the sfp1Uppercase Greek Letter DeltaGALSFP1 experiment and 11 are in the 136 RP genes (8.1%). These genes are colored light blue in Figure 4A. With a P-value threshold of 50, 330/6000 (5.5%) promoters possess PAC elements, of which 56 are in the 116 genes (48.3%) induced early in the sfp1Uppercase Greek Letter DeltaGALSFP1 experiment and 5 are in the 136 RP genes (3.6%). These genes are colored dark blue in Fig. 4A. With a P-value threshold of 100, 283/6000 (4.7%) promoters possess RAP1 elements, of which 3 are in the 116 genes (2.6%) induced early in the sfp1Uppercase Greek Letter DeltaGALSFP1 experiment and 87 are in the 136 RP genes (64.0%). These genes are colored purple in Fig. 4A.

Construction of a nucleolar network
The cell size/nucleolar network was constructed solely with published protein-protein interaction data from two recent high throughput mass spectrometric studies(S10, S11) as well as large scale purifications of proteins interacting with Nop7, Ssf1, and Nug1 (S12S14). Data from large-scale two hybrid screens was not considered. The nucleolar interaction network may contain up to 250 proteins, depending on how the network is defined; we assembled a sub-network containing the 13 cell size control gene products and 106 other highly connected proteins (Fig. 4B). This network was constructed by first connecting the 13 cell size control gene products (Rpa14 , Rpa49, Ssf1, Rpa190, Nop1, Nop2, Nop5, Nog1, Noc1/Mak21, Tif6/Cdc95, Rlp24, Noc2, Rvb2) with interactions, either direct or bridged via an intermediary partner. Rpa14 was not connected to any proteins in the large-scale studies, but is a known constituent of highly purified RNA Polymerase I (S15). Two cell size control gene products implicated in ribosome biogenesis, Tom1 and Sky1 (S3), did not appear in any of the interaction references used and were not included in the network; however, Tom1 interacts by two-hybrid with Krr1, which is a member of the nucleolar network(S16). 59 proteins that interacted most highly with the initial set of nodes were identified. These 72 proteins were then queried versus all of the interactions from the above listed references to determine the proteins which interacted most highly with this core. This process added 47 additional proteins. But for five exceptions, all gene products in Fig.4B interact with at least 4 other network members, with an average of 12 interactions per protein. The five gene products that did not interact with at least 4 other network members in Fig.4B are cell size control gene products (Rpa14, Rpa49, Rvb2, Rlp24) and a protein (Rvb1) required to connect Rvb2, itself a cell size control gene product, to the rest of the network. For construction of the network, no consideration was given to whether or not genes were transcriptional targets of Sfp1. However, 48 of the network components are induced early (t = 30 or 40 min) in the sfp1Uppercase Greek Letter Delta::GALSFP1 dataset (Fig. 4A, S3). 7 more genes (EST1, LOC1, YHR197W, YDR449C, BMS1, YKL041C, YDR324C, NSA1) that were not induced strongly or consistently enough to be presented in Fig. 4A are also clearly induced early in this data set and in replicate experiments (data not shown). Note that all of the genes in the nucleolar network of Fig. 2B and 4A are not present in Fig. 4B. Rather, the �nucleolar network� proteins of Fig. 2B and 4A are so categorized because they fell into an expanded nucleolar network of ~250 members in which only 2 interactions with nucleolar proteins was required for membership (data not shown)


Supplemental Figure 1. Clustering of cell size distributions. (A) Size distributions can predict gene function. Rad50, Mre11, and Xrs2 form the MRX DNA repair complex, Rlr1 and Hpr1 are part of the THO complex involved in transcription-associated recombination, and Pdr13 and Zuo1 form the RAC chaperone complex (S3). Complex components cluster closely together on the dendrogram. (B) Hierarchical clustering of cell size distributions for 1,142 diploid strains heterozygous for deletion of an essential gene reveals clusters of haploinsufficient whi and lge strains.


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Supplemental Figure 2. Cellular processes that influence size homeostasis. (A) The 202 largest and 196 smallest haploid deletion strains compiled according to known cellular role. Possible diploid contaminants in the haploid set were identified by the complete absence of 1N DNA content. (B) The 27 largest and 26 smallest heterozygous diploid deletion strains compiled according to known cellular role.


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Supplemental Figure 3. Magnified view of functional groups from Fig.4A.


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Supplemental Table 1. Daughter and median cell size for the smallest and largest 5% of haploid deletion strains. Data is from the primary screen; size bins from 10-128 fL were used to calculate median cell size. Genes are arranged according to functional category (in bold). A question mark indicates that daughter cell size could not be determined. (A) Smallest 5% of haploid deletion strains. (B) Largest 5% of haploid deletion strains.

Supplementary Table 1A

Gene nameORFDaughter cell size (fL)Median cell size (fL)
wild typeN/A2341
Ribosome biogenesis
RSA1YPL193W1534
LOC1YFR001W1535
RRP8YDR083W1835
SSF1YHR066W1936
RPA49YNL248C1936
DBP3YNL228W1836
RPA14YDR156W1937
Ribosomal subunits
RPL1BYGL135W1332
YDR115W1532
RPS1B YML063W?32
RPL17AYGL076C1433
RPL31AYDL075W1434
RPL35AYLR185W1634
RPL13A YDL082W1734
RPL37BYDR500C1734
RPL4BYLL045C1835
RPL16A YIL133C2335
RPP2BYDR382W1635
RPL17AYBL087C1735
RPL35BYDL136W2435
RPS16BYDL083C1736
RPL19A YBR084C-A1836
RPS6BYBR181C1836
RPL15BYEL054C1836
DBP3YGL078C1836
RPL21BYPL079W1936
RPL18AYMR242C1737
RPL37BYDR500C1737
RPS29BYDL061C1837
RPS18A YDR450W1837
RPS33BYLR264W1837
RPS8AYBL072C1837
RML2YEL050C1838
RPS21AYKR057W1838
RPS0AYGR214W1839
RPS19BYNL302C?39
RPL2BYIL018W1640
RPS0BYLR048W1840
RPS10AYOR293W1841
Translation
SSZ1YHR064C1534
MSM1YGR171C1435
ZUO1YGR285C1635
YER087W1635
DIA4YHR011W1437
MAP1YLR244C1837
Known cell cycle regulators
RCS1YGL071W1437
SWE1YJL187C1837
CDH1YGL003C1640
WHI3YNL197C1745
Glucose signaling
HXK2YGL253W1834
TPS1YBR126C2135
GPA2YER020W2136
Known mitochondrial function
MRPL9YGR220C?26
MRPL38YKL170W?27
CYT1YOR065W?28
MRPL31YKL138C1029
MRP7YNL005C1029
MRPL31YKL138C1829
MRF1YGL143C1830
RIM1YCR028C-A1232
ATP14YLR295C1232
MSK1YNL073W1332
MRPS5YBR251W?32
OXA1YER154W?32
ISM1YPL040C1433
MSE1YOL033W1633
PIF1 YML061C1633
SUV3YPL029W1334
MRPL13YKR006C1434
TOM37YMR060C1534
MRPL23YOR150W1834
MRP1YDR347W1335
MRPL10YNL284C1535
SCO1 YBR037C1535
IFM1YOL023W1435
ATP12YJL180C1435
ATP17YDR377W1435
MSD1YPL104W1535
ATP11YNL315C1535
TUF1YOR187W1535
MGM101YJR144W1535
MRPS28YDR337W1635
ATP4YPL078C1735
MDM12YOL009C1136
MRPL8YJL063C1436
MRP20YDR405W1636
COX10YPL172C1736
ATP13YMR282C1637
MRPS5YBR251W1737
MEF1YLR069C1737
MRPL15YLR312W-A2137
MRPL6YHR147C1337
MDM10YAL010C1637
NAM2YLR382C1737
PPT2 YPL148C1737
POR1YNL055C1837
MRPL3YMR024W1837
PET111YMR257C1837
MSF1YPR047W1538
CBP1YJL209W1738
ATP11YNL315C1838
COX7YMR256C1838
ATP1YBL099W1439
MSR1YHR091C1439
MIP1YOR330C1839
QCR10YHR001W-A1740
MRPL37YBR268W?40
FZO1YBR179C1640
MMM1YLL006W1444
Did not grow on XY 2% glycerol but
no known mitochondrial function
KNH1YDL049C721
AGP2 YBR132C1229
YMR097C?30
YDR065W1331
YDR470C732
YDL062W1332
YML088W1132
QRI5YLR204W1334
YDL063C1434
YGR102C1534
YGR219W?34
MRPL17YNL252C1035
YOR200W1435
YGL107C1535
EUG1 YDR518W1835
HAP4YKL109W1835
YGR150C1135
MEC3YLR288C1235
SSA4YER103W1335
YJR113C1535
MAC1YMR021C1535
YGL129C1735
YLR114C1835
YGL220W1636
YPR099C1636
GLO3YER122C1636
YNL213C1736
YKL169C1936
YPL005W1337
YGL218W1537
YDL057W1837
YLR260W1837
YGL218W1537
YCP4YCR004C1837
YJL046W2037
DHS1YOR033C1739
YGL246C1839
PEP3YLR148W1839
YMR158W1440
PHO85YPL031C1740
RDI1YDL135C1840
ISA2YPR067W1840
YPR100W1841
MNN9YPL050C1546
YGR165W1738
Other function
SCH9YHR205W927
PRS3 YHL011C1832
PHO5 YBR093C1834
SKY1YMR216C2134
YVH1YIR026C1635
MRT4YKL009W1536
LUV1YDR027C1736
KEL1YHR158C2136
SNF12YNR023W1743
Unknown function
YLR074C1624
SFP1YLR403W925
UAF30YOR295W1632
YIL110W1832
YOR083W1932
YCR062W2033
YLR184W1634
FYV9YDR140W1635
YBR266C1635
YOR309C1835
YHR034C1835
YDR101C1835
YML122C1735
YDR417C1835
YNL227C1835
YLR402W1835
PTK2YJR059W1835
LTV1YKL143W1736
YOR309C1836
PDR6YGL016W1836
YER014C-A1637
FYV13YGR160W1637
FYV7YLR068W1737
YNL226W1837
YPL080C2037
YOR078W1838
YKR020W1838
YMR269W1838
YJL188C1838
YGR111W2138
YGR064W2138
FYV5YCL058C1847
YLL044W1739

Supplementary Table 1B

Gene nameORFDaughter cell size (fL)Median cell size (fL)
wild typeN/A2341
Translation and ribosome biogenesis
EAP1 YKL204W4168
RS12YOR369C4466
NCL1YBL024W3763
ASC1YMR116C?53
RPS21B YJL136C3151
DBP7YKR024C2949
RPL15BYMR121C2849
RPL43A YPR043W2649
RPS27BYHR021C2349
RPS4A YJR145C2845
FUN12YAL035W2645
RNA Pol II transcription complexes
ROX3YBL093C7099
SPT10YJL127C3666
RSC1YGR056W3764
CCR4YAL021C4060
SRB5YGR104C3958
SPT7YBR081C?58
ANC1YPL129W?56
POP2 YNR052C2956
CTK3YML112W3855
SNF5YBR289W3454
CDC73YLR418C9153
YLR358C2353
HFI1YPL254W?53
RPB9YBL058W2952
SPT4YGR063C2352
SSN3YPL042C?51
GAL11YOL051W2450
SIN3YOL004W3050
MOT2YER068W?47
RSC2YLR357W?45
RPB4YJL140W?37
Cell cycle (including DNA metabolism and microtubule dynamics)
RNR1YER070W5284
TRF4YOL115W4776
FCL1YOR080W?73
HOF1YMR032W?73
POB1YPR135W?71
CIN8YEL061C3970
RNR4YGR180C3768
SWI4YER111C4466
SPC72YAL047C?65
CLB2YPR119W3862
CIK1YMR198W3460
CLN3YAL040C?59
ACE2YLR131C?59
ALF1YNL148C3259
SWI6YLR182W3058
BCK2YER167W2857
KAR3YPR141C?55
RTS1YOR014W3555
PAC10YGR078C3455
DCC1YCL016C3055
EST2YLR318W?53
BUB3YOR026W2453
DBF2 YGR092W3352
HSL1YKL101W3052
RAD50YNL250W2652
RAD5YLR032W2452
HDF1YMR284W2951
YLR181C2751
BRE1YDL074C2950
CDC50YCR094W3049
CDC26YFR036W2749
EST1YLR233C2346
GIM5YML094W2545
DPB3YBR278W3842
CDC40YDR364C?40
TPD3YAL016W?33
Actin cytoskeleton
ARP5YNL059C?68
BEM1YBR200W3165
YCL061C4163
RHO4YKR055W2957
RVS161YCR009C3155
YOL076W?54
SAC3YDR159W2854
BEM4YPL161C2653
CLA4YNL298W2850
FIG4YNL325C2749
VRP1YLR337C?48
CAP2YIL034C2647
BEM2 YER155C?45
Vesicular trafficking (including ER, Vacoule and Golgi function)
VID31YKL054C?89
AKR1YDR264C?76
MET7YOR241W?74
CYC8 YBR112C?71
REF2YDR195W4470
PRP18YGR006W3469
FAA3YIL009W4668
KEM1YGL173C4168
SYG1YIL047C4165
APG17YLR423C4065
ADK1YDR226W3465
MOG1 YJR074W3564
BUR2YLR226W3164
IMP2' YIL154C3963
ANP1YEL036C2860
YDJ1YNL064C4059
MNN10YDR245W3156
KEX2YNL238W2956
UBP3 YER151C2656
SST2YLR452C2455
RCY1YJL204C3255
PHM8YER037W3155
URA7YBL039C3055
FAB1 YFR019W3054
URE2YNL229C3153
SEC66 YBR171W2453
YBR022W2353
PMP1YCR024C-A3352
RSG1YCR027C3352
SHP1YBL058W3052
HOC1YJR075W2552
HCM1YCR065W3150
PLC1YPL268W3150
IRA2YOL081W2650
VPS34YLR240W?50
MRPL36 YBR122C3150
BUL1YMR275C2950
LSM6YDR378C2750
CWH8YGR036C2350
ECM33YBR078W2350
PPH3YDR075W2948
SEC22YLR268W2848
DEG1YFL001W2748
SHE4YOR035C?46
PDE2YOR360C3142
Unknown function
BUD32YGR262C?82
YBL094C?77
YIL121W4163
MRC1YCL060C4163
YJR054W2763
VID21YDR359C2462
KRE28YDR532C3159
YML014W2659
KRE22YDR433W3358
YLR386W3058
YIL054W2558
YLR320W?58
BUD31YCR063W2956
YPR045C3555
YHR009C3455
YPL055C3155
YIL011W2755
YIL040W3354
YCR025C3354
SHD7YPL180W3154
YKL053W3154
YDL032W2454
KIM2YLL002W2653
YCR087C-A3152
YCR026C3351
YOR073W2351
YCR061W3150
YCR087W3150
YCR045C3150
SLX8YER116C2850
YCR050C3150
YLR422W2950
GIS2YNL255C2850
GIS2YNL255C2849
YNL324W2749
KRE21YLR338W?48
YKL023W3148
YPR044C2947
YGL214W2542
HIT1 YJR055W4640
YBR277C3840
YBL051C28?
0% 1N DNA content
SCP160YJL080C?94
CLC1YGR167W4879
YJR018W5378
CTK2 YJL006C5177
YMR014W5676
PAF1YBR279W5376
TIF3YPR163C?75
YCL005W4475
SLA2YNL243W5674
AAC3 YBR085W5073
VPS53YJL029C4773
HOP2YGL033W4173
YBL006C4572
SRV2YNL138W4871
HNT3YOR258W4071
CDC10YCR002C4170
BDF1YLR399C?69
KGD1YIL125W4669
YPR087W4469
YLR322W?68
RLR1YNL139C4168
NPL6YMR091C?67
PRO1YDR300C3167
KRE6YPR159W?66
HPR1YDR138W4466
YEL044W3064
YPR139C4163
HTL1YCR020W-B?63
BFR1YOR198C3662
GPM2YDL021W2559
ARP8YOR141C1259
YNL171C?58
ADH1YOL086C2656
YKE2YLR200W?54


Supplemental Table 2. Median cell size for the smallest and largest 5% of heterozygous deletion strains. Deletion strains were sized twice to ensure that small or large cell size was reproducible. Data is from the primary screen; size bins from 30 � 120 fL were used to calculate median cell size. Genes are arranged according to functional category (in bold). (A) Haploinsufficient whi mutants. (B) Haploinsufficient lge mutants.

Supplementary Table 2A

Gene nameORFMedian cell size (fL)
wild type N/A70
Ribosome biogenesis
RPA190YOR341W64
NOP1YDL014W65
NOG1YPL093W65
MAK21YDR060W65
NOP2YNL061W65
NOP5YOR310C66
TIF6YPR016C66
RLP24YLR009W66
NOC2YOR206W67
RVB2YPL235W67
Ribosomal subunits
RPL18AYOL120C62
RPS15YOL040C63
RPL5YPL131W65
RPS13YDR064W65
RPL17AYKL180W65
RPL30YGL030W66
RPL15AYLR029C67
Other function
CDC24YAL041W61
MRS5YBR091C63
RPB5YBR154C67
MEX67YPL169C65
Unknown function
YBR190WYBR190W65
YDR531WYDR531W65
YGL047WYGL047W66
YPL142CYPL142C67

Supplementary Table 2B

Gene nameORFMedian cell size (fL)
wild typeN/A70
26S proteasome subunits
RPT2YDL007W103
RPT4YOR259C79
RPT5YOR117W83
RPN7YPR108W79
RPN12YFR052W80
RPN11YFR004W79
Ribosome biogenesis
RPC10YHR143W-A81
NOP8YOL144W79
RRP43YCR035C79
Greek Letter Gamma-tubulin
SPC98YNL126W84
TUB4YLR212C81
Glycolysis
PGI1YBR196C84
PDC2YDR081C81
RNA Pol II transcription
RPB7YDR404C81
RSC6YCR052W81
Other function
GSP1YLR293C91
MCD4YKL165C89
PKC1YBL105C83
CDC47YBR202W83
BET1YIL004C82
SRM1YGL097W82
ACT1YFL039C81
RSP5YER125W80
Unknown function
YDR526CYDR526C86
YGR115CYGR115C85
YGR265WYGR265W83
YJL086CYJL086C83
YJL091CYJL091C79


References and Notes

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S2. B. J. Breitkreutz, P. Jorgensen, A. Breitkreutz, M. Tyers, Genome Biol.2 (2001).

S3. Gene functions can be found at the Saccharomyces Genome Database, http://genome-www.stanford.edu/Saccharomyces/.

S4. A. H. Tong et al., Science294, 2364 (2001).

S5. M. Tyers, G. Tokiwa, B. Futcher, EMBO J.12, 1955 (1993).

S6. D. D. Shoemaker, D. A. Lashkari, D. Morris, M. Mittmann, R. W. Davis, Nat. Genet.14, 450 (1996).

S7. E. A. Winzeler et al., Science285, 901 (1999).

S8. C. Wade, K. A. Shea, R. V. Jensen, M. A. McAlear, Mol. Cell. Biol.21, 8638 (2001).

S9. J. D. Hughes, P. W. Estep, S. Tavazoie, G. M. Church, J. Mol. Biol.296, 1205 (2000).

S10. Y. Ho et al., Nature415, 180 (2002).

S11. A. C. Gavin et al., Nature415, 141 (2002).

S12. P. Harnpicharnchai et al., Mol. Cell8, 505 (2001).

S13. A. Fatica, A. D. Cronshaw, M. Dlakic, D. Tollervey, Mol. Cell9, 341 (2002).

S14. J. Bassler et al., Mol. Cell8, 517 (2001).

S15. A. Smid, M. Riva, F. Bouet, A. Sentenac, C. Carles, J. Biol. Chem. 270, 13534 (1995).

S16. T. Sasaki, E. A. Toh, Y. Kikuchi, Mol. Cell. Biol.20, 7971 (2000).