Supplementary Material

advertisement
Supplementary Material
Supplementary Tables
Table S1 – Media Used in this Study
Table S2 – Expression Data
Table S3 – Quiescent specific and Growth rate dependent genes
Table S4 – Metabolite Data
Table S5 – List of Mutants Defective in Surviving Starvation
Supplementary Figures
Figure S1. Method for analyzing gene expression values.
Figure S2. Overlap of genes exhibiting quiescent specific expression with those affected
by acute heat shock.
Figure S3. Singular value decomposition of starvation expression data.
Figure S4 Discordance in trehalose intermediates and gene expression levels during
starvation.
Table S1 – Media Used in this Study
Batch culture media (per 1L)
Glucose starvation batch media
Compound
Yeast Nitrogen Base w/o
amino acids
Glucose
Synthetic Defined
6.7 g
Glucose starvation
6.7 g
20 g
-
Nitrogen starvation batch media
Compound
Yeast Nitrogen Base w/o
amino acids and ammonium
sulfate
Glucose
Na2SO4
(NH4)2SO4
Nitrogen limited
1.7 g
Nitrogen starvation
1.7 g
20 g
5g
625 mg
20 g
5g
-
Phosphate starvation batch media
Compound
Glucose
1000X vitamins
1000X metals
10X salts
KH2PO4
Phosphate limited
20 g
1 ml
1 ml
100 ml
20 mg
10X Salts for phosphate starvation batch media
10X salts (per 1L)
Ammonium Sulfate
Potassium Chloride
Magnesium Sulfate
Sodium Chloride
Calcium Chloride
50 g
10 g
5g
1g
1g
Phosphate starvation
20 g
1 ml
1 ml
100 ml
-
Chemostat media (per 1L)
Compound
Yeast Nitrogen Base w/o
amino acids and ammonium
sulfate
(NH4)2SO4
Glucose
Glucose limitation
1.7 g
Nitrogen limitation
1.7 g
5g
8g
0.053 g
20 g
Phosphate limitation
Compound
KH2PO4
CaCl2.2H2O
MgSO4.7H2O
KCl
(NH4)2SO4
NaCl
Glucose
1000X vitamins
1000X metals
10 mg
0.1 g
0.5 g
1g
5g
0.1 g
5g
1 ml
1 ml
1000X vitamins and metals (Lu et al. 2009)
1000X vitamins (per
1L)
Biotin
Calcium pantothenate
Folic acid
Myo-inositol
Nicotinic acid
1000X Metals (per 1L)
1 mg
400 mg
2 mg
2000 mg
400 mg
p-aminobenzoic acid
200 mg
Pyridoxine HCl
Riboflavin
Thiamine HCl
400 mg
200 mg
400 mg
Boric acid
Copper Sulfate.5H2O
Potassium Iodide
Ferric Chloride.6H2O
Manganese
Sulfate.H2O
Sodium Molybdate.2
H2O
Zinc Sulfate.7H2O
500 mg
40 mg
100 mg
200 mg
400 mg
200 mg
400 mg
Table S2 – Expression Data (Downloadable Excel Spreadsheet)
The prototrophic W303 derivative, Y3358, and the CEN.PK prototroph, Y3840, were
grown in liquid SD (prior transfer to glucose starvation), nitrogen limited (prior transfer to
nitrogen starvation) or phosphate limited media (prior transfer to phosphate starvation media) to
an OD600 of 0.1 and then filtered onto nylon filters. Filter membranes were incubated on agarose
plates with respective limited media for 2.5 h and then transferred to starvation plates; afterwards
a series of time points was collected. For each of the starvation conditions (glucose, nitrogen or
phosphate) time point 0, collected immediately prior to transfer of remaining filters to starvation
media, was used as reference sample to which the following time points in respective starvation
conditions were compared. For the W303 strain eight time points, besides time point 0 used as a
reference, ranging from 30 minutes (30 min) to 8 days (8 d) were collected for glucose (W303
G), nitrogen (W303 N) and phosphate starvation (W303 P) each. For the CEN.PK strain three
time points were collected for glucose (CEN.PK G), nitrogen (CEN.PK N) and phosphate
starvation (CEN.PK P) ranging from 2 days after transfer to starvation media (2 d) to 4 days after
transfer (4 d). Comparison between the reference samples used: time points 0 in SD (W303 G 0
min), nitrogen limited (W303 N 0 min) and phosphate limited media (W303 P 0 min), with
glucose time point 0 (W303 G 0 min) used as reference, was also conducted. A comparison
between the CEN.PK strain grown in a chemostat in glucose limited medium at dilution rate of
0.25/h (CEN.PK 0.25/h G limited) with CEN.PK grown in SD (CEN.PK G 0 min), nitrogen
limited (CEN.PK N 0 min) and phosphate limited media (CEN.PK P 0 min) at time point 0 was
also conducted, to make possible a comparison between our CEN.PK data and data generated by
Brauer et al. 2008.
Table S3 – Quiescent specific and Growth rate dependent genes (Downloadable Excel
Spreadsheet)
Gene lists with effect size and FDR corrected p-values for testing significant growth-rate or
quiescent specific components. The lists are divided into two sheets: the overall model tests
across all the conditions, and the nutrient specific model tests on a per nutrient basis. Data are
provided for both CEN.PK and W303 strains. The “Saturated?” column denotes whether or not
the probe intensity on the array has been saturated. Lastly, the group column identifies whether
there is “Both” a significant growth-rate and quiescent specific component, “Gr-Dn” or “Gr-Up”
a significant growth-rate component, “Q-Dn” or “Q-Up” a significant quiescent-specific
component, or “None” no significant component identified. The nutrient specific model bins the
results into “CQ-Up/CQ-Dn”, “NQ-Up/NQ-Dn”, and “PQ-Up/PQ-Dn” for quiescent-specific
effects in glucose, nitrogen, and phosphate starvations, respectively.
Table S4 – Metabolite Data (Downloadable Excel Spreadsheet)
This table lists log-2 transformed metabolite concentrations relative to yeasts growing
exponentially in SDMin. The first three rows denote the nutrient perturbation, whether the
condition was nutrient starvation or nutrient limitation, and the stringency of the perturbation,
where starvation units are time in minutes and limitation units are in dilution rate (mL/mL*h).
Values reported for starvation are mean averages of biological replicates (n=3) normalized for
cell count and size. Values for limitation were calculated as in Boer (2010) and re-normalized by
subtracting the ratio of concentrations of an exponentially growing culture and the phosphate
limited reference (D=0.05 h-1).
Table S5 – List of Mutants Defective in Surviving Starvation (Downloadable Excel Spreadsheet)
Sheet 1: Genes by Screen. Genes whose deletion resulted in loss of viability following
starvation of cells for the indicated nutrient are listed and subdivided into those that were
identified in the plate based screen (“plate screen only”) or that showed at least a 10 fold drop in
relative abundance in the liquid based screen (“liquid only”) or that were identified in both
screens (“both”).
Sheet 2: Gene overlap. The genes listed in Sheet 1 are organized by those that appeared in only
one screen (“G only”, “N only” or “P only”), or only in two of the three screens (“G+N only”,
“G+P only” or “N+P only”) or in all three (“G+N+P”).
Figure S1. Method for analyzing gene expression values.
Data for ALD4 expression as a function of growth rate and starvation are used to illustrate the
components of the method used to determine the extent to which a gene exhibits growth rate
specific or quiescence specific gene expression. The figure defines the growth-rate slope,
nutrient-specific effects for carbon, nitrogen, and phosphorus, and a quiescence specific effect
that are applied in the equation shown in Materials and Methods.
Figure S2. Overlap of genes exhibiting quiescent specific expression with those affected by
acute heat shock.
Histogram showing the fraction of genes from a total of 5448 that exhibit a given change in
mRNA levels (log2) 15 min following transfer from 23° to 37° (blue line), taken from Gasch et
al. (2000). Green line: histogram of the fraction of genes identified as repressed in a quiescence
specific manner in this current study (218 total) that exhibit a given change in the Gasch et al
acute heat shock experiment. Red line: histogram of the fraction of genes identified as induced
in a quiescence specific manner in this current study (93 total) that exhibit a given change in the
Gasch et al acute heat shock experiment.
Figure S3. Singular value decomposition of starvation expression data.
Transcription data displayed in Figure 2 was subjected to singular value decomposition and the
major eigengenes as well as the extent to which each eigengene contributes to the variation in the
data are shown. Singular value decomposition (SVD) is a matrix decomposition that is
commonly used as an unsupervised technique for finding the major patterns in a data set. SVD
takes a matrix X as input and returns i) a matrix V of linearly independent vectors of length 1,
here referred to as “eigengenes,” that capture the major signals in the data; ii) a diagonal matrix
of constants or “singular values” S that scale the magnitudes of these eigengenes; and iii) a
matrix U that gives each original row (in this case, the expression data for each gene) in terms of
its contributions from S and V , such that X = USV T . SVD is related to principal-components
analysis or PCA (Wall et al. 2003). A more thorough treatment can be found in Alter, 2000.
Percent signal explained by each eigengene was calculated by taking 100·si / ∑nj=1 sj, where n is
the number of eigengenes (equal to the number of experimental conditions) and si is the singular
value for eigengene i (equivalent to Si,i above). SVD was performed in the R software
environment (R_Development_Core_Team 2009).
Figure S4 Discordance in trehalose intermediates and gene expression levels during
starvation.
On a diagram of the trehalose synthesis and degradation pathways, metabolic intermediates
(ovals) and the genes encoding enzymes (rounded rectangles) catalyzing interconversion of those
intermediates are indicated the change in levels of the metabolites and mRNAs at four days
starvation for glucose (upper panel), nitrogen (middle panel) or phosphate (lower panel) relative
to those in cells growing exponentially on SDmin.
Supplementary References
Boer VM, Crutchfield CA, Bradley PH, Botstein D, Rabinowitz JD. 2010. Growth-limiting
intracellular metabolites in yeast growing under diverse nutrient limitations. Molecular
biology of the cell 21: 198-211.
R_Development_Core_Team. 2009. R: a language and environment for statistical computing. R
Foundation for Statistical Computing, Vienna, Austria.
Wall ME, Rechtsteiner A, Rocha LM. 2003. Singular value decomposition and principal
component analysis. in A practical approach to microarry data analysis (eds. DP Berrar,
W Dubitzky, M Granzow), pp. 91-109. Kluwer, Norwell, MA.
Related documents
Download