Network representation of the “Low G1” group.

<p>The interactions shown are from the gold-standard reference database BioGRID <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#pgen.1002590-Stark1" target="_blank">[54]</a>. The network was constructed with Cytoscape <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#pgen.1002590-Smoot1" target="_blank">[83]</a>, and displayed using an unbiased, force-generated layout. Only the factors that showed interactions (physical or functional) are included. We also included the essential gene <i>CDC28</i> (shown in red), encoding the major yeast Cdk.</p

Cys4p advances START both by promoting cell growth and by a separate function, which does not require CBS's catalytic activity.

<p>A, Rate of cell size increase (shown as growth rate, in fl/min) for the indicated strains was measured assuming linear growth from synchronous elutriated cultures in media that contain galactose and induce expression of the <i>P_GAL</i> alleles (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#s4" target="_blank">Methods</a>). The average value for each strain is shown with a horizontal bar (± sd). Where indicated, the <i>P</i> values shown were calculated from two-tailed <i>t</i> tests. The data used to calculate the values shown in A and B are in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#pgen.1002590.s009" target="_blank">Figure S8</a>. B, The critical cell size of the indicated strains (shown in fl), was measured from the same elutriation experiments shown in A (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#pgen.1002590.s010" target="_blank">Figure S9</a>). The analogous experiments in non-inducing, glucose containing, medium are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#pgen.1002590.s010" target="_blank">Figure S9</a>.</p

Decreased fitness correlates with altered cell cycle progression.

<p>The y-axis shows the fitness values of Giaever et al <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#pgen.1002590-Giaever1" target="_blank">[33]</a>. Higher values indicate reduced fitness. The cutoff for reduced fitness was about <85% of the wild type in that study <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#pgen.1002590-Giaever1" target="_blank">[33]</a>. Thus, strains with possible small reductions in fitness have been assigned a “WT-like” fitness score of 1. Giaever et al <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#pgen.1002590-Giaever1" target="_blank">[33]</a> evaluated fitness of the same strains we used, during growth in rich (YPD-2%Dextrose) liquid media, allowing for a direct comparison with our dataset. We used the non-parametric Spearman test to obtain the correlation (<i>r</i>) values we show. The correlation coefficient for all the strains (<i>r</i>_T) is shown at the bottom right of the graph. We colored the r values for the sub-groups as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#pgen-1002590-g002" target="_blank">Figure 2</a>. For every gene we included in this analysis, the values we used in this correlation are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#pgen.1002590.s001" target="_blank">Dataset S1</a>.</p

Schematic overview of our approach.

<p>For a detailed description of all the protocols we used, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#s4" target="_blank">Methods</a>.</p

Cys4p has a vital, non-catalytic role in cell proliferation.

<p>A, Immunoblots showing the levels of Cys4p in the indicated strains, detected with an antibody against human CBS. We probed the same blot with an antibody against yeast Cdc28p, to indicate loading. B, Growth of the same strains on rich (YPD) and synthetic minimal media (SMM). We added cysteine (at 2.5 mM), to the SMM plate at the bottom. All strains were spotted on plates at 5-fold serial dilutions from liquid cultures, starting at ∼5,000 cells.</p

Phenotypes of ribosomal proteins.

<p>We grouped strains (n = 53) that lack ribosomal proteins of the 60S subunit (RPL), against strains (n = 43) that lack ribosomal proteins of the 40S subunit (RPS). We then compared the two groups based on the %G1 DNA content (this study; A), fitness (data from Giaever et al <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#pgen.1002590-Giaever1" target="_blank">[33]</a>; B), or haploid median cell size (data from Jorgensen et al <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#pgen.1002590-Jorgensen1" target="_blank">[23]</a>; C). The box plots were generated with Microsoft Excel. The box represents the middle 50% of the data range (from the 25th percentile to the 75th percentile). The band within the box is the median, while the cross shows the mean. The ends of the whiskers represent the lowest datum still within 1.5 of the interquartile range (IQR) of the lower quartile, and the highest datum still within 1.5 IQR of the upper quartile. Any data points not included within the whiskers are shown as outliers, displayed as filled circles. For the fitness data in B, the lower quartiles are not visible, because they are equal to 1 (i.e., most strains have fitness values similar to WT). The <i>P</i> values were calculated from <i>t</i> tests.</p

DNA content screen identifies genes required for normal cell cycle progression.

<p>Cumulative histogram displaying the percentage of cells in the G1 phase of the cell cycle (%G1), for homozygous diploid deletion strains. The bin width of the histogram is 1%, with each bin containing all the strains with values within the bin boundaries. The black line superimposed to this histogram is the normal distribution fit of the %G1 values of the reference wild type strain. Bins with values >2 sd from the mean of the wild type distribution are in red (“Low G1” group) and green (“High G1” group).</p

Interactions among the factors of the “High G1” group.

<p>The network of interactions was constructed and displayed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#pgen-1002590-g007" target="_blank">Figure 7</a>. We also included factors with known roles at START (shown in red), which were not identified in our study. Among the G1 cyclins, we only included Cln3p, which is responsible for initiating the positive feedback loop of the large G1/S transcriptional program <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#pgen.1002590-Skotheim1" target="_blank">[10]</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#pgen.1002590-Doncic1" target="_blank">[12]</a>. The other G1 cyclins, Cln1p and Cln2p, are important for this feedback, once it is initiated by Cln3p, but they were not included in this network. 60S ribosomal proteins are in yellow, while 40S ribosomal proteins are in orange. The most highly connected factors among the ones we identified are in green, and Cys4p is in blue.</p

Cell size correlates poorly with DNA content.

<p>We plotted the %G1 (x-axis) from all the deletion strains we examined against the haploid median cell size (in fl, y-axis) data of Jorgensen et al <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#pgen.1002590-Jorgensen1" target="_blank">[23]</a>. The dashed lines indicate the cutoffs used in that study. We calculated and displayed the <i>r</i> values as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#pgen-1002590-g003" target="_blank">Figure 3</a>. For every gene we included in this analysis, the values we used in this correlation are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#pgen.1002590.s001" target="_blank">Dataset S1</a>.</p

Deletion of genes involved in ribosome biogenesis delay START.

<p>A, Rate of cell size increase (shown as growth rate, in fl/min) for the indicated strains was measured from synchronous elutriated cultures, in YPD-2% Dextrose medium. The average value for each strain was calculated assuming linear growth and is shown with a horizontal bar (± sd). Where indicated, the <i>P</i> values shown were calculated from two-tailed <i>t</i> tests. The data used to calculate these values are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#pgen.1002590.s008" target="_blank">Figure S7A</a>. B, The specific rate of cell size increase constant <i>k</i> (in h⁻¹) was measured from the same elutriation experiments shown in A, assuming exponential growth. The data used to calculate these values are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#pgen.1002590.s008" target="_blank">Figure S7B</a>. C, The critical cell size of the indicated strains (shown in fl), was measured from the same elutriation experiments shown in A and B (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#pgen.1002590.s008" target="_blank">Figure S7C</a>).</p