Relationships between copy numbers and fold increase in protein level.

<p>ND: Not done,</p>*<p>The number shown is the plasmid copy number determined plus 1 (genomic copy).</p

Relationships between native and TAP-tagged gene copy number limit.

<p>Genes whose copy numbers varied between native and TAP-tagged are shown. Circles indicate those genes whose copy numbers were determined under −leucine conditions, and squares indicate genes whose copy numbers were determined under +leucine conditions. Copy numbers of native genes were obtained from previously published results <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073319#pone.0073319-Moriya2" target="_blank">[2]</a>. The averages of more than three independent experiments are shown. The original data with standard deviations are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073319#pone.0073319.s004" target="_blank">Table S2</a>.</p

Relationships between Cell Cycle Regulator Gene Copy Numbers and Protein Expression Levels in <i>Schizosaccharomyces pombe</i>

<div><p>We previously determined the copy number limits of overexpression for cell division cycle (<i>cdc</i>) regulatory genes in the fission yeast <i>Schizosaccharomyces pombe</i> using the “genetic tug-of-war” (gTOW) method. In this study, we measured the levels of tandem affinity purification (TAP)-tagged target proteins when their copy numbers are increased in gTOW. Twenty analyzed genes showed roughly linear correlations between increased protein levels and gene copy numbers, which suggested a general lack of compensation for gene dosage in <i>S. pombe</i>. Cdc16 and Sid2 protein levels but not their mRNA levels were much lower than that expected by their copy numbers, which suggested the existence of a post-transcriptional down regulation of these genes. The cyclin Cig1 protein level and its mRNA level were much higher than that expected by its copy numbers, which suggested a positive feedback mechanism for its expression. A higher Cdc10 protein level and its mRNA level, probably due to cloning its gene into a plasmid, indicated that Cdc10 regulation was more robust than that previously predicted.</p></div

Quantifying Cdc–TAP protein levels expressed by a single chromosomal copy with an increase in gene copy number.

<p><b>A</b>. <i>S. pombe</i> strains for determining the increases in protein levels expressed by a single chromosomal copy with an increase in gene copy number. Each <i>cdc–</i>TAP strain was transformed with either an empty vector or the corresponding target plasmid and then cultured in medium with or without leucine (as indicated). <b>B</b>–<b>E</b>. Quantitative results for Cdc16–TAP, Sid2–TAP, Cdc10–TAP, and Cig1–TAP. TAP-tagged protein levels were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073319#s4" target="_blank">Methods</a>. Copy number* is the copy number of a Target plasmid plus 1 (chromosomal copy).</p

Quantifying Cdc–TAP protein levels with increased gene copy numbers.

<p><b>A</b>. <i>S. pombe</i> strains for determining increased protein levels expressed by a TAP plasmid and a chromosomal copy with an increase in gene copy number. Each <i>cdc–</i>TAP strain was transformed with either an empty vector or the corresponding <i>cdc–</i>TAP plasmid and then cultured in medium with or without leucine. <b>B</b>–<b>E</b> are examples of these quantitative results. The levels of TAP-tagged proteins were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073319#s4" target="_blank">Methods</a>. Copy number* indicates the copy number of each TAP plasmid plus 1 (chromosomal copy). Circled numbers indicate the fold-dilutions used to measure the intensity of a Cdc–TAP protein. Total proteins were visualized using Coomassie® G-250 staining. <b>B</b>. Pyp3^1–96–TAP used as a control. <b>C</b>. Csk1–TAP; an example for which the protein level increase and the copy number were well correlated. <b>D</b> and <b>E</b>. Cdc16–TAP and Sid2–TAP; examples for which the protein levels did not increase with an increase in copy number. <b>F</b> and <b>G</b>. Cdc10–TAP and Cig1–TAP; examples for which protein level increases exceeded copy number increases. All Cdc–TAP analyses are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073319#pone.0073319.s002" target="_blank">Figure S2</a> and the quantitative results are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073319#pone-0073319-t001" target="_blank">Table 1</a>.</p

Isolation of the element responsible for the low copy number limit in the <i>DIE2</i> region.

<p></p><p></p><p></p><p>Copy number limits of DNA fragments from the <i>DIE2</i> region. The data were obtained from our previous study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120678#pone.0120678.ref001" target="_blank">1</a>].</p><p></p><p></p><p>Copy number limits of DNA fragments (Frag5 in A) with serial 10-bp deletions every 100 bp. The asterisk indicates that only single experiment was performed.</p><p></p><p></p><p>Locations of the small ORFs (<i>sORF1</i> and <i>sORF2</i>) in the 3′ region of <i>DIE2</i>. The numbers indicate the 10-bp deletions analyzed in B. The deletions shown in white did not affect the toxicity of the DNA fragment, whereas the deletion shown in black disrupted the toxicity.</p><p></p><p></p><p>Copy number limits of DNA fragments with ATG to ATC substitutions in <i>sORF2</i>.</p><p></p><p></p><p>Amino acid sequence of <i>sORF2</i>. The substituted methionines (ATG codons) in C are shown in red. A potential NLS sequence is underlined, and an amino acid sequence predicted to construct a helical structure is shown in bold letters.</p><p></p><p></p><p></p

Structural analysis of sORF2.

<p></p><p></p><p></p><p>Alignment of the <i>sORF2</i> regions of <i>S</i>. <i>cerevisiae</i> and <i>S</i>. <i>paradoxus</i>. Identical nucleotides are shown in yellow. ATG and STOP codons of <i>sORF2</i> are shown in red. A TATA repeat and deletion in the <i>S</i>. <i>paradoxus</i> sequence are indicated in blue. The image is a snapshot from the fungal sequence alignment of SGD (<a href="http://www.yeastgenome.org/cache/fungi/YGR229C.html" target="_blank">http://www.yeastgenome.org/cache/fungi/YGR229C.html</a>). The nucleotide numbers indicate the positions relative to the stop codon of <i>SMI1</i>.</p><p></p><p></p><p>Overexpression of sORF2 without the potential NLS (sORF2 _ΔKKRK). The construct used in this experiment is shown. Cells with pTOW-P_GAL1-sORF2 (<i>P</i>_<i>GAL1</i><i>-sORF2</i>) or pTOW-P_GAL1-sORF2_ΔKKRK (<i>P</i>_<i>GAL1</i><i>-sORF2</i>_{<i>ΔKKRK</i>}) were streaked onto SC-glucose and SC-galactose plates and incubated for indicated days. pTOW40836 (Vector) was used as an empty vector control and pTOW-P_GAL1-GFP (<i>P</i>_<i>GAL1</i><i>-GFP</i>) was used to monitor the <i>P</i>_<i>GAL1</i> induction.</p><p></p><p></p><p></p

Expression analysis of <i>sORF2</i>.

<p></p><p></p><p></p><p>RNAseq analysis of the <i>sORF2</i> region of the strain BY4741 with the control vector (pTOWug2–836) and pTOW-Rear2. Parts of the detected reads are shown. The locations of <i>DIE2</i>, <i>sORF2</i>, and <i>SMI1</i> are indicated.</p><p></p><p></p><p>Western blot analysis of sORF2 using TAPtag. Expression of sORF2-TAP from the genomic region or plasmids was detected using peroxidase anti-peroxidase soluble complex. BY4714 is a negative control strain without any TAP-tagged protein expressed. Vector is another negative control, in which BY4741 harbors an empty vector (pTOWug2–836). Cells of BY4741, sORF2-TAP (genome), and POP5-TAP (genome) were cultivated in YPD medium; cells of Vector and sORF2-TAP (plasmid) were cultivated in SC—Ura medium. Dilution indicates the fold-dilution of the cellular lysate applied to the gel. Red-squared dilutions were used to calculate the expression levels of TAP-tagged proteins. The white arrowhead indicates the expected molecular weight of Pop5-TAP protein (39.6kDa), and the black arrowhead indicates the one of sORF2-TAP (27.1 kDa). Structures of sORF2-TAP constructs are shown.</p><p></p><p></p><p></p

Multicopy suppressors of growth inhibition after increasing the copy number in the <i>DIE2</i> 3′ fragment.

<p></p><p></p><p></p><p>Maximum growth rate of BY4741 cells that harbored both pTOW-Rear2 and the suppressor plasmids (pRS423-<i>UBP7</i> and pRS423-<i>PRM1</i>, and the empty vector, pRS423) in SC—Ura—His medium. The averages and standard deviations from six independent experiments are shown.</p><p></p><p></p><p>Growth curves of the BY4741 cells that harbored both pTOW-Rear2 and the suppressor plasmids in SC—Ura—His medium. One representative data is shown from each experiment.</p><p></p><p></p><p></p

Small Toxic Protein Encoded on Chromosome VII of <i>Saccharomyces cerevisiae</i>

<div><p>In a previous study, we found an unknown element that caused growth inhibition after its copy number increased in the 3′ region of <i>DIE2</i> in <i>Saccharomyces cerevisiae</i>. In this study, we further identified this element and observed that overexpression of a small protein (sORF2) of 57 amino acids encoded in this region caused growth inhibition. The transcriptional response and multicopy suppression of the growth inhibition caused by sORF2 overexpression suggest that sORF2 overexpression inhibits the ergosterol biosynthetic pathway. sORF2 was not required in the normal growth of <i>S</i>. <i>cerevisiae</i>, and not conserved in related yeast species including <i>S</i>. <i>paradoxus</i>. Thus, sORF2 (designated as <i>OTO1</i>) is an orphan ORF that determines the specificity of this species.</p></div