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

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 fold-increases in protein levels and copy numbers.

<p><b>A</b>. A scatter plot between the fold increase in protein level and the copy number. Squares indicate the results of control experiments using Pyp3^1–96–TAP. Genes that showed high variations between protein level increases and copy numbers are indicated. <b>B</b> and <b>C</b>. Fold increase in the gene copy number, the mRNA and protein levels of indicated gene in the +leucine (<b>B</b>) and –leucine (<b>C</b>) conditions. The original data for the gene copy number and protein increase are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073319#pone-0073319-t001" target="_blank">Table 1</a>, and the data of the mRNA increase are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073319#pone.0073319.s005" target="_blank">Table S3</a>.</p

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