Plasmid Construction Using Recombination Activity in the Fission Yeast Schizosaccharomyces pombe

BACKGROUND: Construction of plasmids is crucial in modern genetic manipulation. As of now, the common method for constructing plasmids is to digest specific DNA sequences with restriction enzymes and to ligate the resulting DNA fragments with DNA ligase. Another potent method to construct plasmids, known as gap-repair cloning (GRC), is commonly used in the budding yeast Saccharomyces cerevisiae. GRC makes use of the homologous recombination activity that occurs within the yeast cells. Due to its flexible design and efficiency, GRC has been frequently used for constructing plasmids with complex structures as well as genome-wide plasmid collections. Although there have been reports indicating GRC feasibility in the fission yeast Schizosaccharomyces pombe, this species is not commonly used for GRC as systematic studies of reporting GRC efficiency in S. pombe have not been performed till date. METHODOLOGY/PRINCIPAL FINDINGS: We investigated GRC efficiency in S. pombe in this study. We first showed that GRC was feasible in S. pombe by constructing a plasmid that contained the LEU2 auxotrophic marker gene in vivo and showed sufficient efficiency with short homology sequences (>25 bp). No preference was shown for the sequence length from the cut site in the vector plasmid. We next showed that plasmids could be constructed in a proper way using 3 DNA fragments with 70% efficiency without any specific selections being made. The GRC efficiency with 3 DNA fragments was dramatically increased >95% in lig4Delta mutant cell, where non-homologous end joining is deficient. Following this approach, we successfully constructed plasmid vectors with leu1+, ade6+, his5+, and lys1+ markers with the low-copy stable plasmid pDblet as a backbone by applying GRC in S. pombe. CONCLUSIONS/SIGNIFICANCE: We concluded that GRC was sufficiently feasible in S. pombe for genome-wide gene functional analysis as well as for regular plasmid construction. Plasmids with different markers constructed in this research are available from NBRP-yeast (http://yeast.lab.nig.ac.jp/)

Yeast screening system reveals the inhibitory mechanism of cancer cell proliferation by benzyl isothiocyanate through down-regulation of Mis12

Benzyl isothiocyanate (BITC) is a naturally-occurring isothiocyanate derived from cruciferous vegetables. BITC has been reported to inhibit the proliferation of various cancer cells, which is believed to be important for the inhibition of tumorigenesis. However, the detailed mechanisms of action remain unclear. In this study, we employed a budding yeast Saccharomyces cerevisiae as a model organism for screening. Twelve genes including MTW1 were identified as the overexpression suppressors for the antiproliferative effect of BITC using the genome-wide multi-copy plasmid collection for S. cerevisiae. Overexpression of the kinetochore protein Mtw1 counteracts the antiproliferative effect of BITC in yeast. The inhibitory effect of BITC on the proliferation of human colon cancer HCT-116 cells was consistently suppressed by the overexpression of Mis12, a human orthologue of Mtw1, and enhanced by the knockdown of Mis12. We also found that BITC increased the phosphorylated and ubiquitinated Mis12 level with consequent reduction of Mis12, suggesting that BITC degrades Mis12 through an ubiquitin-proteasome system. Furthermore, cell cycle analysis showed that the change in the Mis12 level affected the cell cycle distribution and the sensitivity to the BITC-induced apoptosis. These results provide evidence that BITC suppresses cell proliferation through the post-transcriptional regulation of the kinetochore protein Mis12

Yeast screening system reveals the inhibitory mechanism of cancer cell proliferation by benzyl isothiocyanate through down-regulation of Mis12

Benzyl isothiocyanate (BITC) is a naturally-occurring isothiocyanate derived from cruciferous vegetables. BITC has been reported to inhibit the proliferation of various cancer cells, which is believed to be important for the inhibition of tumorigenesis. However, the detailed mechanisms of action remain unclear. In this study, we employed a budding yeast Saccharomyces cerevisiae as a model organism for screening. Twelve genes including MTW1 were identified as the overexpression suppressors for the antiproliferative effect of BITC using the genome-wide multi-copy plasmid collection for S. cerevisiae. Overexpression of the kinetochore protein Mtw1 counteracts the antiproliferative effect of BITC in yeast. The inhibitory effect of BITC on the proliferation of human colon cancer HCT-116 cells was consistently suppressed by the overexpression of Mis12, a human orthologue of Mtw1, and enhanced by the knockdown of Mis12. We also found that BITC increased the phosphorylated and ubiquitinated Mis12 level with consequent reduction of Mis12, suggesting that BITC degrades Mis12 through an ubiquitin-proteasome system. Furthermore, cell cycle analysis showed that the change in the Mis12 level affected the cell cycle distribution and the sensitivity to the BITC-induced apoptosis. These results provide evidence that BITC suppresses cell proliferation through the post-transcriptional regulation of the kinetochore protein Mis12

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