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Relationships between cell cycle regulator gene copy numbers and protein expression levels in Schizosaccharomyces pombe.

Chino A, Makanae K, Moriya H - PLoS ONE (2013)

Bottom Line: We previously determined the copy number limits of overexpression for cell division cycle (cdc) regulatory genes in the fission yeast Schizosaccharomyces pombe 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.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.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Science and Technology, Okayama University, Kita-ku, Okayama, Japan ; Research Core for Interdisciplinary Sciences, Okayama University, Kita-ku, Okayama, Japan.

ABSTRACT
We previously determined the copy number limits of overexpression for cell division cycle (cdc) regulatory genes in the fission yeast Schizosaccharomyces pombe 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 S. pombe. 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.

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Quantifying Cdc–TAP protein levels expressed by a single chromosomal copy with an increase in gene copy number.A. S. pombe strains for determining the increases in protein levels expressed by a single chromosomal copy with an increase in gene copy number. Each cdc–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–E. Quantitative results for Cdc16–TAP, Sid2–TAP, Cdc10–TAP, and Cig1–TAP. TAP-tagged protein levels were determined as described in Methods. Copy number* is the copy number of a Target plasmid plus 1 (chromosomal copy).
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pone-0073319-g005: Quantifying Cdc–TAP protein levels expressed by a single chromosomal copy with an increase in gene copy number.A. S. pombe strains for determining the increases in protein levels expressed by a single chromosomal copy with an increase in gene copy number. Each cdc–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–E. Quantitative results for Cdc16–TAP, Sid2–TAP, Cdc10–TAP, and Cig1–TAP. TAP-tagged protein levels were determined as described in Methods. Copy number* is the copy number of a Target plasmid plus 1 (chromosomal copy).

Mentions: We further measured the levels of the 4 proteins mentioned above when they were expressed by the chromosomal genes to confirm if the expression by each gene copy also changed when the gene copy numbers increased. As shown in Figure 5A, we measured the TAP-tagged Cdc proteins expressed by the chromosomal genes of the TAP strains when the target plasmids (without TAP-tag) were introduced. We then compared these protein levels with those expressed by the chromosomal genes of the TAP strain with the empty vector. As shown in Figures 5B and 5C, Cdc16–TAP and Sid2–TAP expressed by the chromosomal genes were dramatically reduced when their gene copy numbers increased, suggesting that the levels of these proteins were compensated by increase in the numbers of their genes. Cig1–TAP protein expressed by the chromosomal gene increased when its copy number increased (Figure 5E), suggesting the existence of positive feedback mechanism for Cig1 protein expression. However, Cdc10–TAP expressed by a chromosomal gene did not show consistent higher levels when the gene copy number increased (Figure 5D), and the PI/CN ratios (0.4 and 3.1) were much lower than the ones expressed from the TAP plasmid (7.03 and 9.20, Figure 3F). Thus, we considered that the high PI/CN ratio observed with Cdc10–TAP plasmid experiment (Figure 3F) was not because of a feedback mechanism but because of an artificial increase in expression after cloning cdc10 on a plasmid.


Relationships between cell cycle regulator gene copy numbers and protein expression levels in Schizosaccharomyces pombe.

Chino A, Makanae K, Moriya H - PLoS ONE (2013)

Quantifying Cdc–TAP protein levels expressed by a single chromosomal copy with an increase in gene copy number.A. S. pombe strains for determining the increases in protein levels expressed by a single chromosomal copy with an increase in gene copy number. Each cdc–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–E. Quantitative results for Cdc16–TAP, Sid2–TAP, Cdc10–TAP, and Cig1–TAP. TAP-tagged protein levels were determined as described in Methods. Copy number* is the copy number of a Target plasmid plus 1 (chromosomal copy).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3760898&req=5

pone-0073319-g005: Quantifying Cdc–TAP protein levels expressed by a single chromosomal copy with an increase in gene copy number.A. S. pombe strains for determining the increases in protein levels expressed by a single chromosomal copy with an increase in gene copy number. Each cdc–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–E. Quantitative results for Cdc16–TAP, Sid2–TAP, Cdc10–TAP, and Cig1–TAP. TAP-tagged protein levels were determined as described in Methods. Copy number* is the copy number of a Target plasmid plus 1 (chromosomal copy).
Mentions: We further measured the levels of the 4 proteins mentioned above when they were expressed by the chromosomal genes to confirm if the expression by each gene copy also changed when the gene copy numbers increased. As shown in Figure 5A, we measured the TAP-tagged Cdc proteins expressed by the chromosomal genes of the TAP strains when the target plasmids (without TAP-tag) were introduced. We then compared these protein levels with those expressed by the chromosomal genes of the TAP strain with the empty vector. As shown in Figures 5B and 5C, Cdc16–TAP and Sid2–TAP expressed by the chromosomal genes were dramatically reduced when their gene copy numbers increased, suggesting that the levels of these proteins were compensated by increase in the numbers of their genes. Cig1–TAP protein expressed by the chromosomal gene increased when its copy number increased (Figure 5E), suggesting the existence of positive feedback mechanism for Cig1 protein expression. However, Cdc10–TAP expressed by a chromosomal gene did not show consistent higher levels when the gene copy number increased (Figure 5D), and the PI/CN ratios (0.4 and 3.1) were much lower than the ones expressed from the TAP plasmid (7.03 and 9.20, Figure 3F). Thus, we considered that the high PI/CN ratio observed with Cdc10–TAP plasmid experiment (Figure 3F) was not because of a feedback mechanism but because of an artificial increase in expression after cloning cdc10 on a plasmid.

Bottom Line: We previously determined the copy number limits of overexpression for cell division cycle (cdc) regulatory genes in the fission yeast Schizosaccharomyces pombe 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.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.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Science and Technology, Okayama University, Kita-ku, Okayama, Japan ; Research Core for Interdisciplinary Sciences, Okayama University, Kita-ku, Okayama, Japan.

ABSTRACT
We previously determined the copy number limits of overexpression for cell division cycle (cdc) regulatory genes in the fission yeast Schizosaccharomyces pombe 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 S. pombe. 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.

Show MeSH
Related in: MedlinePlus