Limits...
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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Related in: MedlinePlus

Relationships between fold-increases in protein levels and copy numbers.A. A scatter plot between the fold increase in protein level and the copy number. Squares indicate the results of control experiments using Pyp31–96–TAP. Genes that showed high variations between protein level increases and copy numbers are indicated. B and C. Fold increase in the gene copy number, the mRNA and protein levels of indicated gene in the +leucine (B) and –leucine (C) conditions. The original data for the gene copy number and protein increase are provided in Table 1, and the data of the mRNA increase are provided in Table S3.
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pone-0073319-g004: Relationships between fold-increases in protein levels and copy numbers.A. A scatter plot between the fold increase in protein level and the copy number. Squares indicate the results of control experiments using Pyp31–96–TAP. Genes that showed high variations between protein level increases and copy numbers are indicated. B and C. Fold increase in the gene copy number, the mRNA and protein levels of indicated gene in the +leucine (B) and –leucine (C) conditions. The original data for the gene copy number and protein increase are provided in Table 1, and the data of the mRNA increase are provided in Table S3.

Mentions: We simultaneously measured the copy number (CN) of TAP plasmid in a cell for each experiment and compared this with the fold-increase in the protein level (protein increase: PI) measured as described above. We then calculated the PI/CN ratio. This corresponded to the protein level expressed by each copy of the gene when the gene copy number increased (Table 1). Figure 4A shows the relationships between copy numbers and protein increases. As expected, the protein levels increased according to the increase in copy number in four control experiments with Pyp31–96–TAP. We did not analyze the protein levels of genes those have the copy number limits less than the copy number of the middle vector (about 55 copies/cell) in −leucine conditions [2], because the cells with the TAP plasmids of these genes did not grow well in −leucine conditions and we thus had difficulties to prepare equal amount of cells and extracted proteins to the control ones for the quantifications of TAP-tagged proteins (data not shown).


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

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

Relationships between fold-increases in protein levels and copy numbers.A. A scatter plot between the fold increase in protein level and the copy number. Squares indicate the results of control experiments using Pyp31–96–TAP. Genes that showed high variations between protein level increases and copy numbers are indicated. B and C. Fold increase in the gene copy number, the mRNA and protein levels of indicated gene in the +leucine (B) and –leucine (C) conditions. The original data for the gene copy number and protein increase are provided in Table 1, and the data of the mRNA increase are provided in Table S3.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3760898&req=5

pone-0073319-g004: Relationships between fold-increases in protein levels and copy numbers.A. A scatter plot between the fold increase in protein level and the copy number. Squares indicate the results of control experiments using Pyp31–96–TAP. Genes that showed high variations between protein level increases and copy numbers are indicated. B and C. Fold increase in the gene copy number, the mRNA and protein levels of indicated gene in the +leucine (B) and –leucine (C) conditions. The original data for the gene copy number and protein increase are provided in Table 1, and the data of the mRNA increase are provided in Table S3.
Mentions: We simultaneously measured the copy number (CN) of TAP plasmid in a cell for each experiment and compared this with the fold-increase in the protein level (protein increase: PI) measured as described above. We then calculated the PI/CN ratio. This corresponded to the protein level expressed by each copy of the gene when the gene copy number increased (Table 1). Figure 4A shows the relationships between copy numbers and protein increases. As expected, the protein levels increased according to the increase in copy number in four control experiments with Pyp31–96–TAP. We did not analyze the protein levels of genes those have the copy number limits less than the copy number of the middle vector (about 55 copies/cell) in −leucine conditions [2], because the cells with the TAP plasmids of these genes did not grow well in −leucine conditions and we thus had difficulties to prepare equal amount of cells and extracted proteins to the control ones for the quantifications of TAP-tagged proteins (data not shown).

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