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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Relationships between native and TAP-tagged gene copy number limit.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 [2]. The averages of more than three independent experiments are shown. The original data with standard deviations are provided in Table S2.
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pone-0073319-g002: Relationships between native and TAP-tagged gene copy number limit.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 [2]. The averages of more than three independent experiments are shown. The original data with standard deviations are provided in Table S2.

Mentions: Because C-terminal TAP-tagging may affect the activity of target proteins, we indirectly evaluated their activities by measuring the copy number limits of tagged genes and compared these with those of native genes. As shown in Figure 2, the tagged genes and the native genes exhibited similar overall copy number limits (Pearson’s r = 0.65). However, the tagged genes dfp1 and slp1 showed increased copy number limits compared with their native genes, which suggested that their activities were reduced. In contrast, the tagged genes cut1, puc1, res2, and rum1 showed reduced copy number limits compared with their native genes, which suggested that their activities were increased or that they had become dominant active.


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 native and TAP-tagged gene copy number limit.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 [2]. The averages of more than three independent experiments are shown. The original data with standard deviations are provided in Table S2.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0073319-g002: Relationships between native and TAP-tagged gene copy number limit.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 [2]. The averages of more than three independent experiments are shown. The original data with standard deviations are provided in Table S2.
Mentions: Because C-terminal TAP-tagging may affect the activity of target proteins, we indirectly evaluated their activities by measuring the copy number limits of tagged genes and compared these with those of native genes. As shown in Figure 2, the tagged genes and the native genes exhibited similar overall copy number limits (Pearson’s r = 0.65). However, the tagged genes dfp1 and slp1 showed increased copy number limits compared with their native genes, which suggested that their activities were reduced. In contrast, the tagged genes cut1, puc1, res2, and rum1 showed reduced copy number limits compared with their native genes, which suggested that their activities were increased or that they had become dominant active.

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