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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 with increased gene copy numbers.A. S. pombe strains for determining increased protein levels expressed by a TAP plasmid and a chromosomal copy with an increase in gene copy number. Each cdc–TAP strain was transformed with either an empty vector or the corresponding cdc–TAP plasmid and then cultured in medium with or without leucine. B–E are examples of these quantitative results. The levels of TAP-tagged proteins were determined as described in Methods. 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. Pyp31–96–TAP used as a control. C. Csk1–TAP; an example for which the protein level increase and the copy number were well correlated. D and E. Cdc16–TAP and Sid2–TAP; examples for which the protein levels did not increase with an increase in copy number. F and G. Cdc10–TAP and Cig1–TAP; examples for which protein level increases exceeded copy number increases. All Cdc–TAP analyses are shown in Figure S2 and the quantitative results are summarized in Table 1.
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pone-0073319-g003: Quantifying Cdc–TAP protein levels with increased gene copy numbers.A. S. pombe strains for determining increased protein levels expressed by a TAP plasmid and a chromosomal copy with an increase in gene copy number. Each cdc–TAP strain was transformed with either an empty vector or the corresponding cdc–TAP plasmid and then cultured in medium with or without leucine. B–E are examples of these quantitative results. The levels of TAP-tagged proteins were determined as described in Methods. 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. Pyp31–96–TAP used as a control. C. Csk1–TAP; an example for which the protein level increase and the copy number were well correlated. D and E. Cdc16–TAP and Sid2–TAP; examples for which the protein levels did not increase with an increase in copy number. F and G. Cdc10–TAP and Cig1–TAP; examples for which protein level increases exceeded copy number increases. All Cdc–TAP analyses are shown in Figure S2 and the quantitative results are summarized in Table 1.

Mentions: We then measured each of the TAP-tagged Cdc proteins expressed in the TAP strains harboring an empty vector or a TAP plasmid cultured in medium with or without leucine using quantitative Western blotting (Figure 3A; procedure shown in Figure S1). Our aim was not to quantify the absolute levels of these target proteins but to measure the fold increase in the protein level when expressed by the TAP plasmids over that expressed by the genomic copy (single copy). To avoid saturation effects with quantitative Western blotting, we prepared serial dilutions of the proteins expressed in the strains with TAP plasmids. Examples of these measurements are shown in Figure 3 and all measurements are shown in Figure S2.


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 with increased gene copy numbers.A. S. pombe strains for determining increased protein levels expressed by a TAP plasmid and a chromosomal copy with an increase in gene copy number. Each cdc–TAP strain was transformed with either an empty vector or the corresponding cdc–TAP plasmid and then cultured in medium with or without leucine. B–E are examples of these quantitative results. The levels of TAP-tagged proteins were determined as described in Methods. 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. Pyp31–96–TAP used as a control. C. Csk1–TAP; an example for which the protein level increase and the copy number were well correlated. D and E. Cdc16–TAP and Sid2–TAP; examples for which the protein levels did not increase with an increase in copy number. F and G. Cdc10–TAP and Cig1–TAP; examples for which protein level increases exceeded copy number increases. All Cdc–TAP analyses are shown in Figure S2 and the quantitative results are summarized in Table 1.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0073319-g003: Quantifying Cdc–TAP protein levels with increased gene copy numbers.A. S. pombe strains for determining increased protein levels expressed by a TAP plasmid and a chromosomal copy with an increase in gene copy number. Each cdc–TAP strain was transformed with either an empty vector or the corresponding cdc–TAP plasmid and then cultured in medium with or without leucine. B–E are examples of these quantitative results. The levels of TAP-tagged proteins were determined as described in Methods. 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. Pyp31–96–TAP used as a control. C. Csk1–TAP; an example for which the protein level increase and the copy number were well correlated. D and E. Cdc16–TAP and Sid2–TAP; examples for which the protein levels did not increase with an increase in copy number. F and G. Cdc10–TAP and Cig1–TAP; examples for which protein level increases exceeded copy number increases. All Cdc–TAP analyses are shown in Figure S2 and the quantitative results are summarized in Table 1.
Mentions: We then measured each of the TAP-tagged Cdc proteins expressed in the TAP strains harboring an empty vector or a TAP plasmid cultured in medium with or without leucine using quantitative Western blotting (Figure 3A; procedure shown in Figure S1). Our aim was not to quantify the absolute levels of these target proteins but to measure the fold increase in the protein level when expressed by the TAP plasmids over that expressed by the genomic copy (single copy). To avoid saturation effects with quantitative Western blotting, we prepared serial dilutions of the proteins expressed in the strains with TAP plasmids. Examples of these measurements are shown in Figure 3 and all measurements are shown in Figure S2.

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