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

Procedures for TAP plasmid and TAP strain construction.We first constructed a “TAP plasmid” from a “Target plasmid” containing one of the target cdc genes [2]. This TAP plasmid was then used as a PCR template for the chromosomal integration of the TAP construct to obtain the “TAP strain.” Red letters indicate the PCR primers, which are listed in Tables S6, S7, S8, and S9. The details for construction are described in the Strains and plasmids section of Methods. The numbers of successfully constructed plasmids and TAP protein detection are also shown.
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pone-0073319-g001: Procedures for TAP plasmid and TAP strain construction.We first constructed a “TAP plasmid” from a “Target plasmid” containing one of the target cdc genes [2]. This TAP plasmid was then used as a PCR template for the chromosomal integration of the TAP construct to obtain the “TAP strain.” Red letters indicate the PCR primers, which are listed in Tables S6, S7, S8, and S9. The details for construction are described in the Strains and plasmids section of Methods. The numbers of successfully constructed plasmids and TAP protein detection are also shown.

Mentions: As targets for this study, we chose 31 cdc genes that we had previously analyzed by gTOW [2]. These are listed in Table S1. Because specific antibodies against most of these Cdc proteins were not available, we used tandem affinity purification (TAP) tagging to detect the target proteins of interest [7]. We first attempted to construct C-terminally TAP-tagged cdc genes on a gTOW plasmid (plasmid construction is shown in Figure 1). We had gTOW vectors with three different maximum plasmid copy numbers; we chose the “middle” vector because it covered the widest range for copy number limits [1]–[3]. Each tagged-target protein was expressed using its native promoter, although each terminator was not native, as we used the terminator for the ADH1 gene from S. cerevisiae. We designated the constructed plasmid TAP plasmid. Of 31 genes that we attempted to analyze, we successfully constructed 29 TAP plasmids and confirmed the expressions of 24 TAP proteins using these plasmids (summarized in Figure 1 and Table S1).


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

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

Procedures for TAP plasmid and TAP strain construction.We first constructed a “TAP plasmid” from a “Target plasmid” containing one of the target cdc genes [2]. This TAP plasmid was then used as a PCR template for the chromosomal integration of the TAP construct to obtain the “TAP strain.” Red letters indicate the PCR primers, which are listed in Tables S6, S7, S8, and S9. The details for construction are described in the Strains and plasmids section of Methods. The numbers of successfully constructed plasmids and TAP protein detection are also shown.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0073319-g001: Procedures for TAP plasmid and TAP strain construction.We first constructed a “TAP plasmid” from a “Target plasmid” containing one of the target cdc genes [2]. This TAP plasmid was then used as a PCR template for the chromosomal integration of the TAP construct to obtain the “TAP strain.” Red letters indicate the PCR primers, which are listed in Tables S6, S7, S8, and S9. The details for construction are described in the Strains and plasmids section of Methods. The numbers of successfully constructed plasmids and TAP protein detection are also shown.
Mentions: As targets for this study, we chose 31 cdc genes that we had previously analyzed by gTOW [2]. These are listed in Table S1. Because specific antibodies against most of these Cdc proteins were not available, we used tandem affinity purification (TAP) tagging to detect the target proteins of interest [7]. We first attempted to construct C-terminally TAP-tagged cdc genes on a gTOW plasmid (plasmid construction is shown in Figure 1). We had gTOW vectors with three different maximum plasmid copy numbers; we chose the “middle” vector because it covered the widest range for copy number limits [1]–[3]. Each tagged-target protein was expressed using its native promoter, although each terminator was not native, as we used the terminator for the ADH1 gene from S. cerevisiae. We designated the constructed plasmid TAP plasmid. Of 31 genes that we attempted to analyze, we successfully constructed 29 TAP plasmids and confirmed the expressions of 24 TAP proteins using these plasmids (summarized in Figure 1 and Table S1).

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