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Development of a heat-shock inducible gene expression system in the red alga Cyanidioschyzon merolae.

Sumiya N, Fujiwara T, Kobayashi Y, Misumi O, Miyagishima SY - PLoS ONE (2014)

Bottom Line: At least a 30-min heat shock is required for the expression of a protein of interest and a 60-min heat shock yields the maximum level of protein expression.After the heat shock, the mRNA level decreases rapidly.Expression of the dominant negative DRP5B resulted in the appearance of aberrant-shaped cells in which two daughter chloroplasts and the cells are still connected by a small DRP5B positive tube-like structure.

View Article: PubMed Central - PubMed

Affiliation: Center for Frontier Research, National Institute of Genetics, Mishima, Shizuoka, Japan; Japan Science and Technology Agency, CREST, Kawaguchi, Saitama, Japan.

ABSTRACT
The cell of the unicellular red alga Cyanidioschyzon merolae contains a single chloroplast and mitochondrion, the division of which is tightly synchronized by a light/dark cycle. The genome content is extremely simple, with a low level of genetic redundancy, in photosynthetic eukaryotes. In addition, transient transformation and stable transformation by homologous recombination have been reported. However, for molecular genetic analyses of phenomena that are essential for cellular growth and survival, inducible gene expression/suppression systems are needed. Here, we report the development of a heat-shock inducible gene expression system in C. merolae. CMJ101C, encoding a small heat shock protein, is transcribed only when cells are exposed to an elevated temperature. Using a superfolder GFP as a reporter protein, the 200-bp upstream region of CMJ101C orf was determined to be the optimal promoter for heat-shock induction. The optimal temperature to induce expression is 50°C, at which C. merolae cells are able to proliferate. At least a 30-min heat shock is required for the expression of a protein of interest and a 60-min heat shock yields the maximum level of protein expression. After the heat shock, the mRNA level decreases rapidly. As an example of the system, the expression of a dominant negative form of chloroplast division DRP5B protein, which has a mutation in the GTPase domain, was induced. Expression of the dominant negative DRP5B resulted in the appearance of aberrant-shaped cells in which two daughter chloroplasts and the cells are still connected by a small DRP5B positive tube-like structure. This result suggests that the dominant negative DRP5B inhibited the final scission of the chloroplast division site, but not the earlier stages of division site constriction. It is also suggested that cell cycle progression is not arrested by the impairment of chloroplast division at the final stage.

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Transcriptional activities of the upstream region of CMJ101C orf before and after heat shock treatment in transiently transformed cells.Several different lengths of the upstream region of CMJ101C orf were respectively fused with sfGFP orf. The plasmids were transiently transformed into C. merolae wild-type cells. After transformation, cells were cultured at 36°C for 1 day and then shifted to 50°C for 1 h. Expression of the GFP protein was examined by fluorescence microscopy and immunoblotting. (A) Schematic diagram of the plasmids used. (B and C) The percentage of the cells with GFP fluorescence was compared before and after the 1-h 50°C heat shock treatment. The red is the autofluorescence of chlorophyll. The error bars represent the standard deviation calculated from three individual experiments (n = 3). 100 cells were counted for each experiment. The scale bar is 10 µm. (D) Immunoblotting with the anti-GFP antibody before (36°C) and after (50°C) the heat shock treatment. CBB staining of the PVDF membrane is shown as a loading control.
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pone-0111261-g003: Transcriptional activities of the upstream region of CMJ101C orf before and after heat shock treatment in transiently transformed cells.Several different lengths of the upstream region of CMJ101C orf were respectively fused with sfGFP orf. The plasmids were transiently transformed into C. merolae wild-type cells. After transformation, cells were cultured at 36°C for 1 day and then shifted to 50°C for 1 h. Expression of the GFP protein was examined by fluorescence microscopy and immunoblotting. (A) Schematic diagram of the plasmids used. (B and C) The percentage of the cells with GFP fluorescence was compared before and after the 1-h 50°C heat shock treatment. The red is the autofluorescence of chlorophyll. The error bars represent the standard deviation calculated from three individual experiments (n = 3). 100 cells were counted for each experiment. The scale bar is 10 µm. (D) Immunoblotting with the anti-GFP antibody before (36°C) and after (50°C) the heat shock treatment. CBB staining of the PVDF membrane is shown as a loading control.

Mentions: Upstream of the CMJ101C orf, the CMJ100C orf locates in the opposite orientation and the interval between the two orfs is 345 bp (Figure 3A). It is well known that there are conserved cis-regulatory promoter elements called heat shock elements in the upstream region of heat shock genes (reviewed in [27]), but there are no typical heat shock elements (at least of the three nGAAn elements) in the 345-bp region. In order to define the region that is optimal for transcription specifically in response to heat shock, we fused a series of CMJ101C upstream sequences of different length to sfGFP orf and the constructs (plasmids; P-100 to P-1,000) were transiently transformed into C. merolae wild-type cells (Figure 3A). The transient transformation of C. merolae cells resulted at most in 2∼10% of transformants in a population [9]. After transformation, cells were cultured at 36°C for 24 h and then the temperature was shifted to 50°C.


Development of a heat-shock inducible gene expression system in the red alga Cyanidioschyzon merolae.

Sumiya N, Fujiwara T, Kobayashi Y, Misumi O, Miyagishima SY - PLoS ONE (2014)

Transcriptional activities of the upstream region of CMJ101C orf before and after heat shock treatment in transiently transformed cells.Several different lengths of the upstream region of CMJ101C orf were respectively fused with sfGFP orf. The plasmids were transiently transformed into C. merolae wild-type cells. After transformation, cells were cultured at 36°C for 1 day and then shifted to 50°C for 1 h. Expression of the GFP protein was examined by fluorescence microscopy and immunoblotting. (A) Schematic diagram of the plasmids used. (B and C) The percentage of the cells with GFP fluorescence was compared before and after the 1-h 50°C heat shock treatment. The red is the autofluorescence of chlorophyll. The error bars represent the standard deviation calculated from three individual experiments (n = 3). 100 cells were counted for each experiment. The scale bar is 10 µm. (D) Immunoblotting with the anti-GFP antibody before (36°C) and after (50°C) the heat shock treatment. CBB staining of the PVDF membrane is shown as a loading control.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4206486&req=5

pone-0111261-g003: Transcriptional activities of the upstream region of CMJ101C orf before and after heat shock treatment in transiently transformed cells.Several different lengths of the upstream region of CMJ101C orf were respectively fused with sfGFP orf. The plasmids were transiently transformed into C. merolae wild-type cells. After transformation, cells were cultured at 36°C for 1 day and then shifted to 50°C for 1 h. Expression of the GFP protein was examined by fluorescence microscopy and immunoblotting. (A) Schematic diagram of the plasmids used. (B and C) The percentage of the cells with GFP fluorescence was compared before and after the 1-h 50°C heat shock treatment. The red is the autofluorescence of chlorophyll. The error bars represent the standard deviation calculated from three individual experiments (n = 3). 100 cells were counted for each experiment. The scale bar is 10 µm. (D) Immunoblotting with the anti-GFP antibody before (36°C) and after (50°C) the heat shock treatment. CBB staining of the PVDF membrane is shown as a loading control.
Mentions: Upstream of the CMJ101C orf, the CMJ100C orf locates in the opposite orientation and the interval between the two orfs is 345 bp (Figure 3A). It is well known that there are conserved cis-regulatory promoter elements called heat shock elements in the upstream region of heat shock genes (reviewed in [27]), but there are no typical heat shock elements (at least of the three nGAAn elements) in the 345-bp region. In order to define the region that is optimal for transcription specifically in response to heat shock, we fused a series of CMJ101C upstream sequences of different length to sfGFP orf and the constructs (plasmids; P-100 to P-1,000) were transiently transformed into C. merolae wild-type cells (Figure 3A). The transient transformation of C. merolae cells resulted at most in 2∼10% of transformants in a population [9]. After transformation, cells were cultured at 36°C for 24 h and then the temperature was shifted to 50°C.

Bottom Line: At least a 30-min heat shock is required for the expression of a protein of interest and a 60-min heat shock yields the maximum level of protein expression.After the heat shock, the mRNA level decreases rapidly.Expression of the dominant negative DRP5B resulted in the appearance of aberrant-shaped cells in which two daughter chloroplasts and the cells are still connected by a small DRP5B positive tube-like structure.

View Article: PubMed Central - PubMed

Affiliation: Center for Frontier Research, National Institute of Genetics, Mishima, Shizuoka, Japan; Japan Science and Technology Agency, CREST, Kawaguchi, Saitama, Japan.

ABSTRACT
The cell of the unicellular red alga Cyanidioschyzon merolae contains a single chloroplast and mitochondrion, the division of which is tightly synchronized by a light/dark cycle. The genome content is extremely simple, with a low level of genetic redundancy, in photosynthetic eukaryotes. In addition, transient transformation and stable transformation by homologous recombination have been reported. However, for molecular genetic analyses of phenomena that are essential for cellular growth and survival, inducible gene expression/suppression systems are needed. Here, we report the development of a heat-shock inducible gene expression system in C. merolae. CMJ101C, encoding a small heat shock protein, is transcribed only when cells are exposed to an elevated temperature. Using a superfolder GFP as a reporter protein, the 200-bp upstream region of CMJ101C orf was determined to be the optimal promoter for heat-shock induction. The optimal temperature to induce expression is 50°C, at which C. merolae cells are able to proliferate. At least a 30-min heat shock is required for the expression of a protein of interest and a 60-min heat shock yields the maximum level of protein expression. After the heat shock, the mRNA level decreases rapidly. As an example of the system, the expression of a dominant negative form of chloroplast division DRP5B protein, which has a mutation in the GTPase domain, was induced. Expression of the dominant negative DRP5B resulted in the appearance of aberrant-shaped cells in which two daughter chloroplasts and the cells are still connected by a small DRP5B positive tube-like structure. This result suggests that the dominant negative DRP5B inhibited the final scission of the chloroplast division site, but not the earlier stages of division site constriction. It is also suggested that cell cycle progression is not arrested by the impairment of chloroplast division at the final stage.

Show MeSH
Related in: MedlinePlus