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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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Duration-dependent effect of 50°C heat shock on the mRNA and protein levels in the stable transformants.The stable S-200 transformant (Figure 4) cultured at 34°C was shifted to 50°C and cultured for 120 min or 24 h. (A) Semi-quantitative RT-PCR showing the GFP mRNA level. TIM13 (CMB148C) was used as a quantitative control. (B) Immunoblotting with the anti-GFP antibody showing the GFP protein level. CBB staining of the PVDF membrane is shown as a loading control. (C) Micrographs showing the GFP fluorescence and autofluorescence of chlorophyll (red). The scale bar is 10 µm. (D) Semi-quantitative RT-PCR showing the GFP mRNA level up to 24 h at 50°C. TIM13 (CMB148C) was used as a quantitative control.
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pone-0111261-g006: Duration-dependent effect of 50°C heat shock on the mRNA and protein levels in the stable transformants.The stable S-200 transformant (Figure 4) cultured at 34°C was shifted to 50°C and cultured for 120 min or 24 h. (A) Semi-quantitative RT-PCR showing the GFP mRNA level. TIM13 (CMB148C) was used as a quantitative control. (B) Immunoblotting with the anti-GFP antibody showing the GFP protein level. CBB staining of the PVDF membrane is shown as a loading control. (C) Micrographs showing the GFP fluorescence and autofluorescence of chlorophyll (red). The scale bar is 10 µm. (D) Semi-quantitative RT-PCR showing the GFP mRNA level up to 24 h at 50°C. TIM13 (CMB148C) was used as a quantitative control.

Mentions: To determine the optimum duration of the 50°C heat shock to induce gene expression in the stable transformants, the S-200 cells were shifted from 34°C to 50°C and cultured for 120 min. GFP mRNA was detected in within 5 min of the temperature shift and reached the maximum level at 30 min (Figure 6A). The GFP protein was detected at 30 min and reached the maximum level at 60 min (Figure 6B). GFP fluorescence-positive cells were detected at 30 min and the strongest fluorescence was detected at 60 min (Figure 6C). Thus, for the expression of GFP, at least 30-min heat shock is required and the protein expression level increased for a further 60 min after the temperature shift. Because GFP mRNA was detected for at least 24 hours in cells cultured at 50°C (Figure 6D), the duration of translational induction is likely to be able to be extended for particular purposes.


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)

Duration-dependent effect of 50°C heat shock on the mRNA and protein levels in the stable transformants.The stable S-200 transformant (Figure 4) cultured at 34°C was shifted to 50°C and cultured for 120 min or 24 h. (A) Semi-quantitative RT-PCR showing the GFP mRNA level. TIM13 (CMB148C) was used as a quantitative control. (B) Immunoblotting with the anti-GFP antibody showing the GFP protein level. CBB staining of the PVDF membrane is shown as a loading control. (C) Micrographs showing the GFP fluorescence and autofluorescence of chlorophyll (red). The scale bar is 10 µm. (D) Semi-quantitative RT-PCR showing the GFP mRNA level up to 24 h at 50°C. TIM13 (CMB148C) was used as a quantitative control.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111261-g006: Duration-dependent effect of 50°C heat shock on the mRNA and protein levels in the stable transformants.The stable S-200 transformant (Figure 4) cultured at 34°C was shifted to 50°C and cultured for 120 min or 24 h. (A) Semi-quantitative RT-PCR showing the GFP mRNA level. TIM13 (CMB148C) was used as a quantitative control. (B) Immunoblotting with the anti-GFP antibody showing the GFP protein level. CBB staining of the PVDF membrane is shown as a loading control. (C) Micrographs showing the GFP fluorescence and autofluorescence of chlorophyll (red). The scale bar is 10 µm. (D) Semi-quantitative RT-PCR showing the GFP mRNA level up to 24 h at 50°C. TIM13 (CMB148C) was used as a quantitative control.
Mentions: To determine the optimum duration of the 50°C heat shock to induce gene expression in the stable transformants, the S-200 cells were shifted from 34°C to 50°C and cultured for 120 min. GFP mRNA was detected in within 5 min of the temperature shift and reached the maximum level at 30 min (Figure 6A). The GFP protein was detected at 30 min and reached the maximum level at 60 min (Figure 6B). GFP fluorescence-positive cells were detected at 30 min and the strongest fluorescence was detected at 60 min (Figure 6C). Thus, for the expression of GFP, at least 30-min heat shock is required and the protein expression level increased for a further 60 min after the temperature shift. Because GFP mRNA was detected for at least 24 hours in cells cultured at 50°C (Figure 6D), the duration of translational induction is likely to be able to be extended for particular purposes.

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