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

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...

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Autores principales:	Nobuko Sumiya, Takayuki Fujiwara, Yusuke Kobayashi, Osami Misumi, Shin-ya Miyagishima
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/b4e164ada98a4abc9ba52a05b5d19963
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spelling	oai:doaj.org-article:b4e164ada98a4abc9ba52a05b5d199632021-11-25T05:55:37ZDevelopment of a heat-shock inducible gene expression system in the red alga Cyanidioschyzon merolae.1932-620310.1371/journal.pone.0111261https://doaj.org/article/b4e164ada98a4abc9ba52a05b5d199632014-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0111261https://doaj.org/toc/1932-6203The 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.Nobuko SumiyaTakayuki FujiwaraYusuke KobayashiOsami MisumiShin-ya MiyagishimaPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 10, p e111261 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Nobuko Sumiya Takayuki Fujiwara Yusuke Kobayashi Osami Misumi Shin-ya Miyagishima Development of a heat-shock inducible gene expression system in the red alga Cyanidioschyzon merolae.
description	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.
format	article
author	Nobuko Sumiya Takayuki Fujiwara Yusuke Kobayashi Osami Misumi Shin-ya Miyagishima
author_facet	Nobuko Sumiya Takayuki Fujiwara Yusuke Kobayashi Osami Misumi Shin-ya Miyagishima
author_sort	Nobuko Sumiya
title	Development of a heat-shock inducible gene expression system in the red alga Cyanidioschyzon merolae.
title_short	Development of a heat-shock inducible gene expression system in the red alga Cyanidioschyzon merolae.
title_full	Development of a heat-shock inducible gene expression system in the red alga Cyanidioschyzon merolae.
title_fullStr	Development of a heat-shock inducible gene expression system in the red alga Cyanidioschyzon merolae.
title_full_unstemmed	Development of a heat-shock inducible gene expression system in the red alga Cyanidioschyzon merolae.
title_sort	development of a heat-shock inducible gene expression system in the red alga cyanidioschyzon merolae.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/b4e164ada98a4abc9ba52a05b5d19963
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Development of a heat-shock inducible gene expression system in the red alga Cyanidioschyzon merolae.

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