Construction of a &lt;i&gt;URA5.3&lt;/i&gt; deletion strain of the unicellular red alga &lt;i&gt;Cyanidioschyzon merolae&lt;/i&gt;: A backgroundless host strain for transformation experiments

Taki, Keiko; Sone, Toshiyuki; Kobayashi, Yuki; Watanabe, Satoru; Imamura, Sousuke; Tanaka, Kan

doi:10.2323/jgam.61.211

Cited by 25 publications

(17 citation statements)

References 8 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As shown in Fig. 2b, SF12 exhibited uracil auxotrophy; in addition, it displayed 5-FOA resistance in a manner similar to the URA5.3 deletion mutant T1 (Taki et al, 2015). In agreement with our previous observation of strain F12 , SF12 strain also exhibited rapamycinsusceptible phenotypes, such as growth inhibition (Fig.…”

supporting

confidence: 90%

“…In a recent study, we demonstrated that the T1 strain, in which the URA5.3 gene is completely deleted, is a 5-fluoroorotic acid (5-FOA)-resistant, backgroundless host for transformation experiments (Taki et al, 2015). In the study reported here, we constructed a plasmid pKTL1, serving as a genomic knock-in cassette for insertion into the URA5.3 gene (Fig.…”

mentioning

confidence: 99%

“…The resulting 7.1-kbp fragment was used in polyethylene glycol (PEG)-mediated transformation of wild-type cells. Transformants were then selected on modified Allen's 2 (MA2) plates (Imamura et al, 2010) containing 5-FOA and uracil following the same methods used for isolation of the T1 strain (Taki et al, 2015).…”

mentioning

confidence: 99%

See 2 more Smart Citations

Construction of a rapamycin-susceptible strain of the unicellular red alga Cyanidioschyzon merolae for analysis of the target of rapamycin (TOR) function

Imamura

Taki

Tanaka

2017

J. Gen. Appl. Microbiol.

Self Cite

View full text Add to dashboard Cite

supporting

confidence: 90%

mentioning

confidence: 99%

See 1 more Smart Citation

Construction of a rapamycin-susceptible strain of the unicellular red alga Cyanidioschyzon merolae for analysis of the target of rapamycin (TOR) function

Imamura

Taki

Tanaka

2017

J. Gen. Appl. Microbiol.

Self Cite

View full text Add to dashboard Cite

“…The complete genome sequences of the nucleus, mitochondria, and chloroplast were determined, and various tools for molecular genetics analyses have been developed (Fujiwara et al, 2013;Imamura et al, 2009;Kobayashi et al, 2010;Kuroiwa, 1998;Matsuzaki et al, 2004;Nozaki et al, 2007;Taki et al, 2015). C. merolae has been extensively used for studying organelle division processes, cell cycle regulation, and structural biology.…”

Section: Introductionmentioning

confidence: 99%

Lability in sulfur acidic cultivation medium explains unstable effects of CDK inhibitors on Cyanidioschyzon merolae cell proliferation

Kobayashi

Tanaka

2018

J. Gen. Appl. Microbiol.

Self Cite

View full text Add to dashboard Cite

We previously showed that nuclear DNA replication (NDR) is regulated by a checkpoint monitoring the occurrence of organelle DNA replication (ODR) in a unicellular red alga Cyanidioschyzon merolae. These analyses depended on the use of chemical CDK inhibitors such as CDK2 inhibitor II and roscovitine, but subsequent analyses yielded conflicting results depending on the experimental conditions. In the present study, we identified significantly short half-lives of the used chemicals in the sulfur acidic cultivation medium, which reconciles the discrepancy among these results.Cyanidioschyzon merolae is a unicellular red alga living in acidic hot springs, and was introduced as a novel model eukaryotic cell to elucidate various fundamental cell processes because it contains the simplest structure among eukaryotic cells. The complete genome sequences of the nucleus, mitochondria, and chloroplast were determined, and various tools for molecular genetics analyses have been developed (Fujiwara et al., 2013;Imamura et al., 2009;Kobayashi et al., 2010;Kuroiwa, 1998;Matsuzaki et al., 2004;Nozaki et al., 2007;Taki et al., 2015). C. merolae has been extensively used for studying organelle division processes, cell cycle regulation, and structural biology.In a previous study, we investigated a mechanism underlying the coordination of organelle DNA replication (ODR) with nuclear DNA replication (NDR) in C. merolae (Kobayashi et al., 2009). Under periodic light-dark cultivation conditions, the algal cell cycle is arrested at the G1 phase in the dark, and initiated by illumination. Detailed analyses revealed that both mitochondria and chloroplast genomes replicate upon the onset of illumination, and then NDR occurs afterward. As the underlying mechanism, we revealed that an increase of intracellular Mg-ProtoIX, an intermediate of chlorophyll biosynthesis produced in the chloroplast, induced by the ODR occurrence was recognized as a retrograde signal from the chloroplast to the nucleus to activate CDKA, responsible for activating NDR.During these experiments, specific inhibitors for cyclin-dependent kinases (CDK) were conveniently used to analyze the underlying mechanism. CDK2 inhibitor II is a commercially available inhibitor for animal CDK2 and plant CDKA (Merck, Darmstadt, Germany) (Davis et al., 2001). As expected, biochemical and physiological analyses revealed that this compound inhibited the relevant CDK (CDKA) activity and NDR in C. merolae (Kobayashi et al., 2009). Another CDK inhibitor used was roscovitine (Merck, Darmstadt, Germany), that is known as a general inhibitor for various CDK types, as revealed by its inhibition of cdc2/cyclin B, cdk2/cyclin A, cdk2/cyclin E, and cdk5/p53 in mammalian cells (Xie et al., 2016). Our results indicated that CDK inhibitor II inhibited only NDR, while roscovitine inhibited both ODR and NDR, and, thus, it was suggested that ODR is under the control of a CDK other than CDKA in C.merolae. Because both CDK inhibitors inhibit NDR at the cell cycle S phase, their addition to the ...

show abstract

“…The parental strain is a uracil-auxotrophic strain, which has a point mutation in the chromosomal URA5.3 gene ( URA ) (strain M4; Minoda et al, 2004) or in which the URA gene is completely deleted (Taki et al, 2015). To produce stable transformants, a transgene conjugated with the URA selection marker is integrated into a chromosome of the M4 strain by homologous recombination ( URA -M4 selection system; Imamura et al, 2010; Fujiwara et al, 2013a).…”

Section: Introductionmentioning

confidence: 99%

Development of a Double Nuclear Gene-Targeting Method by Two-Step Transformation Based on a Newly Established Chloramphenicol-Selection System in the Red Alga Cyanidioschyzon merolae

Fujiwara¹,

Ohnuma

Kuroiwa

et al. 2017

Front. Plant Sci.

View full text Add to dashboard Cite

The unicellular red alga Cyanidioschyzon merolae possesses a simple cellular architecture that consists of one mitochondrion, one chloroplast, one peroxisome, one Golgi apparatus, and several lysosomes. The nuclear genome content is also simple, with very little genetic redundancy (16.5 Mbp, 4,775 genes). In addition, molecular genetic tools such as gene targeting and inducible gene expression systems have been recently developed. These cytological features and genetic tractability have facilitated various omics analyses. However, only a single transformation selection marker URA has been made available and thus the application of genetic modification has been limited. Here, we report the development of a nuclear targeting method by using chloramphenicol and the chloramphenicol acetyltransferase (CAT) gene. In addition, we found that at least 200-bp homologous arms are required and 500-bp arms are sufficient for a targeted single-copy insertion of the CAT selection marker into the nuclear genome. By means of a combination of the URA and CAT transformation systems, we succeeded in producing a C. merolae strain that expresses HA-cyclin 1 and FLAG-CDKA from the chromosomal CYC1 and CDKA loci, respectively. These methods of multiple nuclear targeting will facilitate genetic manipulation of C. merolae.

show abstract

Construction of a <i>URA5.3</i> deletion strain of the unicellular red alga <i>Cyanidioschyzon merolae</i>: A backgroundless host strain for transformation experiments

Cited by 25 publications

References 8 publications

Construction of a rapamycin-susceptible strain of the unicellular red alga <i>Cyanidioschyzon merolae</i> for analysis of the target of rapamycin (TOR) function

Construction of a rapamycin-susceptible strain of the unicellular red alga <i>Cyanidioschyzon merolae</i> for analysis of the target of rapamycin (TOR) function

Lability in sulfur acidic cultivation medium explains unstable effects of CDK inhibitors on <i>Cyanidioschyzon merolae</i> cell proliferation

Development of a Double Nuclear Gene-Targeting Method by Two-Step Transformation Based on a Newly Established Chloramphenicol-Selection System in the Red Alga Cyanidioschyzon merolae

Contact Info

Product

Resources

About