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Masahiro Ishiura

Publications and source records attributed to Masahiro Ishiura.

At least 19 recordsLinked to original sources
PubMed2006

Hexamerization by the N-terminal domain and intersubunit phosphorylation by the C-terminal domain of cyanobacterial circadian clock protein KaiC.

Cyanobacterial clock protein KaiC has a hexagonal, pot-shaped structure composed of six identical dumbbell-shaped subunits. The opposing spherical regions of the dumbbell-shaped structures correspond to the N-terminal and C-terminal domains of KaiC. Previously, we hypothesized that the N-terminal domain of KaiC is responsible for the ATP-induced hexamerization of KaiC while the C-terminal domain is responsible for the phosphorylation of KaiC (Hayashi et al. 2004, J. Biol. Chem. 279, 52331-52337). Here, we tested that hypothesis using the purified protein of each domain. We prepared N-terminal and C-terminal domain proteins (KaiCN and KaiCC, respectively), examined their function by analyzing their ATP- or 5'-adenylylimidodiphosphate (AMPPNP; an unhydrolyzable ATP analog)-induced hexamerization, interactions with KaiA, and phosphorylation, and we demonstrated the following: (1) KaiCN had higher ATP- or AMPPNP-induced oligomerization activity than KaiCC. (2) KaiCc had phosphorylation activity as KaiCWT whereas KaiCN had no activity. (3) KaiCC interacted with KaiA whereas KaiCN did not. (4) The interactions of KaiCC with KaiA did not require that KaiC has a hexamer structure. (5) The interactions of KaiCC with KaiA enhanced the phosphorylation of KaiCC. Furthermore, we presented evidence for the intersubunit phosphorylation of KaiC. KaiCCatE2-, which lacks KaiC phosphorylation activity due to mutations of the catalytic Glu residues, was phosphorylated when it was co-incubated with KaiCC. We propose that the KaiC hexamer consists of a rigid ring structure formed by six N-terminal domains with hexamerization activity and a flexible structure formed by six C-terminal domains with intersubunit phosphorylation activity.

Amino Acid Sequence↗
PubMed2006

Real-time monitoring of chloroplast gene expression by a luciferase reporter: evidence for nuclear regulation of chloroplast circadian period.

Chloroplast-encoded genes, like nucleus-encoded genes, exhibit circadian expression. How the circadian clock exerts its control over chloroplast gene expression, however, is poorly understood. To facilitate the study of chloroplast circadian gene expression, we developed a codon-optimized firefly luciferase gene for the chloroplast of Chlamydomonas reinhardtii as a real-time bioluminescence reporter and introduced it into the chloroplast genome. The bioluminescence of the reporter strain correlated well with the circadian expression pattern of the introduced gene and satisfied all three criteria for circadian rhythms. Moreover, the period of the rhythm was lengthened in per mutants, which are phototactic rhythm mutants carrying a long-period gene in their nuclear genome. These results demonstrate that chloroplast gene expression rhythm is a bona fide circadian rhythm and that the nucleus-encoded circadian oscillator determines the period length of the chloroplast rhythm. Our reporter strains can serve as a powerful tool not only for analysis of the circadian regulation mechanisms of chloroplast gene expression but also for a genetic approach to the molecular oscillator of the algal circadian clock.

Animals↗
PubMed2005

Functionally important substructures of circadian clock protein KaiB in a unique tetramer complex.

KaiB is a component of the circadian clock molecular machinery in cyanobacteria, which are the simplest organisms that exhibit circadian rhythms. Here we report the x-ray crystal structure of KaiB from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. The KaiB crystal diffracts at a resolution of 2.6 A and includes four subunits organized as a dimer of dimers, each composed of two non-equivalent subunits. The overall shape of the tetramer is an elongated hexagonal plate, with a single positively charged cleft flanked by two negatively charged ridges whose surfaces includes several terminal chains. Site-directed mutagenesis of Synechococcus KaiB confirmed that alanine substitution of residues Lys-11 or Lys-43 in the cleft, or deletion of C-terminal residues 95-108, which forms part of the ridges, strongly weakens in vivo circadian rhythms. Characteristics of KaiB deduced from the x-ray crystal structure were also confirmed by physicochemical measurements of KaiB in solution. These data suggest that the positively charged cleft and flanking negatively charged ridges in KaiB are essential for the biological function of KaiB in the circadian molecular machinery in cyanobacteria.

Amino Acid Sequence↗
PubMed2005

RAP, an integrated program for monitoring bioluminescence and analyzing circadian rhythms in real time.

To process the large number of circadian rhythmic bioluminescence data that we generated with specially developed, high-throughput monitoring apparatuses, we developed an integrated rhythm-analyzing program (RAP) with a user-friendly graphical interface. RAP does all of the following in real time: (i) displays the bioluminescence time course, (ii) records time-series data, (iii) analyzes the data, (iv) displays the analyzed results, (v) statistically evaluates the analyzed results, and (vi) provides printouts. Because RAP can import files, it can analyze not only bioluminescence data but also other data such as those obtained by DNA array experiments and by Northern and Western blot analyses. The program is a powerful tool for large-scale investigations of biological rhythmic phenomena in general.

Arabidopsis↗
PubMed2005

An automated apparatus for the real-time monitoring of bioluminescence in plants.

We developed an automated, high-throughput, bioluminescence-monitoring apparatus that can monitor 1920 individual plant seedlings under uniform light conditions. The apparatus is composed of five units: (i) a plate platform that can hold 20 96-well microplates under uniform light conditions, (ii) a scintillation counter, (iii) a robot that conveys plates between the plate platform and a scintillation counter, (iv) a sequence controller, and (v) an external computer that collects and analyzes bioluminescence data automatically. The apparatus gave reproducible and reliable results for both bioluminescence photon counts and period length of bioluminescence rhythms; neither was affected by the well position in a plate or the plate position on the platform. The apparatus is a powerful tool for both large-scale detailed analysis of gene expression and large-scale screening of mutants.

Arabidopsis↗
PubMed2005

Identification of novel clock-controlled genes by cDNA macroarray analysis in Chlamydomonas reinhardtii.

Circadian rhythms are self-sustaining oscillations whose period length under constant conditions is about 24 h. Circadian rhythms are widespread and involve functions as diverse as human sleep-wake cycles and cyanobacterial nitrogen fixation. In spite of a long research history, knowledge about clock-controlled genes is limited in Chlamydomonas reinhardtii. Using a cDNA macroarray containing 10 368 nuclear-encoded genes, we examined global circadian regulation of transcription in Chlamydomonas. We identified 269 candidates for circadianly expressed gene. Northern blot analysis confirmed reproducible and sustainable rhythmicity for 12 genes. Most genes exhibited peak expression at the transition point between day and night. One hundred and eighteen genes were assigned predicted annotations. The functions of the cycling genes were diverse and included photosynthesis, respiration, cellular structure, and various metabolic pathways. Surprisingly, 18 genes encoding chloroplast ribosomal proteins showed a coordinated circadian pattern of expression and peaked just at the beginning of subjective day. The co-regulation of genes bearing a similar function was also observed in genes involved in cellular structure. They peaked at the end of the subjective night, which is when the regeneration of cell walls and flagella in daughter cells occurs. Expression of the chlamyopsin gene, which encodes an opsin-type photoreceptor, also exhibited circadian rhythm.

Animals↗
PubMed2005

PHYTOCLOCK 1 encoding a novel GARP protein essential for the Arabidopsis circadian clock.

Previously, we screened 50 000 seedlings of Arabidopsis thaliana carrying a P(GI)::LUC+ bioluminescence reporter gene mutagenized with ethylmethanesulfonate for mutants with phenotypes of extensively altered circadian rhythms, and identified three loci, PHYTOCLOCK 1 (PCL1), PCL2 and PCL3, whose mutations cause arrhythmia. Here we succeeded to clone the PCL1 gene and show that the PCL1 gene encodes a novel DNA binding protein belonging to the GARP protein family and is essential for a functional clock oscillator in A. thaliana. The PCL1 gene satisfies the requirements for the clock oscillator gene: (i) pcl1 null mutations caused arrhythmia in multiple circadian outputs, including expression of potential clock genes TOC1, CCA1 and LHY, and flowering lacked a photoperiodic response; (ii) PCL1 expression showed circadian rhythm in both constant light and constant dark; (iii) over-expression of the PCL1 gene gradually caused arrhythmicity in all the multiple circadian outputs examined; and (iv) the PCL1 gene controlled its own expression via negative feedback. Therefore, the PCL1 gene is the clock oscillator gene essential to the generation of clock oscillation in the higher plant.

Amino Acid Sequence↗
PubMed2005

Transcriptional regulation of the circadian clock operon kaiBC by upstream regions in cyanobacteria.

In the cyanobacterium, Synechococcus elongatus PCC 7942, the kaiBC operon is upregulated by the KaiA protein and downregulated by the KaiC protein to generate circadian oscillation. We investigated the regulation of kaiBC transcription. A primer extension and deletion analyses of the upstream region mapped the sufficient promoter region (SPR) to base pairs -55 to +1 (the transcription start site, TSS) and identified a constitutive negative regulatory region upstream of the SPR (base pairs -897 to -56) that extended into the coding sequence of kaiA. Base-pair substitution within the SPR identified a sequence from -52 to -28 that was the essential element for transcription. Most of the examined sequences drove rhythmic expression of a luxAB reporter that was similar to the expression driven by the kaiBC promoter (PkaiBC) and responded to the overexpression of kaiA or kaiC, even in a promoter activity range of 1-8000%. These results indicate that circadian feedback regulation by KaiA and KaiC is addressed to a global step preceding transcription driven by PkaiBC. However, increasing or decreasing the intrinsic activity of PkaiBC greatly affected the rhythm, suggesting that constitutive adjustment of PkaiBC activity by the sequences identified here is essential for the oscillator.

Bacterial Proteins↗
PubMed2005

Global analysis of circadian expression in the cyanobacterium Synechocystis sp. strain PCC 6803.

Cyanobacteria are the only bacterial species found to have a circadian clock. We used DNA microarrays to examine circadian expression patterns in the cyanobacterium Synechocystis sp. strain PCC 6803. Our analysis identified 54 (2%) and 237 (9%) genes that exhibited circadian rhythms under stringent and relaxed filtering conditions, respectively. The expression of most cycling genes peaked around the time of transition from subjective day to night, suggesting that the main role of the circadian clock in Synechocystis is to adjust the physiological state of the cell to the upcoming night environment. There were several chromosomal regions where neighboring genes were expressed with similar circadian patterns. The physiological functions of the cycling genes were diverse and included a wide variety of metabolic pathways, membrane transport, and signal transduction. Genes involved in respiration and poly(3-hydroxyalkanoate) synthesis showed coordinated circadian expression, suggesting that the regulation is important for the supply of energy and carbon source in the night. Genes involved in transcription and translation also followed circadian cycling patterns. These genes may be important for output of the rhythmic information generated by the circadian clock. Our findings provided critical insights into the importance of the circadian clock on cellular physiology and the mechanism of clock-controlled gene regulation.

3-Hydroxybutyric Acid↗
PubMed2005

Improvement of the bioluminescence reporter system for real-time monitoring of circadian rhythms in the cyanobacterium Synechocystis sp. strain PCC 6803.

Circadian rhythm is a self-sustaining oscillation whose period length coincides with the 24-hour day-night cycle. A powerful tool for circadian clock research is the real-time automated bioluminescence monitoring system in which a promoter region of a clock-controlled gene is fused to a luciferase reporter gene and rhythmic regulation of the promoter activity is monitored as bioluminescence. In the present study, we greatly improved the bioluminescence reporter system in the cyanobacterium Synechocystis sp. strain PCC 6803. We fused an 805-bp promoter region of the dnaK gene seamlessly to the luxA coding sequence and integrated the P(dnaK)::luxAB fusion gene into a specific intergenic region of the Synechocystis genome (targeting site 1). The resulting new reporter strain, PdnaK::luxAB(-), showed 12 times the bioluminescence intensity of the standard reporter strain, CFC2. Furthermore, we generated strain PdnaK::luxAB(+), in which the P(dnaK)::luxAB fusion gene and the selection-marker spectinomycin resistance gene are transcribed in opposite directions. The PdnaK::luxAB(+) strain showed 19 times the bioluminescence intensity of strain CFC2. The procedures used to increase the bioluminescence intensity are especially useful for bioluminescence monitoring of genes with low promoter activity. In addition, these reporter constructs facilitate bioluminescence monitoring of any gene because the promoter fragments they contain can easily be replaced by digestion with unique restriction enzymes. They would therefore contribute to a genome-wide analysis of gene expression in Synechocystis.

Circadian Rhythm↗
PubMed2004

Roles of two ATPase-motif-containing domains in cyanobacterial circadian clock protein KaiC.

Cyanobacterial clock protein KaiC has a hexagonal, pot-shaped structure composed of six identical dumbbell-shaped subunits. Each subunit has duplicated domains, and each domain has a set of ATPase motifs. The two spherical regions of the dumbbell are likely to correspond to two domains. We examined the role of the two sets of ATPase motifs by analyzing the in vitro activity of ATPgammaS binding, AMPPNP-induced hexamerization, thermostability, and phosphorylation of KaiC and by in vivo rhythm assays both in wild type KaiC (KaiCWT) and KaiCs carrying mutations in either Walker motif A or deduced catalytic Glu residues. We demonstrated that 1) the KaiC subunit had two types of ATP-binding sites, a high affinity site in N-terminal ATPase motifs and a low affinity site in C-terminal ATPase motifs, 2) the N-terminal motifs were responsible for hexamerization, and 3) the C-terminal motifs were responsible for both stabilization and phosphorylation of the KaiC hexamer. We proposed the following reaction mechanism. ATP preferentially binds to the N-terminal high affinity site, inducing the hexamerization of KaiC. Additional ATP then binds to the C-terminal low affinity site, stabilizing and phosphorylating the hexamer. We discussed the effect of these KaiC mutations on circadian bioluminescence rhythm in cells of cyanobacteria.

Adenosine Triphosphatases↗
PubMed2004

Crystal structure of the C-terminal clock-oscillator domain of the cyanobacterial KaiA protein.

KaiA, KaiB and KaiC constitute the circadian clock machinery in cyanobacteria, and KaiA activates kaiBC expression whereas KaiC represses it. Here we show that KaiA is composed of three functional domains, the N-terminal amplitude-amplifier domain, the central period-adjuster domain and the C-terminal clock-oscillator domain. The C-terminal domain is responsible for dimer formation, binding to KaiC, enhancing KaiC phosphorylation and generating the circadian oscillations. The X-ray crystal structure at a resolution of 1.8 A of the C-terminal clock-oscillator domain of KaiA from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 shows that residue His270, located at the center of a KaiA dimer concavity, is essential to KaiA function. KaiA binding to KaiC probably occurs via the concave surface. On the basis of the structure, we predict the structural roles of the residues that affect circadian oscillations.

Amino Acid Sequence↗
PubMed2004

Stoichiometric interactions between cyanobacterial clock proteins KaiA and KaiC.

We determined the stoichiometry of KaiA-KaiC interactions. Using immunoblotting and two-dimensional Native- and SDS-PAGE (2DNS-PAGE) analysis, we demonstrated that the reaction products of KaiA-KaiC interactions in the presence of ATP consisted of only phosphorylated KaiC whereas in the presence of the unhydrolyzable analogue 5'-adenylylimidodiphosphate (AMPPNP) they consisted of KaiA and KaiC. In the presence of ATP, the KE (molar ratio of KaiA dimer to KaiC hexamer giving half saturation in the enhancement of KaiC phosphorylation) was 0.25, and IAsys affinity biosensor analysis demonstrated that 1 molecule of KaiA dimer interacted with 1 molecule of KaiC hexamer. In the presence of AMPPNP, the ratio of KaiA dimer to KaiC hexamer in KaiA-KaiC complexes was determined to be 2 by 2DNS-PAGE, Native-PAGE/Scatchard plot, and IAsys analyses. These results suggest that 2 molecules of KaiA dimer can interact with 1 molecule of KaiC hexamer, and that interactions of at least 1 molecule of KaiA dimer with 1 molecule of KaiC hexamer are enough to enhance the phosphorylation of KaiC by KaiA at an almost saturated level.

Adenylyl Imidodiphosphate↗
PubMed2004

Crystallization and preliminary crystallographic analysis of the circadian clock protein KaiB from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1.

KaiB is a component of the circadian clock oscillator in cyanobacteria, which are the simplest organisms that exhibit circadian rhythms. KaiB consists of 108 amino-acid residues and has a molecular weight of 12 025 Da. KaiB and Cys-substituted KaiB mutants from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 were expressed as GST-fusion proteins in Escherichia coli, purified and crystallized. The crystals of wild-type KaiB belong to the monoclinic space group P2(1), with unit-cell parameters a = 89.6, b = 71.2, c = 106.8 A, beta = 100.1 degrees. While the native crystals diffract to 3.7 A, osmium derivatives, which show an approximately 4 A shrinkage in the b axis, diffract to 2.6 A. The crystals of the singly Cys-substituted mutant T64C with Hg, which show different morphology, diffract to 2.5 A and belong to the monoclinic space group P2, with unit-cell parameters a = 63.7, b = 33.4, c = 93.7 A, beta = 100.1 degrees. Anomalous difference Patterson maps of the Os- and Hg-derivative crystals had significant peaks in their Harker sections, suggesting that both derivatives are suitable for structure determination.

Amino Acid Substitution↗
PubMed2004

Large-scale screening of Arabidopsis circadian clock mutants by a high-throughput real-time bioluminescence monitoring system.

Using a high-throughput real-time bioluminescence monitoring system, we screened large numbers of Arabidopsis thaliana mutants for extensively altered circadian rhythms. We constructed reporter genes by fusing a promoter of an Arabidopsis flowering-time gene - either GIGANTEA (GI) or FLOWERING LOCUS T (FT) - to a modified firefly luciferase gene (LUC(+)), and we transferred the fusion gene (P(GI)::LUC(+) or P(FT)::LUC(+)) into the Arabidopsis genome. After mutagenesis with ethyl methanesulfonate, 50 000 M(2) seedlings carrying the P(GI)::LUC(+) and 50 000 carrying P(FT)::LUC(+) were screened their bioluminescence rhythms. We isolated six arrhythmic (AR) mutants and 29 other mutants that showed more than 3 h difference in the period length or phase of rhythms compared with the wild-type strains. The shortest period length was 16 h, the longest 27 h. Five of the six AR mutants carrying P(GI)::LUC(+) showed arrhythmia in bioluminescence rhythms in both constant light and constant dark. These five AR mutants also showed arrhythmia in leaf movement rhythms in constant light. Genetic analysis revealed that each of the five AR mutants carried a recessive mutation in a nuclear gene and the mutations belonged to three complementation groups, and at least one of which was mapped on a novel locus. Our results suggest that the three loci identified here may contain central clock or clock-related genes, at least one of which may be a novel.

Arabidopsis↗
PubMed2004

Circadian rhythms in the thermophilic cyanobacterium Thermosynechococcus elongatus: compensation of period length over a wide temperature range.

Proteins derived from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1, which performs plant-type oxygenic photosynthesis, are suitable for biochemical, biophysical, and X-ray crystallographic studies. We developed an automated bioluminescence real-time monitoring system for the circadian clock in the thermophilic cyanobacterium T. elongatus BP-1 that uses a bacterial luciferase gene set (Xl luxAB) derived from Xenorhabdus luminescens as a bioluminescence reporter gene. A promoter region of the psbA1 gene of T. elongatus was fused to the Xl luxAB gene set and inserted into a specific targeting site in the genome of T. elongatus. The bioluminescence from the cells of the psbA1-reporting strain was measured by an automated monitoring apparatus with photomultiplier tubes. The strain exhibited the circadian rhythms of bioluminescence with a 25-h period length for at least 10 days in constant light and temperature. The rhythms were reset by light-dark cycle, and their period length was almost constant over a wide range of temperatures (30 to 60 degrees C). Theses results indicate that T. elongatus has the circadian clock that is widely temperature compensated.

Bacteriological Techniques↗
PubMed2004

Determinants of sensitivity and specificity in spotted DNA microarrays with unmodified oligonucleotides.

DNA microarrays with unmodified oligonucleotides are a cost-effective alternative to cDNA microarrays. This study examined how purity, length, homology and GC content of the oligonucleotide probes influence the sensitivity and specificity of the method using cyanobacterial genes. Oligonucleotide purification by high pressure liquid chromatography was omitted without significant reduction in hybridization sensitivity. For two of three genes tested, a reduction in oligonucleotide length did not reduce hybridization sensitivity, and maximum sensitivity was achieved with probes that were 45 nt long. Oligonucleotide probes with <or=71% contiguous sequence identity to non-target DNA cross-hybridized with the sequences at a rate of < 8%. Cross-hybridization decreased as the GC content decreased from 65% to 55% or 35%. These results support the following criteria for selecting unmodified oligonucleotide probes that generate sensitive and specific microarrays: length of 45 nt, <or=71% identity to non-target sequences, and <or=55% GC content. In most of the bacterial species tested, oligonucleotide probes meeting these criteria were successfully designed for more than 95% of genes.

Base Sequence↗