Melanopsin DNA aptamers can regulate input signals of mammalian circadian rhythms by altering the phase of the molecular clock.

DNA aptamers can bind specifically to biomolecules to modify their function, potentially making them ideal oligonucleotide therapeutics. Herein, we screened for DNA aptamer of melanopsin (OPN4), a blue-light photopigment in the retina, which plays a key role using light signals to reset the phase of circadian rhythms in the central clock. Firstly, 15 DNA aptamers of melanopsin (Melapts) were identified following eight rounds of Cell-SELEX using cells expressing melanopsin on the cell membrane. Subsequent functional analysis of each Melapt was performed in a fibroblast cell line stably expressing both Period2:ELuc and melanopsin by determining the degree to which they reset the phase of mammalian circadian rhythms in response to blue-light stimulation. Period2 rhythmic expression over a 24-h period was monitored in Period2:ELuc stable cell line fibroblasts expressing melanopsin. At subjective dawn, four Melapts were observed to advance phase by >1.5 h, while seven Melapts delayed phase by >2 h. Some Melapts caused a phase shift of approximately 2 h, even in the absence of photostimulation, presumably because Melapts can only partially affect input signaling for phase shift. Additionally, some Melaps were able to induce phase shifts in Per1::luc transgenic (Tg) mice, suggesting that these DNA aptamers may have the capacity to affect melanopsin in vivo. In summary, Melapts can successfully regulate the input signal and shifting phase (both phase advance and phase delay) of mammalian circadian rhythms in vitro and in vivo.

Distribution of Phototrophic Purple Nonsulfur Bacteria in Massive Blooms in Coastal and Wastewater Ditch Environments.

The biodiversity of phototrophic purple nonsulfur bacteria (PNSB) in comparison with purple sulfur bacteria (PSB) in colored blooms and microbial mats that developed in coastal mudflats and pools and wastewater ditches was investigated. For this, a combination of photopigment and quinone profiling, pufM gene-targeted quantitative PCR, and pufM gene clone library analysis was used in addition to conventional microscopic and cultivation methods. Red and pink blooms in the coastal environments contained PSB as the major populations, and smaller but significant densities of PNSB, with members of Rhodovulum predominating. On the other hand, red-pink blooms and mats in the wastewater ditches exclusively yielded PNSB, with Rhodobacter, Rhodopseudomonas, and/or Pararhodospirillum as the major constituents. The important environmental factors affecting PNSB populations were organic matter and sulfide concentrations and oxidation‒reduction potential (ORP). Namely, light-exposed, sulfide-deficient water bodies with high-strength organic matter and in a limited range of ORP provide favorable conditions for the massive growth of PNSB over co-existing PSB. We also report high-quality genome sequences of Rhodovulum sp. strain MB263, previously isolated from a pink mudflat, and Rhodovulum sulfidophilum DSM 1374T, which would enhance our understanding of how PNSB respond to various environmental factors in the natural ecosystem.

A strong structural correlation between short inverted repeat sequences and the polyadenylation signal in yeast and nucleosome exclusion by these inverted repeats.

DNA sequences that read the same from 5' to 3' in either strand are called inverted repeat sequences or simply IRs. They are found throughout a wide variety of genomes, from prokaryotes to eukaryotes. Despite extensive research, their in vivo functions, if any, remain unclear. Using Saccharomyces cerevisiae, we performed genome-wide analyses for the distribution, occurrence frequency, sequence characteristics and relevance to chromatin structure, for the IRs that reportedly have a cruciform-forming potential. Here, we provide the first comprehensive map of these IRs in the S. cerevisiae genome. The statistically significant enrichment of the IRs was found in the close vicinity of the DNA positions corresponding to polyadenylation [poly(A)] sites and ~ 30 to ~ 60 bp downstream of start codon-coding sites (referred to as 'start codons'). In the former, ApT- or TpA-rich IRs and A-tract- or T-tract-rich IRs are enriched, while in the latter, different IRs are enriched. Furthermore, we found a strong structural correlation between the former IRs and the poly(A) signal. In the chromatin formed on the gene end regions, the majority of the IRs causes low nucleosome occupancy. The IRs in the region ~ 30 to ~ 60 bp downstream of start codons are located in the + 1 nucleosomes. In contrast, fewer IRs are present in the adjacent region downstream of start codons. The current study suggests that the IRs play similar roles in Escherichia coli and S. cerevisiae to regulate or complete transcription at the RNA level.

Identification of novel long chain N-acylhomoserine lactones of chain length C20 from the marine phototrophic bacterium Rhodovulum sulfidophilum.

Gram-negative bacterial quorum sensing is mainly regulated by an extracellularly produced N-acylhomoserine lactone (AHL). AHL consists of a lactone ring and an acyl chain, which generally varies from C4 to C18 in length and affords species-specific variety. In this study, we developed an ultra-high performance liquid chromatography tandem mass spectrometry system and detected two kinds of long chain AHLs with chain length C20 from the reverse-phase thin layer chromatography-fractionated cultured supernatant of the marine photosynthetic bacterium Rhodovulum sulfidophilum. By fragmentation search analysis to detect compounds with a homoserine lactone ring moiety for data dependent acquisition, a minor AHL, presumed to be 3-OH-C18-homoserine lactone (HSL), was also found. Among the detected C20-HSLs, 3-OH-C20-HSL was structurally identified and 3-OH-C20:1-HSL was strongly suggested. To our knowledge, this is the first report to show a novel AHL with the longest C20 acyl side chain found to date. ABBREVIATIONS: AGC: automatic gain control; AHL: N-acylhomoserine lactone; CD: cyclodextrin; CID: collision induced dissociation; DDA: data dependent acquisition; EPI: enhanced product ion; FISh: fragment ion search; HCD: high energy collisional dissociation; HSL: homoserine lactone; IT: injection time; LC: liquid chromatography; MS: mass spectrometry; PRM: parallel reaction monitoring; RP: reverse phase; SRM: selected reaction monitoring; TLC: thin layer chromatography; UHPLC: ultra high performance liquid chromatography.

Genetic dissection of the signaling pathway required for the cell wall integrity checkpoint.

The cell wall integrity checkpoint monitors synthesis of cell wall materials during the Saccharomyces cerevisiae cell cycle. Upon perturbation of cell wall synthesis, the cell wall integrity checkpoint is activated, downregulating Clb2 transcription. Here, we identified genes involved in this checkpoint by genetic screening of deletion mutants. In addition to the previously identified dynactin complex, the Las17 complex, in particular the Bzz1 and Vrp1 components, plays a role in this checkpoint. We also revealed that the high osmolarity glycerol (HOG) and cell wall integrity mitogen-activated protein kinase (MAPK) signaling pathways are essential for checkpoint function. The defective checkpoint caused by the deficient dynactin and Las17 complexes was rescued by hyperactivation of the cell wall integrity MAPK pathway, but not by the activated form of Hog1, suggesting an order to these signaling pathways. Mutation of Fkh2, a transcription factor important for Clb2 expression, suppressed the checkpoint-defective phenotype of Las17, HOG MAPK and cell wall integrity MAPK mutations. These results provide genetic evidence that signaling from the cell surface regulates the downstream transcriptional machinery to activate the cell wall integrity checkpoint.

Involvement of the response regulator CtrA in the extracellular DNA production of the marine bacterium Rhodovulum sulfidophilum.

The marine bacterium Rhodovulum sulfidophilum is a nonsulfur phototrophic bacterium, which is known to produce extracellular nucleic acids in soluble form in culture medium. In the present paper, constructing the response regulator ctrA-deficient mutant of R. sulfidophilum, we found that this mutation causes a significant decrease in the extracellular DNA production. However, by the introduction of a plasmid containing the wild type ctrA gene into the mutant, the amount of extracellular DNA produced was recovered. This is the first and clear evidence that the extracellular DNA production is actively controlled by the CtrA in R. sulfidophilum.

Extracellular nucleic acids of the marine bacterium Rhodovulum sulfidophilum and recombinant RNA production technology using bacteria.

Extracellular nucleic acids of high molecular weight are detected ubiquitously in seawater. Recent studies have indicated that these nucleic acids are, at least in part, derived from active production by some bacteria. The marine bacterium Rhodovulum sulfidophilum is one of those bacteria. Rhodovulumsulfidophilum is a non-sulfur phototrophic marine bacterium that is known to form structured communities of cells called flocs, and to produce extracellular nucleic acids in culture media. Recently, it has been revealed that this bacterium produces gene transfer agent-like particles and that this particle production may be related to the extracellular nucleic acid production mechanism. This review provides a summary of recent physiological and genetic studies of these phenomena and also introduces a new method for extracellular production of artificial and biologically functional RNAs using this bacterium. In addition, artificial RNA production using Escherichia coli, which is related to this topic, will also be described.

Magnesium chloride and polyamine can differentiate mouse embryonic stem cells into trophectoderm or endoderm.

Magnesium chloride and polyamines stabilize DNA and chromatin. Furthermore, they can induce nucleosome aggregation and chromatin condensation in vitro. To determine the effects of elevating the cation concentrations in the nucleus of a living cell, we microinjected various concentrations of mono-, di- and polyvalent cation solutions into the nuclei of mouse embryonic stem (ES) cells and traced their fates. Here, we show that an elevation of either MgCl2, spermidine or spermine concentration in the nucleus exerts a significant effect on mouse ES cells, and can differentiate a certain population of the cells into trophectoderm, a lineage that mouse ES cells do not normally generate, or endoderm. It is hypothesized that the cell differentiation was most probably caused by the condensation of chromatin including the Oct3/4 locus, which was induced by the elevated concentrations of these cations.

Functional analyses of carnivorous plant-specific amino acid residues in S-like ribonucleases.

Unlike plants with no carnivory, carnivorous plants seem to use S-like ribonucleases (RNases) as an enzyme for carnivory. Carnivorous plant-specific conserved amino acid residues are present at four positions around the conserved active site (CAS). The roles of these conserved amino acid residues in the enzymatic function were explored in the current study by preparing five recombinant variants of DA-I, the S-like RNase of Drosera adelae. The kcat and kcat/Km values of the enzymes revealed that among the four variants with a single mutation, the serine to glycine mutation at position 111 most negatively influenced the enzymatic activity. The change in the bulkiness of the amino acid residue side-chain seemed to be the major cause of the above effect. Modeling of the three dimensional (3D) structures strongly suggested that the S to G mutation at 111 greatly altered the overall enzyme conformation. The conserved four amino acid residues are likely to function in keeping the two histidine residues, which are essential for the cleavage of RNA strands, and the CAS in the most functional enzymatic conformation.

Complete Genome Sequence of Cyanobacterium Geminocystis sp. Strain NIES-3708, Which Performs Type II Complementary Chromatic Acclimation.

To explore the variation of the light-regulated genes during complementary chromatic acclimation (CCA), we determined the complete genome sequence of the cyanobacterium Geminocystis sp. strain NIES-3708. Within the light-regulated operon for CCA, we found genes for phycoerythrin but not phycocyanin, suggesting that this cyanobacterium modulates phycoerythrin composition only (type II CCA).

Complete Genome Sequence of Cyanobacterium Geminocystis sp. Strain NIES-3709, Which Harbors a Phycoerythrin-Rich Phycobilisome.

The cyanobacterium Geminocystis sp. strain NIES-3709 accumulates a larger amount of phycoerythrin than the related NIES-3708 strain does. Here, we determined the complete genome sequence of the NIES-3709 strain. Our genome data suggest that the different copy number of rod linker genes for phycoerythrin leads to the different phycoerythrin contents between the two strains.

Complete Genome Sequence of Rhodovulum sulfidophilum DSM 2351, an Extracellular Nucleic Acid-Producing Bacterium.

Rhodovulum sulfidophilum DSM 2351 is the nonsulfur photosynthetic bacterium that efficiently releases nucleic acids into the extracellular milieu, which leads to flocculation. In this study, we determined the complete genome sequence of R. sulfidophilum DSM 2351, which will provide new insights into the mechanism of its unique nucleic acid release.

The gene transfer agent-like particle of the marine phototrophic bacterium Rhodovulum sulfidophilum.

Gene transfer agents (GTAs) are shaped like bacteriophage particles but have many properties that distinguish them from bacteriophages. GTAs play a role in horizontal gene transfer in nature and thus affect the evolution of prokaryotic genomes. In the course of studies on the extracellular production of designed RNAs using the marine bacterium Rhodovulum sulfidophilum, we found that this bacterium produces a GTA-like particle. The particle contains DNA fragments of 4.5 kb, which consist of randomly fragmented genomic DNA from the bacterium. This 4.5-kb DNA production was prevented while quorum sensing was inhibited. Direct observation of the particle by transmission electron microscopy revealed that the particle resembles a tailed phage and has a head diameter of about 40 nm and a tail length of about 60 nm. We also identified the structural genes for the GTA in the genome. Translated amino acid sequences and gene positions are closely related to those of the genes that encode the Rhodobacter capsulatus GTA. This is the first report of a GTA-like particle from the genus Rhodovulum. However, gene transfer activity of this particle has not yet been confirmed. The differences between this particle and other GTAs are discussed.

Short hairpin RNAs of designed sequences can be extracellularly produced by the marine bacterium Rhodovulum sulfidophilum.

Previously, we proposed a new method for production of RNA aptamers using the marine bacterium Rhodovulum sulfidophilum. A streptavidin RNA aptamer (an RNA which binds to streptavidin) was extracellularly produced by this bacterium containing engineered plasmid. The aptamer had full biological function. As a next step we attempted to produce another functional RNA, short hairpin RNAs (shRNAs) using this bacterial system. We have designed two types of shRNAs targeted to the luciferase gene. Here we report that shRNAs are successfully produced extracellularly by this system. Even if the shRNA has a long stem-loop structure which is thought to interfere with transcription in bacterial cells, the yield of the shRNA is almost the same as that of the streptavidin RNA aptamer. During the course of these experiments, we also found a new type of RNA processing for the double-stranded region of the shRNA.

RNA aptamers inhibit the growth of the fish pathogen viral hemorrhagic septicemia virus (VHSV).

Viral hemorrhagic septicemia virus (VHSV) is a serious disease impacting wild and cultured fish worldwide. Hence, an effective therapeutic method against VHSV infection needs to be developed. Aptamer technology is a new and promising method for diagnostics and therapeutics. It revolves around the use of an aptamer molecule, an artificial ligand (nucleic acid or protein), which has the capacity to recognize target molecules with high affinity and specificity. Here, we aimed at selecting RNA aptamers that can specifically bind to and inhibit the growth of a strain of fish VHSV both in vitro and in vivo. Three VHSV-specific RNA aptamers (F1, F2, and C6) were selected from a pool of artificially and randomly produced oligonucleotides using systematic evolution of ligands by exponential enrichment. The three RNA aptamers showed obvious binding to VHSV in an electrophoretic mobility shift assay but not to other tested viruses. The RNA aptamers were tested for their ability to inhibit VHSV in vitro using hirame natural embryo (HINAE) cells. Cytopathic effect and plaque assays showed that all aptamers inhibited the growth of VHSV in HINAE cells. In vivo tests using RNA aptamers produced by Rhodovulum sulfidophilum showed that extracellular RNA aptamers inhibited VHSV infection in Japanese flounder. These results suggest that the RNA aptamers are a useful tool for protection against VHSV infection in Japanese flounder.

Artificial RNA aptamer production by the marine bacterium Rhodovulum sulfidophilum: improvement of the aptamer yield using a mutated transcriptional promoter.

Noncoding small RNAs and artificial RNA aptamers are now expected to be potential candidates for RNA therapeutic agents. We previously proposed a unique method for economical production of these RNAs using the marine phototrophic bacterium Rhodovulum sulfidophilum. This bacterium does not produce any ribonucleases but does produce extracellular nucleic acids in the culture medium in nature. Using this bacterium and an engineered plasmid containing the rrn promoter for the RNA expression, we developed a method for production of the streptavidin RNA aptamer in the culture medium. However, the yield of this RNA product in the culture medium by this method was not enough for practical use. In the present paper, we improved the yield of this product by modification of the -35 region of the rrn promoter so as to escape from the Fis protein control and the use of a new vector plasmid. Using this system, the extracellular RNA aptamer of approximately 200 ng and the total RNA aptamer (both extra- and intracellular form) of about 20 µg from 1 L culture were accomplished by constitutive expression of the gene.

Knr4 N-terminal domain controls its localization and function during sexual differentiation and vegetative growth.

The Saccharomyces cerevisiae protein Knr4 is composed of a globular central core flanked by two natively disordered regions. Although the central part of the protein holds most of its biological function, the N-terminal domain (amino acids 1-80) is essential in the absence of a functional CWI pathway. We show that this specific protein domain is required for the proper cellular localization of Knr4 at sites of polarized growth during vegetative growth and sexual differentiation (bud tip and 'shmoo' tip). Moreover, Knr4 N-terminal domain is also necessary for cell cycle arrest and shmoo formation in response to pheromone to occur at the correct speed. Thus, the presence of Knr4 at the incipient mating projection site seems important for the establishment of the following polarized growth. Cell wall integrity (CWI) and calcineurin pathways are known to share a common essential function, for which they can substitute for one another. Searching for Knr4 partners responsible for survival in a CWI-defective background, we found that the catalytic subunit of calcineurin Cna1 physically interacts with Knr4 in the yeast two-hybrid assay, in a manner dependent on the presence of the Knr4 N-terminal domain. In addition, we present evidence that Knr4 protein participates in the morphogenesis checkpoint, a safety mechanism that holds the cell cycle in response to bud formation defects or insults in cytoskeleton organization, and in which both the CWI pathway and calcineurin are involved.

Extracellular production of an RNA aptamer by ribonuclease-free marine bacteria harboring engineered plasmids: a proposal for industrial RNA drug production.

Natural noncoding small RNAs have been shown to be involved in a number of cellular processes as regulators. Using the mechanisms thus elucidated, artificial small interfering RNAs (siRNAs), ribozymes, and RNA aptamers are also expected to be potential candidates for RNA therapeutic agents. However, current techniques are too costly for industrial production of these RNAs for use as drugs. Here, we propose a new method for in vivo production of artificial RNAs using the marine phototrophic bacterium Rhodovulum sulfidophilum. Using engineered plasmids and this bacterium, which produces extracellular nucleic acids in nature, we developed a method for extracellular production of a streptavidin RNA aptamer. As the bacterium does not produce any RNases in the culture medium, at least within the cultivation period tested, the designed RNA itself is produced and retained in the culture medium of the bacterium without any specific mechanism for protection against degradation by nucleases. Here, we report that the streptavidin RNA aptamer is produced in the culture medium and retains its specific function. This is the first demonstration of extracellular production of a functional artificial RNA in vivo, which will pave the way for inexpensive production of RNA drugs.

In vivo circular RNA production using a constitutive promoter for high-level expression.

The permuted intron-exon (PIE) method based on group I intron self-splicing is the only methodology currently available for production of circular RNA in vivo. Here, we report improvement of the circular RNA expression method based on an induction-free vector system utilizing the highly efficient constitutive lpp promoter.

Substrate recognition of pre-tRNA by ribonuclease P---subsite model of natural ribozyme originated from Escherichia coli.

Combination of cleavage site analysis and kinetic analysis of a series of shape variant of pre-tRNA substrate, we newly found two subsites which contribute to recognition of shape in substrate binding and catalysis by bacterial RNase P. Our data showed that the ribozyme traps the substrate by 5'- and 3'-end regions to form Michaelis complex, and after that the shape of the substrate is examined by other two subsites in the transition state. In the meeting, we will show the possibility that the S-domain can contribute to stabilize the transition state of the cleavage reaction of a pre-tRNA substrate.