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.     

Near-full-length REV3L appears to be a scarce maternal factor in Xenopus laevis eggs that changes qualitatively in early embryonic development

REV3 is the catalytic subunit of DNA polymerase ζ (pol ζ), which is responsible for the damage-induced mutagenesis that arises during error-prone translesion synthesis in eukaryotes. The related REV3L genes in human and mouse encode proteins of approximately 350 kDa, twice as large as yeast REV3, but full-length REV3L has not been identified in any vertebrate cell. We report that Xenopus laevis REV3L encodes a 352-kDa protein that has high overall amino acid sequence similarity to its mammalian counterparts, and, for the first time in a vertebrate species, we have detected putative REV3L polypeptides of 300 and 340 kDa in X. laevis oocytes. Only the 300-kDa form is stored in eggs, where its concentration of about 65 pM is much lower than those of other replication and repair proteins including the accessory pol ζ subunit REV7. In fertilized eggs, the levels of this polypeptide did not change until neurula; the larger 340-kDa form first appeared at stages after gastrula, suggesting a pattern of regulation during development. These observations indicate the existence of REV3L as a scarce protein, of approximately the full predicted size, whose level may impose severe constraints on the assembly of pol ζ in X. laevis.     

A Non-Native α-Helix Is Formed in the β-Sheet Region of the Molten Globule State of Canine Milk Lysozyme

The native and the molten globule states (N and MG states, respectively) of canine milk lysozyme (CML) were examined by CD spectroscopy and AGADIR algorithm, a helix-coil transition program. It revealed that the helical content of the MG state was higher than that of the N-state, suggesting that non-native alpha-helix is formed in the MG state of CML. Using AGADIR, it indicated the possibility of alpha-helix formation in the third beta-strand region in the MG state. To investigate this possibility, we designed a mutant, Q58P, in which the helical propensity of the MG state was significantly decreased around the third beta-strand region. It appeared that the absolute ellipticity value at 222 nm of the mutant in the MG state was smaller than that of the wild-type protein. It could be assumed that the non-native alpha-helix is formed around the third beta-strand region of wild-type CML in the MG state.     

Human DDX6 effects miRNA-mediated gene silencing via direct binding to CNOT1

MicroRNAs (miRNAs) play critical roles in a variety of biological processes through widespread effects on protein synthesis. Upon association with the miRNA-induced silencing complex (miRISC), miRNAs repress target mRNA translation and accelerate mRNA decay. Degradation of the mRNA is initiated by shortening of the poly(A) tail by the CCR4–NOT deadenylase complex followed by the removal of the 5′ cap structure and exonucleolytic decay of the mRNA. Here, we report a direct interaction between the large scaffolding subunit of CCR4–NOT, CNOT1, with the translational repressor and decapping activator protein, DDX6. DDX6 binds to a conserved CNOT1 subdomain in a manner resembling the interaction of the translation initiation factor eIF4A with eIF4G. Importantly, mutations that disrupt the DDX6–CNOT1 interaction impair miRISC-mediated gene silencing in human cells. Thus, CNOT1 facilitates recruitment of DDX6 to miRNA-targeted mRNAs, placing DDX6 as a downstream effector in the miRNA silencing pathway.     

Rice genetic resources: history,conservation, investigative characterization and use in Japan

Rice has been grown in Japan for about 3000 years. Although both japonica and indica varieties have been grown in Japan, now japonica rices are grown. Japanese rice breeding has used an ecological breeding approach. While emphasis in rice breeding in the 1940's and 1950's focussed on yield in recent decades quality has been of major importance. Consumer preference and name recognition of high quality varieties, such as Koshihikari, has resulted in slow acceptance of new varieties.Rice germplasm was systematically collected throughout Japan between 1962 and 1963. Subsequent acquisition and collecting, in Japan and other countries, has resulted in 28,000 accessions being conserved in the National Genebank, based at the National institute of Agrobiological Resources (NIAR).Research on genetic diversity of rice using a range of techniques, for example esterase isozymes, has revealed clinal variation in rice radiating from the center of diversity of rice in and around southwest China. Newly found genes in traditional rice germplasm, such as genes for non-elongating mesocotyl, are now routinely identified on the rice genome. Pioneering studies on eco-genetic differentiation of species in the genus Oryza in Japan has revealed much about the complex genepool for which rice evolved.Pest and disease resistance sources, particularly to blast, bacterial blight and brown plant hopper, from many countries have been incorporated into Japanese varieties. Cold tolerance at the booting stage was found in the Indonesian variety Silewah. In the future in characterisation of rice germplasm and interaction between rice germplasm specialists and rice molecular scientists, both in Japan and internationally, will be corner stones to securing rice genetic diversity and rice improvement in the next century.     

Mechano-sensitive channels regulate the stomatal aperture in Vicia faba

In the bright fields, stomata of the plants are fully opened to raise the transpiration rate and CO₂ uptake required for photosynthesis. Stomatal opening is driven by the activation of plasma membrane H⁺-ATPase and K⁺_in channels, and the Ca²⁺-dependent inactivation and blockage of both components were supposed to be inevitable function to regulate the stomatal aperture. Although, it is still obscure how these activities are regulated at the open state. Application of an amphipathic membrane creator, trinitrophenol (TNP), instantly generates the convex curvature in the plasma membrane, which occurs in the phases of stomatal opening and closure. TNP surely activates mechanosensitive Ca²⁺-permeable channels and attenuates the promotion of stomatal opening, but does not inhibit and promote stomatal closure. These results suggest that activation of mechanosensitive Ca²⁺-permeable channels regulates the opening phase of stomata in plants.     

Structure of the N-terminal domain of PEX1 AAA-ATPase. Characterization of a putative adaptor-binding domain

Peroxisomes are responsible for several pathways in primary metabolism, including beta-oxidation and lipid biosynthesis. PEX1 and PEX6 are hexameric AAA-type ATPases, both of which are indispensable in targeting over 50 peroxisomal resident proteins from the cytosol to the peroxisomes. Although the tandem AAA-ATPase domains in the central region of PEX1 and PEX6 are highly similar, the N-terminal sequences are unique. To better understand the distinct molecular function of these two proteins, we analyzed the unique N-terminal domain (NTD) of PEX1. Extensive computational analysis revealed weak similarity (<10% identity) of PEX1 NTD to the N-terminal domains of other membrane-related type II AAA-ATPases, such as VCP (p97) and NSF. We have determined the crystal structure of mouse PEX1 NTD at 2.05-A resolution, which clearly demonstrated that the domain belongs to the double-psi-barrel fold family found in the other AAA-ATPases. The N-domains of both VCP and NSF are structural neighbors of PEX1 NTD with a 2.7- and 2.1-A root mean square deviation of backbone atoms, respectively. Our findings suggest that the supradomain architecture, which is composed of a single N-terminal domain followed by tandem AAA domains, is a common feature of organellar membrane-associating AAA-ATPases. We propose that PEX1 functions as a protein unfoldase in peroxisomal biogenesis, using its N-terminal putative adaptor-binding domain.     

Hydrolysis of sphingosylphosphocholine by neutral sphingomyelinases

Sphingosylphosphocholine (SPC), the N-deacylated form of sphingomyelin (SM), is a naturally occurring lipid mediator. However, little is known about the metabolism of SPC. We here report an in vitro assay system for SPC-phospholipase C (PLC). Using this assay system, we demonstrated that nSMase1 and nSMase2, human neutral sphingomyelinases (SMases), are capable of hydrolyzing SPC efficiently under detergent-free conditions. Bacterial and plasmodial neutral SMases also showed SPC-PLC activity. The substrate specificity of neutral SMases that hydrolyze SM, SPC, and monoradyl glycerophosphocholine, but not diradyl glycerophosphocholine, suggested that a hydrogen-bond donor at the C-2 or sn-2 position in the substrate is required for recognition by the enzymes.     

Modulation of the K+ efflux activity of Bacillus subtilis YhaU by YhaT and the C-terminal region of YhaS

The cation/proton antiporter 2 (CPA2) family is a large family of cation transporters and putative channel proteins that are found in bacteria, archaea as well as eukaryotes. Consistent with a K+ efflux capacity that is found in several other CPA2 proteins, it is shown here that the YhaU protein of Bacillus subtilis greatly increased the concentration of K+ required for growth of a K+ uptake-defective mutant of Escherichia coli. No YhaU-dependent K+(Na+)/H+ antiport activity was found in membrane vesicles. Two genes, yhaS and yhaT, are located upstream of yhaU and form an apparent operon with it. The YhaS protein has no reported homologues while the YhaT protein has sequence similarity to a sub-domain of KTN proteins that are associated with potassium-translocating channels and transporters. YhaT and the C-terminal region of YhaS were shown to modulate the K+ transport capacities of YhaU in complementation experiments. Expression studies, conducted by monitoring the beta-galactosidase levels in pMutin-disrupted mutants of the yhaU locus, indicated that yhaU is strongly induced by alkaline pH- plus salt-induced stress and that there are additional sodium-specific responses of yhaS and yhaT.