Molecular breeding of transgenic rice plants expressing a bacterial chlorocatechol dioxygenase gene

The cbnA gene encoding the chlorocatechol dioxygenase gene from Ralstonia eutropha NH9 was introduced into rice plants. The cbnA gene was expressed in transgenic rice plants under the control of a modified cauliflower mosaic virus 35S promoter. Western blot analysis using anti-CbnA protein indicated that the cbnA gene was expressed in leaf tissue, roots, culms, and seeds. Transgenic rice calluses expressing the cbnA gene converted 3-chlorocatechol to 2-chloromucote efficiently. Growth and morphology of the transgenic rice plants expressing the cbnA gene were not distinguished from those of control rice plants harboring only a Ti binary vector. It is thus possible to breed transgenic plants that degrade chloroaromatic compounds in soil and surface water.     

Clostridium thermocellum cellulase CelT,a family 9 endoglucanase without an Ig-like domain or family 3c carbohydrate-binding module

The celT gene of Clostridium thermocellum strain F1 was found downstream of the mannanase gene man26B [Kurokawa J et al. (2001) Biosci Biotechnol Biochem 65:548–554] in pKS305. The open reading frame of celT consists of 1,833 nucleotides encoding a protein of 611 amino acids with a predicted molecular weight of 68,510. The mature form of CelT consists of a family 9 cellulase domain and a dockerin domain responsible for cellulosome assembly, but lacks a family 3c carbohydrate-binding module (CBM) and an immunoglobulin (Ig)-like domain, which are often found with family 9 catalytic domains. CelT devoid of the dockerin domain (CelTΔdoc) was constructed and purified from a recombinant Escherichia coli, and its enzyme properties were examined. CelTΔdoc showed strong activity toward carboxymethylcellulose (CMC) and barley β-glucan, and low activity toward xylan. The V _max and K _m values were 137 μmol min^–1 mg^–1 and 16.7 mg/ml, respectively, for CMC. Immunological analysis indicated that CelT is a catalytic component of the C. thermocellum F1 cellulosome. This is the first report describing the characterization of a family 9 cellulase without an Ig-like domain or family 3c CBM. Electronic Publication     

Promotion of ethylene biosynthesis in peach mesocarp discs by auxin

In this study, we investigated the ability of auxin to promote ethylenebiosynthesis in fruit tissue. Discs prepared from preclimacteric peach fruit(Prunus persica L. Batsch cv.Akatsuki) were incubated for 3 weeks on a solid MS medium containing variousconcentrations of 1-naphthaleneacetic acid (NAA). Higher ethylene productionwasobserved with an increasing concentration of NAA. As the developmental stageprogressed, the time it took for the discs to produce ethylene became shorterand the amount of ethylene became greater. Auxin-induced ethylene productionwaseffectively inhibited by adding 100 M ofCoCl₂ to the medium. The stimulatory effect of auxin on anthocyaninformation was not affected by Co²⁺, although softening wasinhibited, suggesting that the effect of auxin on softening is mediated byethylene.     

Scheduled conversion of replication complex architecture at replication origins of Saccharomyces cerevisiae during the cell cycle

Replication of DNA within Saccharomyces cerevisiae chromosomes is initiated from multiple origins, whose activation follow their own inherent time schedules during the S phase of the cell cycle. It has been demonstrated that a characteristic replicative complex (RC) that includes an origin recognition complex is formed at each origin and shifts between post- and pre-replicative states during the cell cycle. We wanted to determine whether there was an association between this shift in the state of the RC and firing events at replication origins. Time course analyses of RC architecture using UV-footprinting with synchronously growing cells revealed that pre-replicative states at both early and late firing origins appeared simultaneously during late M phase, remained in this state during G(1) phase, and converted to the post-replicative state at various times during S phase. Because the conversion of the origin footprinting profiles and origin firing, as assessed by two-dimensional gel electrophoresis, occurred concomitantly at each origin, then these two events must be closely related. However, conversion of the late firing origin occurred without actual firing. This was observed when the late origin was suppressed in clb5-deficient cells and a replication fork originating from an outside origin replicated the late origin passively. This mechanism ensures that replication at each chromosomal locus occurs only once per cell cycle by shifting existing pre-RCs to the post-RC state, when it is replicated without firing.     

Interaction between a type-II dockerin domain and a type-II cohesin domain from Clostridium thermocellum cellulosome

The interaction between the type-II dockerin domain of the scaffoldin protein CipA and the type-II cohesin domain of the outer layer protein SdbA is the fundamental mechanism for anchoring the cellulosome to the cell surface of Clostridium thermocellum. We constructed and purified a dockerin polypeptide and a cohesin polypeptide, and determined affinity constants of the interaction between them by the surface plasmon resonance method. The dissociation constant (K(D)) value was 1.8 x 10(-9) M, which is a little larger than that for the combination of a type-I dockerin and a type-I cohesin.     

Improved expression of novel red- and green-emitting luciferases of Phrixothrix railroad worms in mammalian cells

Luciferases are widely used for the quantitative monitoring of gene expression in a variety of organisms. We successfully expressed novel red- and green-emitting luciferases of Phrixothrix railroad worms in mammalian cells in combination with the Kozak sequence and the CAG promoter. The characteristic properties of these luciferases indicate that they are appropriate reporter genes for the simultaneous monitoring of two gene expressions.     

Hydrogen gas generation from refuse-derived fuel (RDF) under wet conditions

An explosion has recently occurred at a silo containing refuse-derived fuels (RDF) in Japan. There is a possibility that microorganisms are involved in generation of combustible gas from RDF and this study was aimed at showing the presence of bacteria that can ferment RDF pellets. All RDF samples tested contained a relatively high number of viable bacterial cells, 1.4x10(5) to 3.2x10(6) viable cells/g. These bacteria in the RDF samples fermented them to generate heat and hydrogen gas.     

Cloning and characterization of the cDNA for the Brazilian Cratomorphus distinctus larval firefly luciferase: similarities with European Lampyris noctiluca and Asiatic Pyrocoelia luciferases

Studies on firefly (Lampyridae) luciferases have focused on nearctic species of Photinus and Photuris and Euroasiatic species of Lampyris, Luciola, Hotaria, and Pyrocoelia. Despite accounting for the greatest diversity of fireflies in the world, no molecular studies have been carried out on the highly diverse genera from the neotropical region. Here we report the luciferase cDNA cloning for the larva of the Brazilian firefly Cratomorphus distinctus. The cDNA has 1978 bp and codes for a 547-residue-long polypeptide. Noteworthy, sequence comparison as well as functional properties show the highest degree of similarity with Lampyris noctiluca (93%) and Pyrocoelia spp. (91%) luciferases, suggesting a close phylogenetic relationship despite the geographical distance separating these species. The bioluminescence emission spectrum peaks at 550 nm and, as expected, is sensitive to pH, shifting to 605 nm at pH 6. The kinetic properties of the recombinant luciferase were similar to those of other firefly luciferases.     

Isothermal titration calorimetric studies on the binding of a family 6 carbohydrate-binding module of Clostridium thermocellum xynA with xlylooligosaccharides

The family 6 carbohydrate-binding module (CBM) of Clostridium thermocellum XynA was expressed, and the binding equilibria of the CBM with xylooligosaccharides (degree of polymerization DP = 2-8) were observed by isothermal titration calorimetry (ITC) at pH 8. The association constant, Ka, increased with increasing DP from 5 x 10(3) M(-1) (DP = 2) to approximately 5 x 10(5) M(-1) (DP = 5-8) at 20 degrees C. The Ka values at 60 degrees C were about 1/10 of those at 20 degrees C. The binding was found to be an enthalpy-driven reaction. The DP dependence of the thermodynamic parameters of the binding reaction suggested the size of the ligand-binding site to be 5 xylose units long.     

Suppression of ethanol and lipopolysaccharide-induced liver injury by extracts of Hydrangeae Dulcis Folium in rats

In female SD rats that were injected with 4 g/kg BW ethanol p.o. followed by a 5 mg/kg BW lipopolysaccharide (LPS) i.v. injection, serum glutamic pyruvic transaminases (GPT) activity increased to about eight times that of normal rats. In this model, rats that had been fed a diet containing 1% Hydrangeae Dulcis Folium (HDF) extracts for fifteen days showed significantly lower serum GPT activity (380.0+/-58.2 IU/l) than the control group (3527.0+/-774.1 IU/l). HDF's efficacy was far superior to milk thistle in this model (2950.0+/-915.9 IU/l). When mouse macrophages were treated with HDF extracts at 50 microg/ml, TNF-alpha production induced by LPS was suppressed to about 10% of the control. Rat serum TNF-alpha levels induced by LPS was decreased to 58.7% of the control by administering 1000 mg/kg BW HDF extract p.o. These results indicate that HDF prevents alcohol-induced liver injury through the inhibition of TNF-alpha production.