Cyclization reaction catalyzed by glycogen debranching enzyme (EC 2.4.1.25/EC 3.2.1.33) and its potential for cycloamylose production

Glycogen debranching enzyme (GDE) has 4-alpha-glucanotransferase and amylo-1,6-glucosidase activities in the single polypeptide chain. We analyzed the detailed action profile of GDE from Saccharomyces cerevisiae on amylose and tested whether GDE catalyzes cyclization of amylose. GDE treatment resulted in a rapid reduction of absorbance of iodine-amylose complex and the accumulation of a product that was resistant to an exo-amylase (glucoamylase [GA]) but was degraded by an endo-type alpha-amylase to glucose and maltose. These results indicated that GDE catalyzed cyclization of amylose to produce cyclic alpha-1,4 glucan (cycloamylose). The formation of cycloamylose was confirmed by high-performance anion-exchange chromatography, and the size was shown to range from a degree of polymerization of 11 to a degree of polymerization around 50. The minimum size and the size distribution of cycloamylose were different from those of cycloamylose produced by other 4-alpha-glucanotransferases. GDE also efficiently produced cycloamylose even from the branched glucan substrate, starch, demonstrating its potential for industrial production of cycloamylose.     

tfdA-like genes in 2,4-dichlorophenoxyacetic acid-degrading bacteria belonging to the Bradyrhizobium-Agromonas-Nitrobacter-Afipia cluster in alpha-Proteobacteria

The 2,4-dichlorophenoxyacetate (2,4-D)/alpha-ketoglutarate dioxygenase gene (tfdA) homolog designated tfdAalpha was cloned and characterized from 2,4-D-degrading bacterial strain RD5-C2. This Japanese upland soil isolate belongs to the Bradyrhizobium-Agromonas-Nitrobacter-Afipia cluster in the alpha subdivision of the class Proteobacteria on the basis of its 16S ribosomal DNA sequence. Sequence analysis showed 56 to 60% identity of tfdAalpha to representative tfdA genes. A MalE-TfdAalpha fusion protein expressed in Escherichia coli exhibited about 10 times greater activity for phenoxyacetate than 2,4-D in an alpha-ketoglutarate- and Fe(II)-dependent reaction. The deduced amino acid sequence of TfdAalpha revealed a conserved His-X-Asp-X(146)-His-X(14)-Arg motif characteristic of the active site of group II alpha-ketoglutarate-dependent dioxygenases. The tfdAalpha genes were also detected in 2,4-D-degrading alpha-Proteobacteria previously isolated from pristine environments in Hawaii and in Saskatchewan, Canada (Y. Kamagata, R. R. Fulthorpe, K. Tamura, H. Takami, L. J. Forney, and J. M. Tiedje, Appl. Environ. Microbiol. 63:2266-2272, 1997). These findings indicate that the tfdA genes in beta- and gamma-Proteobacteria and the tfdAalpha genes in alpha-Proteobacteria arose by divergent evolution from a common ancestor.     

Bacterial Distribution and Phylogenetic Diversity in the Changjiang Estuary before the Construction of the Three Gorges Dam

The bacterial community structure in the Changjiang estuary was studied for comparison with future changes, related to the construction of the Three Gorges Dam. Population densities of bacteria in the surface water at station C1 estimated by CFU on marine agar plates and by DAPI direct count, were 2.8 x 10(4) ml(-1) and 4.2 x 10(5) ml(-1), respectively. Physicochemical properties of water, such as temperature and salinity, suggested that station C1 was affected by freshwater from the Changjiang River. Cluster analysis of the PCR-RFLP patterns obtained from 9 samples showed that the bacterial community structure at station C1 was different from the structure at the other stations. Bacterial diversity in the surface water at station C1 was studied based on the genotypes of the 250 clones of 16S rRNA, and on the phenotypes generated on Biolog GN plates for 70 isolates. Sequences of bacteria from two common marine groups, alpha- and gamma-Proteobacteria, were frequently observed. Some other divisions, including the beta-Proteobacteria, C/F/B group, low G+C gram positive, high G+C gram positive, chloroplasts, and relatives of Verrucomicrobia were also observed. The putative dominant species based on both genotype and phenotype analyses were close relatives of Alteromonas macleodii or Roseobacter spp. These results reflected the nutrient-rich environment at station C1.     

Genome-wide transcriptional orchestration of circadian rhythms in Drosophila

Circadian rhythms govern the behavior, physiology, and metabolism of living organisms. Recent studies have revealed the role of several genes in the clock mechanism both in Drosophila and in mammals. To study how gene expression is globally regulated by the clock mechanism, we used a high density oligonucleotide probe array (GeneChip) to profile gene expression patterns in Drosophila under light-dark and constant dark conditions. We found 712 genes showing a daily fluctuation in mRNA levels under light-dark conditions, and among these the expression of 115 genes was still cycling in constant darkness, i.e. under free-running conditions. Unexpectedly the expression of a large number of genes cycled exclusively under constant darkness. We found that cycling in most of these genes was lost in the arrhythmic Clock (Clk) mutant under light-dark conditions. Expression of periodically regulated genes is coordinated locally on chromosomes where small clusters of genes are regulated jointly. Our findings reveal that many genes involved in diverse functions are under circadian control and reveal the complexity of circadian gene expression in Drosophila.     

KIF1Bbeta2, capable of interacting with CHP,is localized to synaptic vesicles

Kinesin family proteins are microtubule-dependent molecular motors involved in the intracellular motile process. Using a Ca2+ -binding protein, CHP (calcineurin B homologous protein), as a bait for yeast two hybrid screening, we identified a novel kinesin-related protein, KIF1Bbeta2. KIF1Bbeta2 is a member of the KIF1 subfamily of kinesin-related proteins, and consists of an amino terminal KIF1B-type motor domain followed by a tail region highly similar to that of KIF1A. CHP binds to regions adjacent to the motor domains of KIF1Bbeta2 and KIF1B, but not to those of the other KIF1 family members, KIF1A and KIF1C. Immunostaining of neuronal cells showed that a significant portion of KIF1Bbeta2 is co-localized with synaptophysin, a marker protein for synaptic vesicles, but not with a mitochondria-staining dye. Subcellular fractionation analysis indicated the co-localization of KIF1Bbeta2 with synaptophysin. These results suggest that KIF1Bbeta2, a novel CHP-interacting molecular motor, mediates the transport of synaptic vesicles in neuronal cells.     

Identification and characterization of the UL56 gene product of herpes simplex virus type 2

The UL56 gene product of herpes simplex virus (HSV) has been shown to play an important role in viral pathogenicity. However, the properties and functions of the UL56 protein are little understood. We raised rabbit polyclonal antisera specific for the UL56 protein of HSV type 2 (HSV-2) and examined its expression and properties. The gene product was identified as three polypeptides with apparent molecular masses ranging from 32 to 35 kDa in HSV-2-infected cells, and at least one species was phosphorylated. Studies of their origins showed that the UL56 protein of HSV-2 is also translated from the upstream in-frame methionine codon that is not present in the HSV-1 genome. Synthesis was first detected at 6 h postinfection and was not abolished by the viral DNA synthesis inhibitor phosphonoacetic acid. Indirect immunofluorescence studies revealed that the UL56 protein localized to both the Golgi apparatus and cytoplasmic vesicles in HSV-2-infected and single UL56-expressing cells. Deletion mutant analysis showed that the C-terminal hydrophobic region of the protein was required for association with the cytoplasmic membrane and that the N-terminal proline-rich region was important for its translocation to the Golgi apparatus and cytoplasmic vesicles. Moreover, the results of protease digestion assays and sucrose gradient fractionation strongly suggested that UL56 is a tail-anchored type II membrane protein associated with lipid rafts. We thus hypothesized that the UL56 protein, as a tail-anchored type II membrane protein, may be involved in vesicular trafficking in HSV-2-infected cells.     

Molecular cloning and overexpression of fleA gene encoding a fucose-specific lectin of Aspergillus oryzae

A protein from the cell lysate of Aspergillus oryzae was purified by column chromatography immobilized with a ferrichrysin (Fcy), which is one of the siderophores of A. oryzae. It is produced only in an iron-deficient culture and its molecular weight is estimated as 35,000 by SDS-PAGE. Two internal amino acid sequences of the protein obtained by lysylendopeptidase digestion were analyzed. Molecular cloning shows that it encodes 310 putative amino acid residues separated by 4 introns and is designated as fleA. It shows approximately 26% similarity with the gene encoding a fucose-specific lectin of Aleuria aurantia (AAL). The gene was overexpressed under control of the melO promoter in a submerged culture of A. oryzae. The fleA gene product showed hemagglutination activity against rabbit erythrocytes. A hemagglutination inhibition assay of monosaccharides showed that this lectin specifically binds to L-fucose and weakly reacts with mannose and N-acetyl-neuraminic acid.     

Splash seed dispersal by raindrops

Splash seed dispersal by raindrops was investigated for plants in southern Japan. Nine families, 10 genera and 19 species were confirmed as raindrop-dispersed plants. The 10 genera were Gentiana, Gratiola, Chrysosplenium, Mazus, Mitella, Ophiorrhiza, Sagina, Sedum, Trigonotis and Veronica. The method of splash rain dispersal in these species was clarified. Raindrop-dispersed species were all small herbaceous plants with a vertical pedicel and an apically opening fresh capsule when the seeds mature. Open capsules were cup-shaped or boat-shaped and can accommodate raindrops easily. The raindrops splashed the seeds from the capsule. In general, the seeds weighed very little, but they were heavier than powder or dust seeds dispersed by wind. A strong negative correlation was found between seed weight and the number of seeds per capsule. In the case of Trigonotis brevipes (Maxim.) Maxim., raindrops were received into the cup-shaped calyx-tube and dispersed the fruitlets. Some species, such as Gentiana thunbergii (G. Don) Griseb., Gentiana zollingeri Fawcett and Ophiorrhiza japonica Blume, had hydroscopic movement capsules that opened widely only when wet. Raindrop-dispersed plants were found in various habitats. For example, some plants grew together on rocks along the mountain torrents where splash water could easily be caught. The results of the laboratory and field experiments indicated that the dispersal distance of seeds by raindrops was 1m or less. For small herbaceous plants, splash dispersal by rain might be an effective and advantageous method of seed dispersal because dispersal is not affected by plant height.     

<Emphasis Type="Italic">HrNodal</Emphasis>, the ascidian <Emphasis Type="Italic">nodal</Emphasis>-related gene,is expressed in the left side of the epidermis,and lies upstream of <Emphasis Type="Italic">HrPitx</Emphasis>

The nodal-related genes are well known for their fundamental roles during vertebrate development, including mesoderm induction, neural induction, and left-right axis formation, as several nodal-related genes show left-sided expression in mesodermal lineages. We have isolated the first non-vertebrate nodal-related gene, HrNodal, from the ascidian Halocynthia roretzi. During the late cleavage and gastrula stages, HrNodal is transiently and bilaterally expressed in several different cell lineages. Expression at the tailbud stage is observed asymmetrically in the left side, but unexpectedly only in the epidermis of the embryo. We also demonstrate the relationship of HrNodal with HrPitx, a Halocynthia homologue of the Pitx2 gene. HrNodal overexpression results in the disturbance of left-sided HrPitx expression. Our results demonstrate that left-right specification during ascidian embryogenesis involves the HrNodal gene, and that the left-sidedness of the expression is evolutionarily conserved throughout the chordate clade.