Phenylalanine Ammonia-Lyase Genes Involved in Anthocyanin Synthesis and the Regulation of its Expression in Suspension Cultured Carrot Cells

D and is also induced rapidly and transiently by transfer of cells to fresh medium and lowering the cell density. From the carrot genomic library, four clones of PAL genes, gDcPAL1,2,3 and 4, were obtained. Analyses of nucleotide sequences revealed that only the gDcPAL3 gene is responsible for the induction of anthocyanin synthesis by 2,4-D. Several cis-elements, boxes M, P, A, L, and G, exist in the proximal promoter region of gDcPAL3. Transient expression experiments in carrot protoplasts using deletion mutants of the proximal promoter region of gDcPAL3 gene showed that boxes M and L, both of which contain core sequences of the Myb binding sites, might play an important role in gDcPAL3 promoter activity. Four myb cDNAs, Dcmyb8,10,12 and 14 were obtained from a carrot subtracted cDNA library and their structure and expression patterns were analyzed. In addition to the analysis of the proximal region of gDcPAL3 promoter, the possibility of the regulation of gene expression by genomic DNA structure and chromatin modification in metabolic differentiation is discussed. Received 10 June 2000/ Accepted in revised form 1 July 2000     

A new comprehensive method for detection of livestock-related pathogenic viruses using a target enrichment system

We tested usefulness of a target enrichment system SureSelect, a comprehensive viral nucleic acid detection method, for rapid identification of viral pathogens in feces samples of cattle, pigs and goats. This system enriches nucleic acids of target viruses in clinical/field samples by using a library of biotinylated RNAs with sequences complementary to the target viruses. The enriched nucleic acids are amplified by PCR and subjected to next generation sequencing to identify the target viruses. In many samples, SureSelect target enrichment method increased efficiencies for detection of the viruses listed in the biotinylated RNA library. Furthermore, this method enabled us to determine nearly full-length genome sequence of porcine parainfluenza virus 1 and greatly increased Breadth, a value indicating the ratio of the mapping consensus length in the reference genome, in pig samples. Our data showed usefulness of SureSelect target enrichment system for comprehensive analysis of genomic information of various viruses in field samples.     

Color Preference and Associative Color Learning in a Parasitoid Wasp, <Emphasis Type="Italic">Ascogaster reticulata</Emphasis> (Hymenoptera: Braconidae)

Natural enemies of agricultural pests, such as parasitoids and predators, often use chemical and visual cues in search of their hosts and prey, and they can learn the association between the cues and the host and prey presence. The braconid, egg-larval endoparasitoid wasp Ascogaster reticulata is a promising biological control agent for tortricid pests, such as Adoxophyes honmai, in tea plantations. Although previous studies revealed that A. reticulata uses contact chemicals released by tea plants in response to tortricid egg oviposition and that it can learn the associated cues, the diurnal wasp is also expected to use visual cues, especially color. Therefore, in this study, we investigated the innate color preference and associative color learning ability of A. reticulata. When a green paper and a paper of a different color (black, blue, red or yellow) was offered together to naive females of the wasp, the females spent less time on a black and blue papers. However, wasps trained to associate black or blue with the presence of a host egg-mass showed increased preference for these colors, whereas red- and yellow-trained wasps did not show changes in preference. Our findings indicate that A. reticulata uses colors, in addition to chemical cues, in host searching behavior and has the ability to learn colors associated with host presence.     

THE CHITINOUS NATURE OF FILAMENTS EJECTED BY PHAEOCYSTIS (PRYMNESIOPHYCEAE)1

Filaments ejected by Phaeocystis globosa Scherffel, organized in star-like structures, were observed and analyzed before and after their discharge from cells. Ultrastructural observations obtained after cryofixation and cryosubstitution led to a model for their storage within the cell and for their ejection from the cell. Electron diffraction analysis on the ejected filaments demonstrated their chitinous composition. This technique indicated without ambiguity that each filament was in fact a whisker-like α-chitin crystal, with the axes of the corresponding polymer chains aligned with the filament's axis. X-ray microanalysis of the mats of filaments indicated that the silica content suggested by earlier workers was an artifact resulting from the filtration procedure.     

Successful Development of Air-Dried Microplates (HP-Plates) for Susceptibility Testing against Helicobacter pylori Isolates

We have successfully developed and evaluated a new susceptibility testing procedure against Helicobacter pylori strains using air-dried microplates “HP-Plates” containing eight serially-diluted anti-H. pylori agents. HP-Plate wells were reconstituted by the inoculation of 100 μl of H. pylori cell suspensions. After incubation at 37 C for 48 hr under humidified microaerophilic conditions, HP-Plates were read visually with a circular mirror. We investigated the within-day reproducibility tests of HP-Plates using the six quality control (QC) strains we proposed. Of the 20 testings, determining the minimum inhibitory concentrations (MICs) of all the QC strains fell within ± 1 log2 dilution ranges. When 200 clinical isolates were tested with HP-Plates and compared with the results obtained with the modified broth macrodilution method of NCCLS, more than 90% of the MICs also fell within ±1 log2 dilution ranges. We concluded that the HP-Plate susceptibility test method is a practical and easily applicable alternative of susceptibility testing for clinical microbiology laboratories in determining the MICs of H. pylori isolates.     

The cytotaxonomy and origin ofRanunculus yaegatakensis,an endemic taxon of Yakushima Island

Ranunculus yaegatakensis, a close relative of the widespreadR. silerifolius, is endemic to the alpine region (ca. 1,600 m alt.) of Yakushima Island. This species differs morphologically fromRanunculus silerifolius mainly due to its dwarfism and leaf shape, but has an identical karyotype with one (Karatsu-type) of the four cytotype ofR. silerifolius. Ranunculus yaegatakensis is highly cross-compatible with the Karatsu-type plants ofR. silerifolius. Their F₁ hybrids show regular meiotic behaviors and set seeds successfully. In addition, the Karatsu-type ofRanunculus silerifolius is found in individuals that are collected not only from the lowland of Yakushima Island but from its neighboring islands. It is supposed to be more specialized than other cytotypes. This evidence suggests thatRanunculus yaegatakensis has been derived from the Karatsu-type plants ofR. silerifolius. We suggest thatRanunculus yaegatakensis be reduced to a variety ofR. silerifolius.     

Structural studies on bacterial system used in the recognition and uptake of the macromolecule alginate

Alginate is an acidic heteropolysaccharide produced by brown seaweed and certain kinds of bacteria. The cells of Sphingomonas sp. strain A1, a gram-negative bacterium, have several alginate-degrading enzymes in their cytoplasm and efficiently utilize this polymer for their growth. Sphingomonas sp. strain A1 cells can directly incorporate alginate into their cytoplasm through a transport system consisting of a “pit” on their cell surface, substrate-binding proteins in their periplasm, and an ATP-binding cassette transporter in their inner membrane. This review deals with the structural and functional aspects of bacterial systems necessary for the recognition and uptake of alginate.     

Proton pumping mechanism of bovine heart cytochrome c oxidase

X-ray structures of bovine heart cytochrome c oxidase at 1.8/1.9 A resolution in the oxidized/reduced states exhibit a redox coupled conformational change of an aspartate located near the intermembrane surface of the enzyme. The alteration of the microenvironment of the carboxyl group of this aspartate residue indicates the occurrence of deprotonation upon reduction of the enzyme. The residue is connected with the matrix surface of the enzyme by a hydrogen-bond network that includes heme a via its propionate and formyl groups. These X-ray structures provide evidence that proton pumping occurs through the hydrogen bond network and is driven by the low spin heme. The function of the aspartate is confirmed by mutation of the aspartate to asparagine. Although the amino acid residues of the hydrogen bond network and the structures of the low spin heme peripheral groups are not completely conserved amongst members of the heme-copper terminal oxidase superfamily, the existence of low spin heme and the hydrogen bond network suggests that the low spin heme provides the driving element of the proton-pumping process.