Detection and Identification of Yersinia pestis by Polymerase Chain Reaction (PCR) Using Multiplex Primers

A PCR method for detection of Yersinia pestis-virulence determinants by the use of multiplex primers was developed. Four pairs of oligonucleotide primers were designed from each gene of three kinds of virulent plasmids and a chromosomal DNA; 60-Md plasmid-located gene (caf1) encoding Y. pestis-specific capsular antigen fraction 1, a Y. pestis-specific region of a yopM gene encoded on 42-Md virulent plasmid, a plasminogen activator gene (pla) encoded on Y. pestis-specific 7-Md plasmid and an invasin protein gene (inv) encoded on chromosomal DNA. This multiplex-primer system was specific for the detection of Y. pestis among pathogenic Yersinia species and other enterobacteriaceae having antigens common to Y. pestis. Since this method is simple and safe, it will be useful to identify and confirm Y. pestis in cases of emergency and for the surveillance of epidemics.     

Serological Characterization of Trichosporon cutaneum and Related Species

Recent molecular biological, chemical, physiological and morphological studies indicate that Trichosporon cutaneum and related species should be reclassified. In this study, antigenic characteristics of the species were determined. The results of adsorption experiments revealed that there were at least three serological types: I, II and III. Specific factor sera I, II and III were prepared on the basis of adsorption experiments and isolates were serotyped by cell slide agglutination (CSA). Since the CSA test was difficult to read in some strains, the results of the CSA test were compared with the findings from an enzyme-linked immunosorbent assay (ELISA). For the ELISA, crude polysaccharide antigens prepared from the culture supernatant were used as the antigen. The types determined by ELISA correlated well with those determined by the CSA test. These data suggest that T. cutaneum and related species have at least three serological types, and that the typing can be done by either CSA or ELISA.     

Cloning and chromosomal mapping of the mouse DNA-dependent protein kinase gene

 Severe combined immune deficiency (scid) mice are assumed to have two types of abnormalities: one is high radiosensitivity and the other is abnormal recombination in immunoglobulin and T-cell receptor genes. The human chromosome 8 q1.1 region has an ability to complement the scid aberrations. Moreover, the localization of the subunit DNA-dependent protein kinase [DNA-PK_cs] participating in DNA double-strand break repair in the same locus was clarified. In scid mouse cells, the number of DNA-PK_cs products and extent of DNA-PK activity remarkably decrease. These observations gave rise to the assumption that DNA-PK_cs is the scid factor itself. In order to determine whether the DNA-PK _cs gene is the scid gene, we isolated the mouse DNA-PK _cs gene and investigated its chromosomal locus by fluorescence in situ hybridization (FISH). Consequently, it became clear that the mouse DNA-PK _cs gene existed in the centromeric region of mouse chromosome 16, determined by cross-genetic study, as a scid locus. This finding strongly suggests that mouse DNA-PK _cs is the scid gene. Received: 22 March 1996     

Salivacin 140, a novel bacteriocin from Lactobacillus salivarius subsp. salicinius T140 active against pathogenic bacteria

K. ARIHARA, S. OGIHARA, T. MUKAI, M. ITOH AND Y. KONDO. 1996. Fifteen of 353 environmental isolates of lactic acid bacteria consistently showed activity against Listeria monocytogenes, Streptococcus mutans, Actinomyces viscosus , and/or Propionibacterium acnes . Strain T140, isolated from the surface of Japanese pampas grass leaves and identified as Lactobacillus salivarius subsp. salicinius , also had activity against several Lactobacillus species, Staphylococcus aureus and Yersinia enterocolitica . Since the antagonistic factor(s) produced by T140 was sensitive to a proteolytic enzyme, it was concluded that a bacteriocin (named salivacin 140) was involved in the inhibition activity. Strain T140 required a high initial pH (7.5–8.5) in agar plates for bacteriocin production.     

Dibromothymoquinone (DBMIB) Replaces the Function of QA at 77 K in the Isolated Photosystem II Reaction Center (D1-D2-Cytochrome b559) Complex: Difference Spectrum of the P680+(DBMIB-) State

Transient absorbance changes of the primary electron donor chlorophylla (P680) and acceptor pheophytin a (H) were measured at 77 Kby nanosecond laser spectroscopy in the D1-D2-cytochrome b₅₅₉photosystem II reaction center complex containing dibromomethylisopropylbenzoquinone (DBMIB). After the laser excitation of the reactioncenter in the presence of DBMIB, only the P680⁺-(DBMIB^-) statewas detected. P680⁺ mainly decayed with a t_1/e of 11 ms. Inthe absence of DBMIB, the excitation produced the P680⁺H^- radicalpair. The radical pair produced the triplet state (P680^T) witha t_1/e of 50 ns, and P680^T then decayed with a t_1/e of 2.1 ms.It was concluded that H_- was oxidized by DBMIB in a time rangefaster than the detecting time resolution (3.5 ns) even at 77K. The rapid oxidation of H^- by DBMIB was also confirmed bythe suppression of delayed fluorescence with a decay t_1/e of50 ns. The P680⁺(DBMIB^-)/P680(DBMIB) difference spectrum exhibiteda Q_y, band with a peak at 682 nm with a shoulder at 673 nm.The spectral shape was almost temperature insensitive between77 and 265 K. The feature of this spectrum in the wavelengthrange between 330 and 720 nm was compared with that of P680_T/P680or H^-/H at 77 K. (Received May 8, 1996; Accepted June 24, 1996)     

New cellulose synthesizing complexes (terminal complexes) involved in animal cellulose biosynthesis in the tunicateMetandrocarpa uedai

Summary Subcellular compartments comprising the endomembrane system in filamentous fungi are poorly characterized with most showing significant morphological differences from eukaryotic cells. For example, many filamentous fungi lack stacked Golgi-body cisternae, but contain Golgi equivalents — single cisternae or tubules which appear to serve the same functions. To help identify fungal endomembrane compartments and interrelationships between them we used a pharmacological agent, brefeldin A, known to affect specific endomembrane organelles in other organisms, most prominently the Golgi apparatus. At 10 g/ml brefeldin A, radial hyphal growth of the rice blast pathogenMagnaporthe grisea on solid agar medium was reduced by 96% over an initial 48 h, but recovered and was reduced by only 20% over a subsequent 72 h exposure. Light microscopic examination of individual living hyphae showed that apical elongation generally halted within 1 min after exposure to brefeldin A. Acute effects of 14 g/ml brefeldin A were characterized ultrasiructurally in cells prepared by freeze substitution. These included the appearance of two types of cisternae with unusual morphology, associated with ca. 45 nm diameter vesicles, as well as the unexpected persistence and increase in complexity of the Golgi equivalents. Also observed were (1) reduced numbers of apicale vesicles and disruption of Spitzenkörper organization, (2) apical clusters of 30–35 nm diameter microvesicles and associated tubular arrays, (3) dilation of rough endoplasmic reticulum, (4) packets of membrane-bounded electron-opaque cell wall inclusions, and (5) altered morphology of some vacuolar compartments. The distribution of concanavalin A binding sites, previously mapped to particular endomembrane compartments, was documented to aid the interpretation of these results. We conclude that brefeldin A effects on cells ofM. grisea differ from those reported with plant and animal cells, perhaps reflecting underlying differences in the endomembrane systems among these eukaryotes.Abbreviations BFA brefeldin A - ConA concanavalin A - ER endoplasmic reticulum - PDA potato dextrose agar - RER rough endoplasmic reticulum     

PHYLOGENETIC RELATIONSHIPS WITHIN THE COLONIAL VOLVOCALES (CHLOROPHYTA) INFERRED FROM CLADISTIC ANALYSIS BASED ON MORPHOLOGICAL DATA1

A cladistic analysis was used to deduce the phylogenetic relationships within the colonial Volvocales. Forty-one pairs of characters related to gross morphology and ultrastructure of vegetative colonies as well as asexual and sexual reproduction were analyzed based on parsimony, using the PAUP 3.0 computer program, for 25 species belonging to nine volvocacean and goniacean genera of the colonial Volvocales. Chlamydomonas reinhardtii Dangeard was the outgroup. The strict consensus tree indicated the presence of two monophyletic groups, one composed of all the volvocacean species analyzed in this study and the other containing the goniacean species except for the four-celled species Gonium sociale (Dujardin) Warming. In addition, these two groups constitute a large monophyletic group, to which G. sociale is a sister group. A new combination Tetrabaena socialis (Dujardin) Nozaki et Itoh and a new family Tetrabaenaceae Nozaki et Itoh are thus proposed for G. sociale. In addition, the analysis suggests that the volvocacean genera Eudorina and Pleodorina are paraphyletic groups, respectively, and that the monotypic genus Yamagishiella has no autapomorphic characters and represents primitive features of the anisogamous and oogamous genera of the Volvocaceae. Phylogenetic relationships within the Volvocaceae and the Goniaceae, as well as the various modes of sexual reproduction exhibited by these organisms, are discussed on the basis of the analysis.     

A new yeast gene, HTR1, required for growth at high temperature, is needed for recovery from mating pheromone-induced G1 arrest

A new temperature-sensitive mutant of Saccharomyces cerevisiae was isolated. Arrested cells grown at the nonpermissive temperature were of dumb-bell shape and contained large vacuoles. A DNA fragment was cloned based on its ability to complement this temperature sensitivity. The HTR1 gene encodes a putative protein of 93 kDa without significant homology to any known proteins. The gene was mapped between ade5 and lys5 on the left arm of chromosome VII. The phenotype of the gene disruptant appeared to be strain-specific; disruption of the gene in strain W303 caused the cells to become temperature sensitive. The arrested phenotype here was similar to that of the original is mutant and cells in G2/M phase predominated at high temperature. Another disruptant in a strain YPH background grew slowly at high temperature due to slow progression through G2/M phase, and morphologically abnormal (elongated) cells accumulated. A single-copy suppressor that alleviated the temperature-sensitive defects in both strains was identified as MCS1/SSD1. The wild-type strains W303 and YPH are known to carry defective MCS1/SSD1 alleles; hence HTR1 may function redundantly with MCS1/SSD1 to suppress the temperature-sensitive phenotypes. In addition, based on a halo bioassay, the disruptant strains appeared to be defective in recovery from, or adaptive response to G1 arrest mediated by mating pheromone, even at the permissive temperature. Thus the gene has at least two functions and is designated HTR1 (required for high temperature growth and recovery from G1 arrest induced by mating pheromone).     

The glycinin box: a soybean embryo factor binding motif within the quantitative regulatory region of the 11S seed storage globulin promoter

The soybean embryo factor binding sequence in the glycinin A₂B_1a gene promoter was delimited to an A/T-rich 9 bp sequence, 5-TAATAATTT-3, designated as the glycinin box, by DNA footprinting and gel mobility shift assay using synthetic oligonucleotides. It was shown that the interaction with the factor takes place at a defined DNA sequence rather than at random A/T-rich sequence blocks in the glycinin 5 flanking region. There are four glycinin boxes in the quantitative regulatory region between positions – 545 and – 378 of the glycinin A₂B_1a promoter. Multiple nonamer motifs similar to the glycinin box were also found in the equivalent regions of other glycinin and legumin promoters, suggesting that they must be conserved as a binding site for the embryo factor that activates the differential and stage-specific expression of seed 11S globulin genes in leguminous plants.     

Isolation of the Photoactive Reaction Center Complex that Contains Three Types of Fe-S Centers and a Cytochrome c Subunit from the Green Sulfur Bacterium Chlorobium limicola f. thiosulfatophilum, Strain Larsen

The photoactive reaction center (RC) complex from the greensulfur bacterium Chlorobium limicola f. thiosulfatophilum, strainLarsen, was isolated after solubilization and ammonium sulfatefractionation followed by ion-exchange chromatography. The spectrumof the complex was almost identical with that of the similarRC complex isolated by Feiler et al. [(1992) Biochemistry 31:2608–2614] except for the presence of cytochrome c₅₅₁instead of c₅₅₃ in the latter study. A molecular ratio of BChla to P840 of the isolated RC complex was assayed to be 25–35.SDSPAGE analysis revealed that the isolated complex containedthree major polypeptides with apparent molecular masses of 68,41 and 21 kDa, respectively. The 21-kDa polypeptide was identifiedto be a heme-binding protein by staining the gel for peroxidaseactivity. The cytochrome c₅₅₁ was oxidized by flash light ina biphasic manner with half times of 90 and 390 µs, respectively,that coincided with the reduction half times of P840⁺. Threedistinct iron-sulfur centers assigned to F_A, F_B and F_x, respectively,from their g-values were detected by EPR spectroscopy at cryogenictemperature. These results suggest that the present preparationcontains a minimal functional unit of the RC of this bacterium,and that this complex appears to lie on a evolutionary linebetween RC's of purple bacteria and photosystem I. (Received August 18, 1992; Accepted October 28, 1992)