Cloning of a Vero toxin (VT2) gene from a VT2-converting phage isolated from Escherichia coli 0157: H7

Abstract A Vero toxin (VT2 or Shiga-like toxin II)-converting phage was isolated from Escherichia coli 0157: H7 strain J-2. Nontoxigenic E. coli C600 produced VT2 when lysogenized with the toxin-converting phage. Eco RI fragments of the phage DNA were ligated with Eco RI-digested pBR322 or pUC118 and were transformed into E. coli MC1061 or MV1184. Transformants exhibiting VT2 production commonly contained a 4.6 kb Eco RI fragment. It was found that a 2.3 kb Kpn I- Sph I fragment coded VT2 production and that this fragment hybridized weakly with the 2.1 kb fragment encoding VT1.     

Molecular cloning and expression ofThiobacillus ferrooxidans chromosomal ribulose bisphosphate carboxylase genes inEscherichia coli

The genes coding ford-ribulose-1,5-bisphosphate carboxylase (RuBPCase) from an iron-oxidizing bacterium,Thiobacillus ferrooxidans, were cloned into anEscherichia coli plasmid, pUC18. The recombinant plasmid, termed pTR11, contained a 4.0-kb PstI fragment including the entire coding regions for both large and small subunits of RuBPCase.Escherichia coli carrying pTR11 did not show any CO₂-fixing activity. However, a derivative plasmid with an appropriate deletion, which was placed under the control of atac promoter, conferred ribulose bisphosphate-dependent CO₂-fixing activity on the host cell. Analysis of gel-filtration chromatography of the RuBPCase synthesized inE. coli revealed that it had a hexadecameric form like the native enzyme ofT. ferrooxidans.     

Effects of Extracellular Potassium on Acid Release and Motility Initiation in Toxoplasma gondii

The internal pH (pH_i) of Toxoplasma gondii was estimated by measuring the accumulation of the weak base 9-aminoacridine in buffers with various ionic compositions. The pH_i of the metabolizing parasite increased when the extracellular K⁺ was elevated in alkaline medium or when the external pH (pH_c) was substantially increased in medium employing high external K⁺ (90 mM). The parasite in mouse peritoneal fluid, or in potassium sulfate buffer (pH 8.2), where the pH_i was demonstrated to be increased to 7.9, became motile when acidic buffer was substituted for the original suspension medium. This acid-induced independent movement subsided within 5 min but was repeatedly induced if the pH_c was serially lowered to 6.0. Basic buffers, on the other hand, abolished motility when applied to the moving parasites. Nigericin, which is known to collapse pH gradients across the membrane, also abolished motility.     

Restriction Enzyme Analysis of Mitochondrial DNA of Acanthamoeba Strains in Japan

ABSTRACT. Eight isolates, identified as either Acanthamoeba castellanii or A. polyphaga from human eye infections, contact lens containers, and soil in Japan, were characterized by restriction fragment length polymorphisms (RFLP) of mitochondrial DNA (mtDNA). Mitochondrial DNA was digested with either Bgl II, Eco R I, Hind III, Hpa I, Sca I or Xba I, electrophoresed in agarose gels, and stained with ethidium bromide. Four distinct RFLP phenotypes that refer to the collection of six fragment size patterns obtained for a single strain with six enzymes, were discovered among the eight strains used in this study. Three strains morphologically classified as A. polyphaga share a single RFLP phenotype with the Ma strain of A. castellanii. The interspecific sequence differences of 7.06–12.74% in DNA nucleotide were estimated from the proportion of DNA fragments shared by each pair of mtDNA.     

RFLP analysis for species separation in the generaBipolaris andCurvularia

Restriction fragment length polymorphism (RFLP) of the total DNA ofBipolaris andCurvularia species was analysed using arbitrarily chosen genomic clones of DNA fromCurvularia lunata andBipolaris maydis as probes. Clear differences among species in both genera, resulting in different banding positions, were obtained with some probe-enzyme combinations. Intraspecific polymorphism in banding positions with these probe-enzyme combinations was slight. These analyses allow discrimination between the species. DNA fingerprinting with intrageneric probes is a potentially useful tool for species separation and identification inBipolaris andCurvularia when coupled with another characteristic such as conidial morphology.Curvularia aeria comb. nov. was proposed forCurvularia lunata var.aeria on the basis of differences in RFLP banding patterns and differences in conidial morphology.     

Occurrence of a novel group of hemagglutinins extractable by Pronase treatment in marine algae

Nine species of marine algae have been assessed for the presence of novel hemagglutinins not extractable with buffer, unless the algal tissue was pretreated with Pronase. All species examined contained hemagglutinins, indicating the existence of a novel group of hemagglutinins which differed from those reported previously in marine algae.     

Identification and characterization of ispA, a Shigella flexneri chromosomal gene essential for normal in vivo cell division and intercellular spreading

The virulent phenotype of Shigella requires loci on the chromosome as well as on the large virulence plasmid, and is regulated via a complex web of interactions amongst various chromosomal and large plasmid genes. To further investigate the role of chromosomal loci in virulence, we performed random Tn 10 mutagenesis in Shigella flexneri YSH6000T, and isolated an avirulent mutant (V3404) incapable of spreading throughout an epithelial cell monolayer. Although V3404 initially spread intercellularly at the same rate as the wild-type, it gradually slowed down and ceased spreading as a result of increasing defects in cell division, leading to the formation of long filamentous bacteria lacking septa, trapped within cells. In addition, the mutation affected the ability of V3404 to polymerize actin, a prerequisite for intra- and inter-cellular spreading ability. Sequencing of Tn 10 -flanking DNA revealed that the mutated gene, designated ispA (intracellular septation), was equivalent to a previously sequenced but uncharacterised gene of Escherichia coli located between trp and tonB . Using E. coli sequence data, we cloned the ispA gene from the YSH6000T chromosome and found that it complemented the V3404 mutation. Nucleotide sequencing and in vitro expression experiments revealed that ispA coded for a small (21 kDa), very hydrophobic protein. These results thus show that ispA is an essential virulence gene affecting several functions of the virulence process.     

A modulating role of mercurials-sensitive sulfhydryl groups in synaptic GABA receptor binding

The specific binding of GABA (γ-aminobutyric acid) agonist ³H-muscimol, to synaptic membranes from the rat brain showed a significant increase, when the membranous preparations were treated with a low concentration (10^?4–10^?5M) of mercurial sulfhydryl reagents such as p-chloromercuribenzoate and mercuric chloride. This activation in GABA receptor binding was bicuculline-sensitive, and was partially restored by subsequent treatments with 10 mM cysteine, penicillamine, or mercaptoethanol. Scatchard analysis of the binding revealed that this activation was due to the increase in the affinity of both high and low affinity bindings sites but not in the B_max values. On the other hand, the treatment of synaptic membranes with hydrophilic sulfhydryl reagents such as N-ethylmaleimide and iodoacetate had no effect on the binding. These hydrophilic sulfhydryl reagents, however, induced an increase of the binding following the pretreatment of synaptic membranes with 0.01% Triton X-100 or 0.5 U/mg prot. of phospholipase A₂ (EC 3.1.1.4.). These results suggest that mercurials-sensitive sulfhydryl groups, which are normally masked by membrane lipids, may play a modulating role in GABA receptor binding at central synapses.     

ROLE OF GLUCOCORTICOIDS IN TERMINAL DIFFERENTIATION AND TISSUE-SPECIFIC FUNCTION (A REVIEW)

Glucocorticoids (GCs) are required by many kinds of organs, tissues and cells for initiation or maintenance of their specific functions in vivo and in vitro. It is noticeable that most of these GC actions can be induced at much lower levels of dosage or concentration than the well-known actions of the steroids in gluconeogenesis, immune suppression and anti-inflammation. Such "differentiation-stimulating" actions of GC are regarded as main physiological roles of the steroid.