Cloning of the nupC gene of Escherichia coli encoding a nucleoside transport system, and identification of an adjacent Insertion element, IS 186

Escherichia coli is known to contain more than one active transport system for nucleoside uptake. In the present study we report the sequence of a gene encoding a second nucleoside transport system, nupC (in addition to nupG.) An open reading frame (ORF) of 1200bp was identified that codes for a hydrophobic polypeptide of 43 560 Da and an NupC fusion protein was shown to be membrane associated. The native NupC protein is also identified, following over-expression. NupC exhibits short regions of homology to several membrane-associated proteins, including LacY and Cyd. Analysis of the nupC promoter region revealed the presence of at least two putative CRP-binding sites, centred at–40bp and–89bp, which probably flank a CytR-binding site. In addition, an adjacent IS186 element was identified and found to reside within a putative terminator structure, downstream from the nupC ORF. This arrangement is shown to reflect the previously established gene order on the E. coli chromosome.     

OPTIMIZATION OF AN IMMUNOFLUORESCENT ASSAY OF THE INTERNAL ENZYME RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE (RUBISCO) IN SINGLE PHYTOPLANKTON CELLS1

Immunochemical probes are widely used to identih different species and to quantify and understand the role that different antigens play within cells. We optimized a single-cell immunofluorescent assay for the carbon fixation enzyme ribulose-1,5-bisphosphate carboxylase (Rubisco) in order to quantify the enzyme by flow cytometry in phytoplankton cells. The criteria for optimization of the immunofluorescent assay for Rubisco in single cells included maximization of Rubisco immunogenicity, minimization of Rubisco diffusion out of the cells, minimization of cell breakage, and maximization of the cell labeling. Several fixatives (cross-linkers and denaturing) and permeabilizing agents were tested on 26 species of phytoplankton. The only fixative / permeabilizing agent that fulfilled the criteria established for the assay was 96% ethanol. Phytoplankton cells collected from the field needed further treatment with a strong oxidant to permeabilize ethanolfixed cells and thus allow the antibody probe to access the Rubisco antigen. This study should have a general applicability to the study of other soluble photosynthetic antigens in single phytoplankton cells.     

Chilling-sensitive mutants of arabidopsis

Twenty-one mutants ofArabidopsis thaliana were isolated that developed chlorosis or necrosis upon incubation at low temperature (10°C to 15°C). Crosses among mutants in different phenotypic classes showed that mutants in three of four classes were found in a small number of loci. This article is reproduced fromWeeds World, vol. 1. For electronic access toWeeds World, see PMBR 12(4):302–303.     

Endothelial-dependent relaxant effects of vaso-active intestinal polypeptide and arachidonic acid in rat aortic strips

The relaxant actions of vaso-active intestinal polypeptide (VIP), acetylcholine (ACh), histamine and papaverine have been compared using circular muscle strips of rat aorta contracted with noradrenaline (NA). Arachidonic acid (AA) in a low dose (6.7 × 10 − 7M) also relaxed the aorta. The relaxant actions of all these substances except papaverine were abolished by removal of the endothelial cells. Higher doses of AA (6.7–13.4 × 10–6M) contracted aortic strips in the absence of NA but the con tractile effect “faded” while AA was still present in the bathing fluid. De-endothelialisation abolished this “fade” portion of the response leaving a sustained contracture. Indomethacin inhibited the contractile effect of AA revealing a weak inhibitory effect. However, it did not affect the relaxations induced by VIP, ACh, histamine or papaverine. ETYA abolished the relaxant actions of all these substances except papaverine. The results are consistent with the hypothesis that VIP, ACh and histamine relax the rat aorta via an endothelial-dependent mechanism which may involve the synthesis of a lipoxygenase product.     

Isolation and Characterization of a Novel Thermophilic, Freshwater Methanogen

A novel thermophilic, coccoid methanogen isolated from nonthermal freshwater sediments is described. Hydrogen plus carbon dioxide and formate were substrates for methanogenesis, and methane production was stimulated by yeast extract, Casamino Acids, and tryptose. Growth also occurred autotrophically. Elevated levels of sodium chloride were not required for maximum growth and were inhibitory above 2%. The minimum doubling time occurred at 57°C, and the upper and lower limits for methane production were 62 and 26°C, respectively. The optimum pH for growth was between 7.0 and 7.5. Inhibitory antibiotics included metronidazole, anisomycin, chloramphenicol, and lasalocid. Colonies were circular, dark yellow, shiny, and convex with entire edges. Cells were 1 to 2.5 μm in diameter, nonmotile, occurring singly or in pairs, and fimbriated. Cells were lysed by pronase or trypsin digestion, glass-distilled water, and 1% sodium dodecyl sulfate. Electron micrographs of thin sections showed a monolayered cell wall ca. 20 nm thick. The DNA base ratio was 49.2 mol% guanine plus cytosine. The whole cell protein pattern differed from that of other named coccoid methanogens.