Transformation and allelic replacement in Francisella spp.

We describe methods for transposon mutagenesis and allelic replacement in the facultative intracellular pathogen Francisella. Recombinant clones were constructed by insertion of partially cut F. tularensis or F. novicida DNA into pUC19 and then mutagenized with a mini-Tn10-Km transposon. F. novicida could be transformed with these plasmids either by a chemical transformation method or by electroporation, whereas F. tularensis could be transformed only by electroporation. Transformation of F. tularensis by electroporation was enhanced in the absence of the capsule. Southern blot analysis showed that the KmR marker was rescued either by integration of the plasmid into the Francisella chromosome or by allelic replacement. Allelic replacement was found to be the mechanism underlying a site-specific mutation affecting FopA, an outer-membrane protein of Francisella. F. novicida could also be transformed with chromosomal DNA carrying the KmR marker and the transformation frequency obtained using chromosomal DNA was generally greater than that obtained using plasmid DNA. F. novicida was also transformed by an IncQ plasmid containing an F. novicida DNA insert, which replicated autonomously in this host.     

Genome modifications in protoplast-derived tobacco plants: Contents of repetitive DNA sequences

Plasticity of the tobacco genome was studied by testing the DNAs of protoplast-derived regenerants with three different repetitive DNA sequences by the method of quantitative DNA/DNA hybridizations. A large population of 91 regenerants belonging to 35 different protoclones was analysed and a high degree of heterogeneity in the contents of the different DNA repeats was detected. The contents of middle repetitive sequences of two types were more stable or changed in the same direction, while the highly repetitive sequence varied independently and displayed a significant reduction in comparison with the two other sequences. Comparing the variation within the subpopulations of plants of the same clonal origin and the variation among the protoclones led to a conclusion that the pre-existing DNA variability in the starting plant material and/or thein vitro stress during the very early stages of protoclone regeneration played a decisive role in the formation of modified genomes in regenerants.     

Microcarrier culture of bowes melanoma cells in serum-free medium with Human plasma fraction IV-4+ V

Bowes melanoma cells were cultivated successfully in a serum-free medium which was constructed by the concept of maximum retention of proteins from fractionated human plasma having growth stimulatory activities. The cells could be cultivated in the serum-free medium without any adaptation period. The major serum-free component of the medium was the fraction IV-4 + V of the Cohn fractionation process of human plasma. Approximately six times increase of tissue-type plasminogen activator (t-PA) activity as compared with that in serum-free medium even though the cell growth was much slower. In addition, the growth stimulatory activities of thrombin and fibronectin were investigated during the cultivation of Bowes melanoma cells in this serum-free medium. These proteins contributed significantly to the enhanced growth of cells by reducing doubling time to 25 and 35 h as compared with 55 h in the serum-free medium without them. Especially, fibronectin supported cells to propagate near to the maximum cell density achieved in the medium with 10% FBS.     

Protection against acute paraquat toxicity by ambroxol

An expression vector for G-CSF, pASLB3-3, was constructed and introduced into Namalwa KJM-1 cells (Hosoi et al., 1988), and cells resistant to 100 nM of methotrexate (MTX) were obtained. Among them, the highest producer, clone SC57, was selected and the productivity of this clone was further characterized. The maximal production of G-CSF was at the most 1.8 g/ml/day using a 25 cm² tissue culture flask, even though the cell number was above 7×10⁵ cells/ml. The limiting factors at high density were analyzed as the deficiency of nutrients, such as glucose, cysteine and serine, and pH control. The depression of specific G-CSF productivity per cell under the batch culture conditions was overcome by using a perfusion culture system, Biofermenter^TM (Sato, 1983) with modifications of nutrients supplementation by a dialysis membrane and/or dissolved oxygen (DO) supplementation by microsilicone fibers. ITPSGF medium was modified to elevate concentrations of amino acids and glucose by 2.0- and 2.5-times, respectively. Under the control of pH at 7.4 and DO at 3 ppm, the specific G-CSF productivity was not depressed even at high cell density (above 1×10⁷ cells/ml), and the amount of G-CSF reached 41 g/ml. These results indicated the possibility of finding the optimum culture conditions for the production of recombinant proteins by Namalwa KJM-1 cells.Abbreviations ABTS 2,2-Azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid - BSA Bovine Serum Albumin - BSA-PBS Phosphate-buffered Saline without Ca²⁺ and Mg²⁺ containing Bovine Serum Albumin - dhfr Dihydrofolate Reductase - DO Dissolved Oxygen - G-CSF Granulocyte Colony-stimulating Factor - HEPES 4-(2-Hydroxyethyl)-1-piperazineethansulfonic Acid - IFN Interferon - MTX Methotrexate - PBS(-) Phosphate-buffered saline without Ca²⁺ and Mg²⁺ - Tween-PBS Phosphate-buffered saline without Ca²⁺ and Mg²⁺ containing 0.05% of Tween 20     

The reactions of thiouridines and thiouracils with chloroacetaldehyde; mechanistic considerations.

2-Thiouridine, 4-thiouridine and the corresponding thiouracils were quantitatively modified with aqueous chloroacetaldehyde /37 degrees C, pH 3.0--6.5/. The rate-pH dependence found for the disappearance of the substrates suggested initial S-alkylation. The unstable S-acetaldehydyl intermediates were not detected due to their further rapid transformations. The following possibilities of such transformations are discussed: 1. intramolecular addition of the endocyclic nitrogen atom to the aldehyde carbonyl group to form the "hydroxyethano" bridged compounds, 2. hydrolysis to the corresponding "oxo" analogues of the substrates, 3. hydrolysis of the N-glycoside bond. The structures of new compounds formed in these reactions were assigned on the basis of their FD-MS, UV, IR and PMR spectra. The reaction rates were similar to those found for modification of adenosine and cytidine with chloroacetaldehyde.     

Influence of clay minerals on sorption of bacteriolytic enzymes

Myxobacteria presumably produce extracellular bacteriolytic enzymes when they are growing in soil. In order to study their ecological significance, adsorption experiments were performed with lytic enzymes produced byMyxococcus virescens in casitone media. Different soils as well as montmorillonite and kaolinite can rapidly adsorb the bacteriolytic but not the proteolytic enzymes. About 1 gm of montmorillonite per liter of cell-free culture solution is enough for the adsorption of 97% of the bacteriolytic enzymes. The adsorption per unit weight is about 100 times greater on montmorillonite than on kaolinite. About 40% of the adsorbed enzymes can be eluted with solutions of high pH or high ionic strength. The only desorbed bacteriolytic enzyme is the alanyl-∈-N-lysine endopeptidase.     

Staining of Some Specific Regions of Human Chromosomes,particularly the Secondary Constriction of No. 9

SEVERAL procedures have been described recently which produce specific patterns of differential staining in human chromosomes^1–9. Techniques which involve DNA denaturation and reannealing reveal deeply stained areas on centromere and secondary constriction regions which have been equated with constitutive heterochromatin⁹.     

The mode of action of thidiazuron: auxins,indoleamines, and ion channels in the regeneration of <Emphasis Type="Italic">Echinacea purpurea</Emphasis> L.

The biochemical mechanisms underlying thidiazuron (TDZ)-induced regeneration in plant cells have not been clearly elucidated. Exposure of leaf explants of Echinacea purpurea to a medium containing TDZ results in undifferentiated cell proliferation and differentiated growth as mixed shoot organogenesis and somatic embryogenesis. The current studies were undertaken to determine the potential roles of auxin, indoleamines, and ion signaling in the dedifferentiation and redifferentiation of plant cells. E. purpurea leaf explants were found to contain auxin and the related indoleamine neurotransmitters, melatonin, and serotonin. The levels of these endogenous indoleamines were increased by exposure to TDZ associated with the induction of regeneration. The auxin-transport inhibitor 2,3,5-triiodobenzoic acid and auxin action inhibitor, p-chlorophenoxyisobutyric acid decreased the TDZ-induced regeneration but increased concentrations of endogenous serotonin and melatonin. As well, inhibitors of calcium and sodium transport significantly reduced TDZ-induced morphogenesis while increasing endogenous indoleamine content. These data indicate that TDZ-induced regeneration is the manifestation of a metabolic cascade that includes an initial signaling event, accumulation, and transport of endogenous plant signals such as auxin and melatonin, a system of secondary messengers, and a concurrent stress response.