Sequential elution of denatured proteins, hydrolyzed RNA, and plasmid DNA of bacterial lysates adsorbed onto stacked DEAE-cellulose membranes.

Modified membrane chromatography is emerging as a widely applicable technique for the separation of macromolecules. The use of DEAE-cellulose membranes (MemSep) for the purification of bacterial plasmid DNA has been assessed. Cleared bacterial lysates were injected directly onto the membranes without any prior sample cleanup. A single phenol extraction of adsorbed DNA was carried out and about 60 times less phenol was required to achieve the same extent of deproteination for DNA adsorbed onto the membranes as compared to DNA in solution. After chloroform and ethanol wash, (oligo)ribonucleotides resulting from RNase treatment were desorbed with 0.3 M LiCl, 5 mM LiOH. Finally, DNA was eluted with 0.5 M NaCl, 5 mM NaOH. The complete elution of DNA required NaOH in addition to NaCl and the latter salt was a better eluent than LiCl. Without RNase treatment, plasmid DNA-RNA complex required 2 M NaCl, 5 mM NaOH to be completely desorbed. The whole procedure took less than 40 min. The DEAE-cellulose membranes can withstand more than 100 cycles of regenerations and uses without any noticeable decrease of their binding capacity. No cross-contamination of successive DNA preparations was observed. Plasmid DNA was a good substrate for DNA endonucleases. Restriction fragments repurified by this procedure were amenable to ligation. Transformation of Escherichia coli and Saccharomyces cerevisiae with plasmid DNA was observed.

A procedure for the preparation of bacterial DNA that employs dimethyl sulfoxide to induce the lysis of cells.

A protocol for the preparation of DNA from Escherichia coli and Bacillus subtilis without the use of lysozyme as a permeabilizing agent is described. This preliminary step is carried out by treating the cells with dimethyl sulfoxide. A 5-min incubation of the cell pellet in the pure solvent, followed by the treatment with sodium dodecyl sulfate, is sufficient to induce cell lysis. The plasmid DNAs obtained by this method were equivalent in purity and quantity to the material prepared from lysozyme-digested cells and amenable to restriction and ligation. Transformation by plasmid and genomic DNAs prepared from dimethyl sulfoxide-treated cells was demonstrated.

Kinetics of spheroplasts formation from selected yeast strains.

The formation of spheroplasts from several Kluyveromyces and Saccharomyces strains was studied with the Coulter counter technique. When yeasts were incubated with zymolyase, the number of intact cells decreased according to first-order kinetics after an initial lag. In the concentration ranges studied, the rates were proportional to the cell-lytic enzyme concentrations. The rate (-0.099 min-1) of the most susceptible strain, K. fragilis, was 80-fold higher than that of S. cerevisiae CBS 5495. Compared to the cell sizing technique, the spectrophotometric determination leads to the underestimation of the reaction course.

[Changes of Peroxidase, Catalase, and Superoxide Dismutase Activities in Ozone-fumigated Spinach Leaves]. / Modifications de l'activité des Peroxydase, catalase et superoxyde dismutase dans des feuilles d'épinard traité à l'ozone.

A sensitive spinach variety, cultivated on nutrient solution, was exposed to lethal and sublethal ozone concentrations for 4 hours. Peroxidase, catalase, and superoxide dismutase (SOD) activities were measured immediately and after one, two, four, and seven days. A significant peroxidase stimulation was observed for seven days at 250 and 125 p.p.b. ozone. Catalase activity increased with the lethal concentration and decreased with the sublethal ones. A superoxide dismutase stimulation still occured after two, four, and seven days, at 60-70, 125, and 250 p.p.b. respectively. In our present experiments, an SOD activity higher than 30 U/g leaves may be considered as a indicator of atmospheric pollution.