Efficient DNA transformation of Bradyrhizobium japonicum by electroporation.

Intact cells of Bradyrhizobium japonicum USDA 110 were transformed with a 30-kilobase plasmid to efficiencies of 10(6) to 10(7) transformants per microgram by high-voltage electroporation. The technique was reliable and simple, with single colonies arising from transformed cells within 5 days of antibiotic selection. Plasmid DNA from B. japonicum transformed the Bradyrhizobium (Arachis) sp. with high efficiency, while the same plasmid extracted from Escherichia coli transformed B. japonicum at very low efficiency. The electrical conditions that resulted in the highest efficiencies were high voltage (10.5 to 12.5 kV/cm) and short pulse length (6 to 7 ms). A linear increase in the number of transformants was observed as DNA concentration was increased over 4 orders of magnitude; saturation appeared to begin between 120 ng/ml and 1.2 micrograms/ml. This novel method of transformation should enhance B. japonicum genetic research by providing a valuable alternative to conjugal mating, which is currently the only efficient, widely used means of introducing DNA into this organism.     

High-efficiency transformation of bacterial cells by electroporation.

We have developed a method for efficiently generating transient pores in the outer membranes of Escherichia coli K-12 derivatives by using a new type of electroporation apparatus. The pores are large enough and persist long enough to facilitate the equilibration of plasmid molecules between the intracellular and extracellular spaces. The method has been used to transform bacterial cells with an efficiency greater than 10(9) transformants per microgram of plasmid. It has also been used to extract intact plasmid from transformed cells with efficiencies comparable to those of the traditional alkaline lysis or CsCl equilibrium density gradient techniques. The technique is simple and rapid, allowing a transformation or the preparation of microgram quantities of plasmid to be accomplished in minutes.     

In vivo electroporation and stable transformation of skin cells of newborn mice by plasmid DNA.

The skin cells of newborn mice were stably transformed in vivo with the aid of electroporation. The plasmid DNA was introduced subcutaneously followed by high-voltage pulses applied to the skin pleat. NEO-resistant colonies were found in primary cell cultures obtained from the treated skin. The experiments show that in vivo electroporation can be used for the introduction of plasmid DNA into skin cells of mouse.     

Efficient DNA transformation of Bradyrhizobium japonicum by electroporation

Intact cells of Bradyrhizobium japonicum USDA 110 were transformed with a 30-kilobase plasmid to efficiencies of 10(6) to 10(7) transformants per microgram by high-voltage electroporation. The technique was reliable and simple, with single colonies arising from transformed cells within 5 days of antibiotic selection. Plasmid DNA from B. japonicum transformed the Bradyrhizobium (Arachis) sp. with high efficiency, while the same plasmid extracted from Escherichia coli transformed B. japonicum at very low efficiency. The electrical conditions that resulted in the highest efficiencies were high voltage (10.5 to 12.5 kV/cm) and short pulse length (6 to 7 ms). A linear increase in the number of transformants was observed as DNA concentration was increased over 4 orders of magnitude; saturation appeared to begin between 120 ng/ml and 1.2 micrograms/ml. This novel method of transformation should enhance B. japonicum genetic research by providing a valuable alternative to conjugal mating, which is currently the only efficient, widely used means of introducing DNA into this organism.     

Efficient transformation of the osmotolerant yeast Zygosaccharomyces rouxii by electroporation

A rapid and simple electroporation method to transform osmotolerant yeast Zygosaccharomyces rouxii has been developed and conditions for efficient transformation of mutants derived from different Z. rouxii wild-type strains optimized.     

Fractionation of a bacterial cell population by adsorption to erythrocytes and yeast cells

Abstract Fluorochrome-labeled antibodies specific for either S or type-1 fimbriae of Escherichia coli were used to show that in broth culture the two fimbrial types of strain 3040 mostly occurred on different cells. 12% of the cells were nonfimbriated. A fractionation procedure that involved adsorption of bacterial cells onto erythrocytes and yeast cells was developed to isolate homogeneous subpopulations (S-fimbriated, type-1-fimbriated, and non-fimbriated) of. E. coli. The level of contamination in each isolated subpopulation was 4% at the highest. The method is useful in obtaining homogeneous bacterial populations for adherence studies and for purification of specific fimbrial antigens.     

Genetic transformation of the yeast Rhodotorula gracilis ATCC 26217 by electroporation

We examined the genetic transformation of the biotechnologically relevant yeast Rhodotorula gracilis ATCC 26217 by electroporation. To evaluate the yeast transformation, we created a genomic integration cassette that was targeted to the yeast orotidine-5′-phosphate decarboxylase gene (URA3 gene) locus and composed of the zeocin-resistant gene (Sh ble gene) with the URA3 promoter and terminator of the yeast. The yeast was unable to grow on medium containing 2.0 μg/mL zeocin, even with the inoculation of a large number of cells (approximately 1.0 × 10⁸ cells/plate). Using the integrative cassette and zeocin-containing medium, we successfully obtained yeast transformants by electroporation, and the highest transformation efficiency of approximately 40 colony-forming units/μg DNA was obtained with a 0.6-kV electrical pulse. No homologous integration of the cassette at the URA3 gene locus was detected by the analyses of uracil auxotrophy and genomic PCR of transformants, suggesting that this method is a useful tool for randomly mutating the yeast genome.     

Calcium-mediated cell surface changes in Chinese hamster cells.

Scanning electron microscopy (SEM) revealed quantitative differences in both surface characteristics and spreading behavior of young and senescent WI38 human diploid fibroblasts. Measurement of spreading behavior involved the determination of the rate of cell elongation beyond an axial ratio of 1.5 after time intervals of 1.5, 3, 6, 9, and 12 h following subculture. Early passage cells exhibited a 44–52% increased spreading rate compared with later passage cells, both obeying zero-order kinetics. Surface microvilli and blebs were also found to vary in number and size after those same time intervals following subculture. In young cells, microvilli density was found initially to be very low but then increased steadily with time. Later passage cells exhibited an initial high density of microvilli, followed by a gradual disappearance with time. The density of cellular blebs did not, however, vary significantly with time following subculture. After approx. 24 h, both early and late passage cells resembled each other. These studies do show that physical membrane characteristics of young and senescent cells differ under certain conditions, and suggest the importance of that structure in the phenomena of senescence.     

Co-transfer of restriction endonucleases and plasmid DNA into mammalian cells by electroporation: effects on stable transformation

The antimutagenic principle of the green fruits of Momordica charantia was shown by the micronucleus test to be an intractable mixture of novel acylglucosylsterols. The antimutagens were extracted from the green fruits with ethanol and isolated from the bioactive petroleum ether and carbon tetrachloride extracts by repeated and sequential flash column chromatography. The major component of the mixture is

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and the minor component is the stearyl derivative (Guevara, 1989). At a dosage range in mice of 50–12.5 μg extract/g, the mixture reduced by about 80% the number of micronucleated polychromatic erythrocytes induced by the well-known mutagen mitomycin C. Structure-activity correlation studies suggested that the antimutagenic activity may reside in the peculiar lipid-like structure of the acylglucosylsterols. Ingestion of these compounds may result in their absorption in the plasma membrane lipid bilayer which could adversely affect the membrane permeability towards mitomycin C and disrupt the cellular activity of the latter.     

High-efficiency transformation of Rhizobium leguminosarum by electroporation.

Electrotransformation of Rhizobium leguminosarum was successfully carried out with a 15.1-kb plasmid, pMP154 (Cmr), containing a nodABC-lacZ fusion by electroporation. The maximum transformation efficiency, 10(8) transformants/microg of DNA, was achieved at a field strength of 14 kV/cm with a pulse of 7.3 ms (186 Omega). The number of transformants was found to increase with increasing cell density, with no sign of saturation. In relation to DNA dosage, the maximum transformation efficiency (5.8 x 10(8) transformants/microg of DNA) was obtained with 0.5 microg of DNA/ml of cell suspension, and a further increase in the DNA concentration resulted in a decline in transformation efficiency.     

Plasmid transformation of Bacteroides spp. by electroporation

Transformation of Bacteroides spp. with a variety of plasmid DNAs was accomplished using electroporation. The standard transformation assay system used to deduce the optimal electroporation parameters employed a 50-to 100-fold concentrated cell suspension of mid-logarithmic phase Bacteroides fragilis strain 638 and the 5.4-kb clindamycin resistance (Ccr) vector, pBI191. A variety of electroporation buffers were used successfully in transformation experiments but of these, 1 mM MgCl2 in 10% glycerol was superior. The incorporation of MgCl2 was essential for optimum viability prior to electroporation and for optimum transformation. Transformants were routinely obtained using 5-ms pulses over a range of field strengths from 5 to 12.5 kV/cm, with a maximum of greater than 10(6) micrograms-1 DNA at 12.5 kV/cm. The number of transformants increased linearly with respect to DNA concentration over the range 0.01-2 micrograms tested. Recovery of transformants required an expression period of up to 2.5 h following exposure to the electric field. This period, however, was dependent on the antibiotic resistance marker used for selection of transformants, with a significantly shorter incubation required when chloramphenicol rather than clindamycin was used in the selective medium. The effect of the DNA source on transformation was tested using the shuttle vector pFD288. Plasmid DNA isolated from Bacteroides uniformis, Bacteroides ovatus, or Bacteroides thetaiotaomicron transformed B. fragilis 638 at frequencies 7.5- to 12.5-fold less than those observed for controls with homologous DNA. Further reductions were seen with Escherichia coli purified pFD288, which transformed at 1000-fold lower frequencies. Finally, using homologous pFD288 or pBI191 isolated from strain 638, several strains of B. fragilis, B. uniformis, and B. ovatus were transformed successfully without modification of the standard assay system. Two strains each of B. thetaiotaomicron and Bacteroides ruminicola were not transformed using the methods described here.     

High-efficiency transformation of Listeria monocytogenes by electroporation of penicillin-treated cells

A procedure has been developed for electroporation-mediated transformation of Listeria monocytogenes with plasmid DNA. The method was optimized for intact cells of L. monocytogenes 23074 by determining the effects of field strength, cell density, and plasmid DNA topology. Transformation efficiencies were dramatically increased when cells were treated with penicillin. Optimum frequencies of transformation (4 x 10(6) transformants/microgram DNA) were obtained when cells were grown in 10 micrograms/ml of penicillin G and electroporated at a field strength of 10 kV/cm. Using this procedure, transformation of relaxed plasmid DNA from ligation reactions provided 1 x 10(4) transformants/microgram DNA, allowing direct molecular cloning of DNA into this organism.     

Efficient transformation of mutant cells of Chlamydomonas reinhardtii by electroporation

Cells of CW-15 mutant of Chlamydomonas reinhardtii without a cell wall were transformed by electroporation. The hpt gene of hygromycin phosphotransferase was used as a selective marker. Optimal conditions of transformation were observed in the middle of the logarithmic growth phase at the density of suspension 10⁶ cells/ml, electric field intensity 1 kV/cm, and pulse duration 2 ms. Under these conditions up to 10³ hygromycin-resistant clones of trasformants per 10⁶ recipient cells were obtained that was 100 times higher than at the usage of wild-type cells. Exogenic DNA integrated into the genome of the nucleus C. reinhardtii was constantly inherited for more than 350 generations. The use of mutants without a cell wall and certain selective systems enable the efficiency of transformant yield to be doubled problems on unstable expression of geterologous genes to be investigated, and ways of obtaining super producers of foreign proteins using the alga C. reinhardtii investigated.