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Electric parameters and solvent conditions are known to influence the efficiency of DNA transfection of cells by a pulsed electric field (PEF). A previous study (Neumann, E., M. Schaefer-Ridder, Y. Wang, and P. H. Hofschneider. 1982. EMBO (Eur. Mol. Biol. Organ.) J. 1:841-845) has indicated that DNA topology is also an important determinant. We report an investigation of the PEF induced uptake, stability, and expression of three different topological isomers, circular supercoiled (scDNA), circular relaxed (crDNA), and linearized (lnDNA) forms of the plasmid pBR322, by Escherichia coli strain JM105. Monomeric pBR322 prepared by the electroelution from an agarose gel was in the supercoiled form. Treatment of the scDNA with wheat germ topoisomerase I removed the superhelicity and the DNA assumed the relaxed circular form. Treatment of scDNA by a restriction endonuclease, EcoRI or Hind III, linearized the DNA. The MgCl2-dependent bindings of all three forms of DNA to the cell surface were indistinguishable. So was the PEF induced cell uptake. In contrast, the transfection efficiency (TE) for the scDNA and the crDNA were high (approximately 2 x 10(8) micrograms-1 DNA at neutral pH), whereas that for the lnDNA was approximately five orders of magnitude lower (less than 1 x 10(3) micrograms-1 DNA). Analysis by agarose gel electrophoresis indicated that the PEF loaded ln DNA was degraded by the host cell within 3 h. However, the loaded scDNA and the crDNA were stable and expressed in the cytoplasm. We conclude that first, the PEF induced DNA entry into E. coli did not depend on the topology of the DNA. As cellular uptake of DNA also correlated with the surface binding, these data support electrophoresis of surface bound DNA as the dominating mechanism for the DNA entry. Second, the variations of TE for different topological forms of DNA reflected their relative stability in the host cells. Third, since the loaded DNA could be either rapidly degraded by the host enzyme or expressed, they were unlikely coated with a layer of protective lipid membrane. Thus, PEF induced cellular uptake of DNA is unlikely by the endocytotic mechanisms as was reported previously for the liposomes (Chernomordik, L. V., A. V. Sokolov, and V. G. Budker. 1990.Biochim. Biophys. Acta. 1024:179-183).

A solid state pulsed X-band source (crystal oscillator-multiplier type) yielding low transient phase distortion and high on/off ratio is described. Design tradeoffs are discussed, and it is shown that placing the RF switch before the last frequency multiplier is a suitable compromise design. Low switch VSWR and good bias stability in the switched stages are necessary for satisfactory transient phase performance.

The ribonucleotide reductase genes (nrd) are induced by thymine starvation. Deletion analysis of the sequences upstream of the cloned nrd genes was used to identify several regulatory regions. The start of transcription (nrdP) was mapped 110 bp upstream of nrdA, the first structural gene. A site required for positive regulation of nrd was mapped 135 bp upstream of nrdP in a region with two direct repeat sequences as well as potential secondary structure. Two other sites (one upstream of nrdP, the other downstream) were identified as sequences whose deletion markedly increase expression. These latter sites show sequence homology and probably interact since the effects of their individual deletion are not additive when combined.

Collin's synthesis of the equal ripple transmission line is shown to be computationally identical with Klopfenstein's synthesis. A simple computer program for computing impedance versus length of a tapered line is given, based on Collin's work.

Impedance versus distance data are provided on a -8.34 dB tapered coupler suitable for use in a broad-band -3 dB magic tee, correcting data previously presented by Fuchs.