We have developed a method for efficiently generating transient pores in the outer membranes ofEscherichia 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 extraceilular spaces. The method has been used to transform bacterial cells with an efficiency greater than 109 transformants per ,ug 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.

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Abstract— The isolation and partial characterization of several photoadducts formed between 8‐methoxypsoralen (8‐MOP) and cytosine is described. The formation of these adducts was analysed in E. coli DNA containing 3H‐labeled cytosine and/or 14C‐labeled thymine, and in oligonucleotides of defined sequence. The major initial adduct has been identified as an 8‐MOP cytosine monoadduct, most likely forming at the pyrone end of the 8‐MOP molecule. Further irradiation converts this adduct to several other species, including both cytosine:cytosine and cytosine:thymine diadducts, as well as a number of derivative monoadducts. One isomer of the C:T diadduct appears to undergo a reversible isomerization under the conditions normally used to analyse adduct mixtures by HPLC. The isomerization can cause this adduct to exhibit a retention time on reversed‐phase HPLC closely resembling either that of a thymine‐thymine crosslink or a thymine monoadduct.

We describe a new approach for quantitating furocoumarin adducts in DNA using enzymatic hydrolysis followed by resolution and recovery of the adduct molecules by electrophoresis on polyacrylamide tube gels. The resolution of this method approaches that of high pressure liquid chromatography but at a considerably lower cost. Digestion conditions using DNase II and spleen phosphodiesterase II to yield mononucleotides and adducted bases were worked out for DNA containing 8‐methoxypsoralen or 4′‐hydroxymethyl‐4,5′,8‐trimethylpsoralen. The phosphatase activity of DNase II was shown to be much more active on dNMPs than on adducted bases. This can be exploited to reduce the background of radioactivity from labeled dNMP's trailing into the adduct peaks, thereby increasing the effective sensitivity of the technique when quantitating adducts made with non‐radioactive drug in labeled DNA. Since resolution of adducts requires dense (30%) acrylamide gels, we have devised a method for making extremely uniform high density gels by polymerization under static pressure. A continuous collection apparatus was constructed to recover material from gels. The identity of the resolved species was determined by several different methods, including analysis by HPLC.