In lysogenic bacteria, a specific system of repression, genetically determined by the prophage, prevents the expression of the phage genes necessary for phage replication and the production of infectious particles. With some prophage strains, treatment with ultraviolet (UV) or X radiation or with various chemical agents causes the repression system to break down and allows the production of phage. Mutant strains of bacteriophage with a thermosensitive repression system have been described.'-3Recently a bacterial thermosensitive mutant has been isolated.4 The mutant strain, when lysogenic for an inducible prophage, grows relatively normally in complete medium at 300, but at 400 phage production is induced. This paper describes some of the properties of this mutant in which elevated temperature alters conditions within the bacterium and thereby leads to prophage induction. This strain, when cured of prophage, forms filaments due to a defect in the formation of crosssepta. The conditions for filament formation are similar to those for induction of prophage in the lysogenic strain.Materials and Methods.-Bacteria and bacteriophage: Escherichia coli K-12, C-600, thr, leu, thi, lysogenic for X, was the parent of the strains used in this study. The lysogenic and nonlysogenic strains of C-600 were used as controls for experiments with the thermoinducible strain, whose isolation is described below. AB1899 ion, a strain of E. coli which forms filaments after low doses of UV light was obtained from Dr. P. Howard-Flanders. Hfr A-235 was obtained from Dr. S. E. Luria.Media and chemicals: Minimal medium M-635 was supplemented with 0.2% glucose, 0.2% Difco casamino acids (fortified with 0.01% each L-tryptophan, Lserine, and L-threonine), and 0.01% thiamin hydrochloride. Nutrient broth was prepared with either 16 gm. per liter of Difco nutrient broth powder or with 5 gm of meat extract, 10 gm of peptone, and 5 gm of NaCl per liter with the pH adjusted to 7.4 with NaOH. Hadacidin was provided by Dr. Shigeura, Merck, Sharp, and Dohme, Rahway, N.J.Growth experiments: Cultures which had been grown overnight at room temperature were diluted into M-63 medium containing 0.2% glucose, 0.2% fortified casamino acids, 0.01% thiamin HC1, and 0.01% guanosine and cytidine. The cultures were then shaken at 26-28°until in log phase. They were quickly chilled and centrifuged and the bacteria were washed and resuspended in cold M-63 medium containing glucose, casamino acids, and thiamin. Ten-ml samples of these suspensions were added to 125-ml nephelometer flasks containing the appropriate additions. The cultures were then shaken at 400, and the optical density of the cultures was followed at 660 m1A with a Klett colorimeter. Sensitivity to UV irradiation: Cells were grown in nutrient broth until in log phase, then chilled and resuspended in cold M-63 minimal medium' to a density 1903

Two enzymes have been partially purified from Escherichia coli and designated 3-methyladenine DNA glycosylases I and II. The glycosylase I is that described by Riazuddin & Lindahl [Riazuddin, S., & Lindahl, T. (1978) Biochemistry 17, 2110-2118]. The apparent molecular weight of glycosylase I is 20 000, and that of II is 27 000. Glycosylase I releases 3-methyladenine (3-MeA) while II releases 3-MeA, 3-methylguanine (3-MeG), 7-methylguanine (7-MeG), and 7-methyladenine (7-MeA). The rate of release of 3-MeA by glycosylase II is 30 times that of 7-MeG. Glycosylase I is missing in mutants tag 1 and tag 2 [Karran, P., Lindahl, T., Ofsteng, I., Evenson, G. B., & Seeberg, E. (1980) J. Mol. Biol. 140, 101-127]. In crude extracts, the 3-MeA activity of II is approximately 10% of the total 3-MeA activity. A 50% inactivation at 48 degrees C required 5 min for I and 65 min for II. The apparent Km for 3-MeA residues for glycosylase I was 1.4 x 10(-8) M. The enzyme was inhibited noncompetitively by 3-MeA with an average apparent Ki of 1.6 mM. The apparent Km for 3-MeA, for glycosylase II, was 9.2 x 10(-9)M, and it was not inhibited by 3-MeA. The 3-MeA and 7-MeG activities of the glycosylase II preparation could not be separated by isoelectric focusing, by chromatography on DEAE, Sephadex G-100, phosphocellulose, DNA-cellulose, or carboxymethylcellulose, or by heating at 50 degrees C. The apparent Km for 7-MeG was 1.1 x 10(-8)M. Glycosylase II released N1-(carboxyethyl)adenine and N7-(carboxymethyl)guanine from DNA treated with beta-[3H]propiolactone but did not release the aflatoxin B-1 adduct at N-7 of guanine.

Highly purified preparations of the DNAdependent RNA polymerase obtained from Escherichia coli contain about 2 g-atoms of tightly bound zinc per mol (molecular weight 370,000) of enzyme. When the purified enzyme is fractionated on Sephadex G-150 or G-200, corre-lation is observed between the zinc and enzymic activity. Although some of the preparations examined also contain iron, copper, and magnesium, the content of these metal ions show no consistent correlation with RNA polymerase activity.Initiation of RNA synthesis is specifically inhibited by 1,10-phenanthroline. Less-effective inhibition is observed for other chelating agents or for a nonchelating phenanthroline analog. The analog also exhibits a pattern of inhibition differing from that characteristic of 1,10-phenanthroline. Binding of purine nucleoside triphosphates at the lower-affinity (Kd = 0.15 mM) site may also be prevented by the addition of 1,10-phenanthroline. One or both of the bound zinc atoms may, therefore, participate in the initiation of RNA synthesis.DNA-dependent RNA polymerase (EC 2.7.7.6) purified from Escherichia coli has been shown to possess a specific binding site for purine nucleoside triphosphates, with a dissociation constant of about 0.15 mM (1, 2). Interaction of nucleoside triphosphates at this site occurred in the absence of added divalent metal ion, and was blocked by the addition of rifamycin. It was proposed that this site might be responsible for binding the 5'-terminal nucleoside triphosphate that is involved in the initiation of RNA synthesis (2, 3). The absence of a metal requirement for binding at this site suggested that the enzyme might contain a bound metal ion and, hence, that chelating agents might inhibit RNA polymerase in the presence of added Mg2+, which is required both for RNA synthesis (1) and for binding of nucleoside triphosphates at a second site (Kd = 0.015 mM) (2). Our results indicate that RNA polymerase contains 2 atoms of bound zinc per molecule of enzyme, and that the chelating agent, 1,10-phenanthroline, specifically inhibits the initiation of RNA synthesis. Slater et al. (4)

Previous studies from this and other laboratories have shown that angiotensin II (AII) induces [Ca2"j transients in proximal tubular epithelium independent of phospholipase C. AII also stimulates formation of 5,6-epoxyeicosatrienoic acid (5,6-EET) from arachidonic acid by a cytochrome P450 epoxygenase and decreases Na' transport in the same concentration range. Because 5,6-EET mimics AII with regard to Na' transport, its effects on calcium mobilization were evaluated. ICa2i, was measured by video microscopy with the fluorescent indicator fura-2 employing cultured rabbit proximal tubule. AII-induced ICa2+i transients were enhanced by arachidonic acid and attenuated by ketoconazole, an inhibitor of cytochrome P450 epoxygenases. Arachidonic acid also elicited a ICa2i1, transient that was attenuated by ketoconazole. 5,6-EET augmented (Ca2'], similar to that seen with AII, but was unaffected by ketoconazole. By contrast, the other regioisomers (8,9-, 11,12-, and 14,15-EET) were much less potent. ICa2+i, transients resulted from influx through verapamil-and nifedipine-sensitive channels. These results suggest a novel mechanism for AII-induced Ca mobilization in proximal tubule involving cytochrome P450-dependent arachidonic asid metabolism and Ca influx through voltage-sensitive channels. (J. Clin. Invest. 1991.

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