Growth of bacteriophage f2 in E. coli treated with rifampicin.

Fromageot, H. P. M.; Zinder, Norton D.

doi:10.1073/pnas.61.1.184

Cited by 55 publications

(31 citation statements)

References 17 publications

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“…Gels were stained and destained as described (17). Radioactivity in gel bands of radioactive samples was determined by slicing frozen cylindrical gels into 1-mm horizontal slices and counting as described by Fromageot and Zinder (18).…”

Section: Methodsmentioning

confidence: 99%

Highly Purified Colicin E3 Contains Immunity Protein

Jakes

Zinder

1974

Proc. Natl. Acad. Sci. U.S.A.

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Colicin E3, even when highly purified, still contains about one molar equivalent of a second protein, "E3 immunity protein." The two proteins are bound together in a complex that can be dissociated only under strongly denaturing conditions, such as electrophoresis in sodium dodecyl sulfate-polyacrylamide gels or by gel filtration in 6 M guanidine-hydrochloride.Colicin and immunity protein were separated by preparative electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The immunity protein prepared in this way was shown to be functionally and immunologically identical to immunity protein purified from colicinogenic cells by other methods. Colicin E3 that had been freed of immunity protein was much more active than complexed colicin in inhibiting protein synthesis in vitro.Colicin E3 is a protein antibiotic produced by certain strains of coliform bacteria, which kills other related bacterial strains (1). The colicin first attaches to specific receptors on the surface of sensitive cells (2, 3). Its ultimate effect is to inhibit protein synthesis by causing the cleavage of a fragment from the 3' end of the 16S ribosomal RNA (4,5). This colicininduced cleavage can also be observed in vitro (6, 7), so it is likely that the colicin penetrates cells to reach its target. Ribosomes from cells treated with colicin E3 in vivo, as well as ribosomes so treated in vitro, show reduced activity in protein synthesis invitro (6, 7).Colicin E3 is produced by strains carrying a specific plasmid DNA, the Col E3 factor (2, 8). Cells carrying this plasmid are also immune to colicin E3 (2, 9). This immunity is distinct from the "resistance" to colicin exhibited by bacteria that lack colicin receptors on their cell surfaces. Immunity to colicin E3 is due to the presence of an acidic protein of 10,000 molecular weight (E3 immunity protein), which can be isolated only from cells carrying the Col E3 factor (10, 11). When added in vitro to mixtures of colicin E3 and ribosomes, immunity protein prevents the inactivation of the ribosomes by E3. We have previously reported that immunity protein can be readily purified to homogeneity, since it can be separated from the colicin on columns of DEAE-Sephadex. However, highly purified colicin E3 contains a component that migrates with immunity protein on sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gels (10). We will now show that this component of colicin E3 is immunity protein and that it is present in the colicin E3 in molar equivalent amounts. MATERIALS AND METHODSColicin E3 and its immunity protein were purified from mitomycin C-induced cultures of W3110 (E3), as described (10, 12). Samples of purified colicin E3 were also generously provided by J. Sidikaro (Univ. of Wisconsin), D. Helinski (Univ. of Calif., San Diego), and S. Kerr (Univ. of Colorado, Boulder), for comparison with our own preparations.[3H]Leucinelabeled colicin E3 was prepared from 1 liter of cells grown to a density of 5 X 108/ml in minimal medium (13) containing 200 ,ug/ml of casamino acids.[3H]Leucine ...

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Section: Methodsmentioning

confidence: 99%

Highly Purified Colicin E3 Contains Immunity Protein

Jakes

Zinder

1974

Proc. Natl. Acad. Sci. U.S.A.

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“…and electrophoretic behavior of the f2 and MS2 proteins has received much attention (19,25,26). Of the three known viral proteins, the largest has been identified as the product of the replicase cistron and is known to be hyperproduced by coat mutants of MS2 and f2 (25,27).…”

Section: Resultsmentioning

confidence: 99%

Structure of the Poly(G) Polymerase Component of the Bacteriophage f2 Replicase

Fedoroff

Zinder

1971

Proc. Natl. Acad. Sci. U.S.A.

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A rifampicin-resistant poly(G) polymerase has been purified from f2 sus 11-infected cells. The poly (G) polymerase is believed to represent part of the f2 replicase on the basis of several criteria. It is present only in infected cells and shares the characteristic rifampicin resistance of crude f2 replicase activity. Partially purified poly(G) polymerase preparations exhibit replicase activity, synthesizing f2 'plus' strand RNA from denatured, partially doublestranded f2 RNA template. Highly purified poly(G) polymerase preparations, although lacking replicase activity, contain a protein which is electrophoretically identical to the protein product of the viral replicase cistron.The replicative cycle of RNA bacteriophages, particularly those of the f2 serotype, has been analyzed in some detail (1, 2). The structure of the RNA species produced during the infectious cycle is known, as is the temporal sequence of their synthesis (2). Studies of RNA metabolism in cells infected with various phage mutants indicate that the enzyme or enzyme complex responsible for phage RNA synthesis contains both bacterial and viral components (1, 3). In vitro studies of the f2-type phage RNA replicases have, however, been hampered by the instability of these enzymes and the difficulty in obtaining enzyme preparations free of endogenous template (2, 4-6). Thus, almost all of the currently available information on the structure and enzymology of phage replicase has come from studies of the more stable enzyme of the serologically unrelated RNA phage QB. The Q,3 enzyme appears to be quite complex, requiring several bacterial proteins in addition to a phage-specified protein to carry out net synthesis of phage 'plus'* strand RNA from 'plus' strand template (7-9). Several laboratories have shown that the Q, replicase contains a simpler 'core' enzyme defined by its ability to utilize phage 'minus't strands or poly(C) as template and its inability to use phage 'plus' strand template (7-10). Our studies of the f2 replicase were predicated on the assumption that the f2 enzyme is similarly subdivisible and that the 'core' enzyme would prove more amenable to purification and analysis than the complete enzyme.We report here a purification of the f2 replicase based on its rifampicin-resistant poly(C)-dependent poly(G) polymerase activity. The slurry was allowed to stand for 1 hr at room temperature, diluted 50-fold with cold absolute ethanol, filtered under suction through Whatman No. 3 paper, and allowed to dry. The powder was suspended in 20 ml of absolute ethanol/500 mg of cellulose, made 10 mM in magnesium acetate, and UV-irradiated for 30 min as described by Litman (17). The ethanol was removed by filtration and the powder was dried at room temperature. Unbound RNA was removed

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“…The development of the RNA bacteriophages such as fl, QP, and MS2 in E. coli is unaffected if rifampicin is added at 4 min or later after infection (FROMAGEOT and ZINDER 1968;JOCKUSCH et al 1970). Addition of rifampicin before or shortly after infection (at 50-100 J.lgjml) interferes with the intracellular growth ofthe bacteriophages (FROMAGEOT and ZINDER 1968;PASSENT and KAESBERG 1971; ENGEL-524 C. GURGO et al BERG 1972;MEIR and HOFSCHNEIDER 1972;ROTHWELL and YAMAZAKI 1972) and release of mature particles (ENGELBERG and SOUDRY 1971 a).…”

Section: H Friedman and S Spectermentioning

confidence: 99%

Chemotherapy of Viral Infections

Jones¹

1982

Handbook of Experimental Pharmacology

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Growth of bacteriophage f2 in E. coli treated with rifampicin.

Cited by 55 publications

References 17 publications

Highly Purified Colicin E3 Contains Immunity Protein

Highly Purified Colicin E3 Contains Immunity Protein

Structure of the Poly(G) Polymerase Component of the Bacteriophage f2 Replicase

Chemotherapy of Viral Infections

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