Endonuclease Assays. Reaction mixtures (0.05 ml) were as described by Henry and Knippers (7), containing 0.1 M NaCl, and DNA and A protein as indicated. Samples were analyzed by agarose gel electrophoresis, alkaline sucrose gradient (8), and/or propidium diiodide (PrdI2)-CsCI density gradient centrifugation.Agarose Gel Electrophoresis. Slab agarose gels (1.4%) were prepared by dissolving agarose (Miles) in 86 mM Tris, 83 mM boric acid, 3 mM EDTA (Tris-borate buffer).Electrophoresis was carried out at 100 V for 4 hr at 25°; gels were then soaked in 0.5 ,g/ml of ethidium bromide in Trisborate buffer for 10-15 min and stained bands were photographed under a shortwave UV lamp. Negatives were traced in a Joyce-Loebl microdensitometer.PrdI2-CsCI Density Gradient Centrifugation. Samples (2.2 ml) were mixed with 2.2 g of solid CsCI, and 0.2 ml of 2 mg/ml PrdI2 solution (initial density = 1.568 g/ml) and centrifuged for 40 hr at 40,000 rpm in the Spinco SW 50.1 rotor at 200. RESULTSPurification of A Protein. A protein was purified from E. coli H514 infected with OX am3, based on the complementation assay described in Materials and Methods (Table 1). The A protein was purified 4000-fold with a yield of 10% (step VI). This activity (and endonuclease activity) was completely heat labile (2 min at 600) and N-ethylmaleimide sensitive (Table 2). All attempts to analyze A protein in nondenaturing gels were unsuccessful; concentrated solution aggregated and did not penetrate neutral gels. Sodium dodecyl sulfate (NaDodSO4) polyacrylamide gels of step VI contained trace contaminating proteins which were removed by further purification; the conditions used were the same as in step IV, except that a smaller column (1 ml, packed volume) was used and 1 mg of protein of step VIP was applied and eluted with a 50 ml of 0.3-2.0 M NaCI gradient. Active fractions (step VII, specific activity 3120 units/mg) showed a single band in NaDodSO4 t In this case, 100 g (wet weight) of cells were used for step VI of the isolation.
Extracts of Escherichia coli strains infected with bacteriophage 4X174 catalyze DNA synthesis dependent on double-stranded, circular kX174 replicative form I (OX RFI) by a semiconservative process. The Replication of /X174 single-stranded, circular DNA occurs in three stages: synthesis of (a) parental replicative form (RF), (b) progeny RF, and (c) progeny single-stranded, circular DNA. In stage a, parental RF synthesis depends solely upon host proteins, and a cell-free system catalyzing this reaction has been developed (1, 2). Complementation assays with this system allowed the isolation of Escherichia coli dnaB, C(D), G, and Z gene products (3, 4) and other proteins required for conversion of OX174 DNA to RFII (5-7). This system is specific for OX174 DNA and is inactive with duplex OXRF forms.In avo, progeny RF and/or progeny qX174 DNA synthesis also depends upon E. coli dnaB (8), dnaC(D) (9), dnaE (9), dnaG (10), dnaZ (11,12), and rep gene (13) products in addition to the kX174 gene A product (14).To study the mechanism of double-stranded DNA replication, we have developed a cell-free system dependent upon added OX RFI DNtA. This communication describes the requirements for this system catalyzed by crude fractions of E. coli. MATERIALS AND METHODSBacterial and Phage Strains. E. coli and bacteriophage qX174 used and their sources were: E. coli H560 (pol Al, Su+). All other strains were Su-, including BT1029 (pol A1, dnaBts), BT1026 (polAi, dnaEts), and BT1040 (polAj, dnaEts) (Dr. J. (prepared from OX-infected cells described below). Reaction mixtures were incubated for 40 min at 300 (unless specified), and acid-insoluble radioactivity was determined (3). When products were characterized by sedimentation analyses, samples were treated either as described by Sakakibara and Tomizawa (20) or by addition of EDTA (24 mM) and Sarkosyl (final 2%) followed by incubation at 420 for 10 min. RESULTSProperties of Conversion of OX RF to Progeny RF In Vitro.Crude fractions from 4X174 infected E. coli incorporated dTMP into an acid-insoluble, alkali-resistant, RNase-resistant, DNase-sensitive product when supplemented with crude fractions from uninfected E. coli*. The reaction depended on added OX RFI DNA, provided small amounts of fraction II was used (Table 1 , Fig. 1); omission of ATP, dNTPs, or Mg++ also abolished activity. The optimum concentrations of ATP and Mg++ required were approximately 1 mM and 10 mM, respectively. N-Ethylmaleimide abolished DNA synthesis, whereas rifampicin had no effect. Nalidixic acid and novobiocin, specific inhibitors of bacterial DNA synthesis (21, 22), markedly inhibited DNA synthesis (Table 1 , Fig. iD); in contrast, formation of OX RFII in vitro from X174 DNA was insensitive to these drugs (data not shown). Replication of OX RFI was inhibited 50% by 50mM KCI, 15% sucrose (final), or 8.5% glycerol (final).The rate of synthesis was proportional to the amount of fraction II and template DNA added (Fig. 1A, B, and C). Fraction II prepared from cells grown and infected at 300 re...
We wish to report the nucleotide sequence of leucine transfer RNA (tRNALeu) from E. coli B.The particular tRNALeu labelled with 32P was first purified by electrophoresis in a polyacrylamide gel of RNA extracted from E. coli B grown in the presence of 32P-phosphate ( fig. 1). Examination of the various bands in the gel revealed that the particular leucine tRNA species was present in the slowest moving transfer RNA band (a), which in addition contains only one other, as yet, unknown tRNA. Final purification of the tRNALeu was achieved by chromatography on a benzoylated DEAE cellulose column [ 1 ] , the tRNALm eluting at a NaCl concentration around 0.7 M. The unknown tRNA species is eluted with 1 M NaCl containing 10% ethanol. Enzymatic digestions of the isolated tRNALeu and subsequent sequence determinations of the resulting oligonucleotides were carried out as previously described in detail by us [2][3][4] . Fig. 2 shows a two-dimensional fractionation of a ribonuclease T, digest of tRNALeu and an accompanying diagram giving the sequences of the nucleotides. A pancreatic ribonuclease digest was fractionated in a similar way and the sequences of the oligonucleotides determined. To overlap the end products large fragments were prepared by partial Tr or pancreatic ribonuclease digestions and separated by homochromatography [5]. The sequences of the partial fragments were determined from their complete T, and pancreatic ribonuclease products. From the large number of partial digestion products a unique sequence could be deduced ( fig. 3). As is the case for all other tRNAs known, the sequence can be arranged in the "clover leaf" pattern.The tRNA,,, consists of 87 nucleotides and is thus the largest tRNA sequenced so far. We have not 168 Fig. 1. Purification of tRNALeu by electrophoretic fractionation on a polyacrylamide gel. The gel (9.5% acrylamine, 0.5% bisacrylamide) was prepared as described by Adams et al.[lo] except that the buffer used was 0.09 M tris, boricsyid pH 8.3 containing 0.025 M EDTA. RNA labelled with P was isolated from E. co/i B grown in the presence of 32P-phosphate by phenol extraction. Electrophoresis was carried out at 40' for 16 hr at 350 V and 30 mA. North-Holland Publishing Company -Amsterdam
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.