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...

The messenger ribonucleic acid (mRNA) pattern of log-phase and sporulating cells of Bacillus subtilis has been analyzed by deoxyribonucleic acid (DNA)-RNA hybrid studies with the complementary-strand fragments of DNA. Approximately 80% of the mRNA of log-phase and sporulating cells from stages I, III, and IV hybridizes with the heavy DNA fragments, and 20% hybridizes with the light DNA fragments. Hybrid competition studies indicated that there was either a greatly reduced rate of transcription or a turn-off of some log-phase genes during the sporulation stages. However, a significant amount of log-phase gene transcription occurred even at late stages of sporulation. Similar studies indicate a significantly increased rate of transcription or a turn-on of sporulation phase genes during the latter stages of sproulation. There is a sequential increase in the amount of sporulation-specific transcription from both complementary-strand fragments of DNA. These results indicate that the RNA polymerase population in sporulating cells can transcribe both log-phase and sporulation-phase genes. MATERIALS AND METHODS Bacterial strain and media. B. subtilis 168 wild type was grown at 37 C in a modified Schaeffer medium (2 x SG) (10) for all experiments except for the preparation of DNA. For this purpose, cells were 775

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Rifampin-resistant mutants ofBacillus subtilis that are conditionally temperature sensitive during sporulation have been isolated and characterized. The mutants can grow at the same rate as the wild type at the nonpermissive temperature but cannot sporulate. Depending on the mutation, they are blocked at either stage 0 to I, II, II to III, or IV of sporulation. The mutants showed an altered pattern of RNA synthesis after the stage at which they were blocked. The effect of rifampin on the activity of enzymes from mutant vegetative cells and sporulating cells was significantly different, suggesting that the RNA polymerase from sporulating cells was different from the RNA polymerase of vegetative cells. These results suggest that the conformation of the RNA polymerase core plays an important role in determining correct transcription during sporulation.

A cell-free system that catalyzes 4X174 replicative form I (supercoiled circular duplex, RFI)dependent 4X174 DNA synthesis has been isolated from Escherichia coli infected with 4X174 phage. The products formed with such preparations are viral strands as judged by hybridization to poly(U,G) followed by equilibrium centrifugation in CsCl. This 4X174 DNA- Reaction mixtures (0.05 ml) contained, in order of addition: 20 mM Tris-HCl (7.5), 10 mM MgCl2, 4 mM dithiothreitol, 1 mM ATP, 0.2 mM NAD+, 40MAM each of dATP, dGTP, dCTP, and [a-32P]dTTP (200-700 cpm/pmol), 0.1 mM each of CTP, UTP, and GTP, 1 nmol of kX174 [3H]RFI (1-6 cpm/pmol), 0.2 mg of ammonium sulfate fraction from E. coli strain BT1029, 11 Mg of fraction II for RF replication or 23 Mg of fraction II for ss DNA synthesis, and 0.1 unit of dna C. Reaction mixtures were incubated as indicated and acid-insoluble radioactivity was measured (6). In the case of the ss DNA system, reactions were stopped by addition of 10 mM EDTA (final) and 0.2% sodium dodecyl sulfate (final) and heated at 650 for 15 min followed by a 4 hr incubation at 370 with autodigested proteinase K (1 mg/ml). The mixtures were dialyzed against 50 mM Tris-HCl, pH 8.0/5 mM EDTA for 12 hr. Viral strand formation was measured after hybridization to poly(U,G) followed by CsCl isopycnic centrifugation for 70 hr as described by Baas et al. (9). The amount of viral strand that banded at the expected density is the value reported above. Under these conditions, >90% of the incorporated 32p was recovered in this fraction. Reference (30,ug/ml) and purified by two phenol extractions followed by pancreatic RNase treatment (1 hr at 370 with 2 ,ug per A260 unit); the DNA was further purified by isopycnic banding in propidium diiodide/ CsCI followed by neutral sucrose gradient centrifugation.Preparation of Protein Fractions. Unless indicated, protein fractions were as described (6), and growth of E. coli and X174 infection were carried out at 300. Ammonium sulfate fractions from uninfected E. coli were as described (6) 4195The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.