James A. Wechsler

Mutation and identification of the F plasmid locus determining resistance to acridine orange curing

Abstract A region in plasmid F responsible for resistance to acridine orange curing has been identified. Insertion of the transposable element for ampicillin resistance Tn 3 at the 45.8- or 46.35-kilobase coordinate in mini-F plasmids results in resistance to curing by acridine orange and also results in increased plasmid copy numbers. In contrast, copy number mutants of mini-F induced by chemical mutagenesis are sensitive to acridine-induced loss of plasmid. Hence, the Tn 3 -induced acridine orange resistance does not represent a suppression of sensitivity because of elevated plasmid copy numbers. General hypotheses to explain our results are presented.

The isolation and characterization of Escherichia coli dnaB::Tn10 insertion mutations

SummaryExploitation of the ability of the ban protein encoded by phage P1 to compensate for dnaB-defective host mutations, allowed the isolation of dnaB::Tn10 insertion mutations. The presence of P1bac prophage was required for survival of dnaB::Tn10 mutants, and such lysogens were cryosensitive. The insertions were shown to map in dnaB by transduction and this was confirmed by complementation analysis. The dnaB::Tn10 (P1bac) strains were non-permissive for λ growth but did support the growth of λ-dnaB+specialized transducing phage. No antigenically active dnaB product could be detected by immunologic assays using either of two methods. In addition, it was shown that the observed cryosensitivity of P1bac suppression was a direct result of reversible inactivation of the ban protein at low temperature.

Growth of phages λ and φX174 under PIban protein control in the absence of host dnaB function

Abstract The absence of dnaB product in dnaB ::Tn 10 insertion mutants was exploited to examine the ability of coliphages λ and φX174 to use the dnaB analog protein ( ban ) of phage P1. A dnaB ::Tn 10 (P1 bac ) lysogen grew at 37° but not at 25° and did not support growth of λ at any temperature. Progeny phage were produced if λ infection was at high multiplicities and this production was shown to be dependent on recombination. In contrast to λ, φX174 utilized ban efficiently at all temperatures including 25°. If the level of ban was increased by using P1 bac crr lysogens, the host survived at 25° and λ also grew though the latent period was extended and the rate of phage production was greatly reduced. It was also shown that mutant dnaB 252, which is defective for initiation of chromosome replication, severely restricted φX174 growth at 42° and was somewhat restrictive even at 30°. Kinetic experiments demonstrated that the block to φX174 growth in this mutant was complete at 5 min after infection but by 20 min postinfection the dnaB function was no longer required.

RNA polymerase is required for DNA initiation in vitro

SummaryWe have previously reported in vitro complementation assays for chromosome initiation that enable dnaA and dnaC mutant extracts to synthesize DNA. To examine the role of RNA polymerase in chromosome initiation, inhibitors of the enzyme and anti-RNA polymerase antibody were used. Though rifampicin failed to efficiently inhibit ribonucleoside triphosphate polymerization under the assay conditions, both streptolydigin and anti-RNA plymerase antibody abolished ribonucleic acid synthesis completely. Antibody effectively inhibited chromosome initiation in the dnoA mutant based reaction but streptolydigin did not. Neither streptolydigin nor antibody affected the dnaC-dependent assay. It was concluded that RNA polymerase is required for initiation but not necessarily to polymerize a polyribonucleotide. A scheme for the sequence of initiation events is presented.

Initiation of chromosomal DNA synthesis in vitro

SummaryAn in vitro complementation assay for initiation of chromosomal DNA replication is described. The initiation reaction is dependent upon extract from either of two hybrid-plasmid containing strains. Each hybrid plasmid carries a suppressor of dnaA-ts mutations. The in vitro DNA synthesis is heavily biased toward the origin region, and the origin of replication (oriC) is replicated as determined by DNA-DNA hybridizations.

High-yield purification of small, single-stranded DNA

Abstract Several simple methods that aid in the high-yield purification of small, single-stranded DNA are described. The methods rely initially on the silicone coating of laboratory ware with a particular brand of commercial silicone coating. The quantitative precipitation of DNA with cetyltrimethylammonium bromide and the fractionation of the DNA by molecular-sieve chromatography allowed purification based on length of the polynucleotide strands. For purification of density-labeled DNA, Cs 2 SO 4 isopycnic centrifugation and precipitation by cetyltrimethylammonium bromide gave good purification of the DNA of interest even at sizes as small as ∼200 nucleotides.

DNA replication intermediates synthesized by lysates of dnaB, dnaG and dnaB dnaG mutants in vitro.

SummaryIsogenic dnaB, dnaG, and dnaB dnaG mutants were constructed and used as extracts in the cellophane-disc in vitro DNA replication system. The increased proportion of 5S DNA characteristic of the dnaB extract and the lack of Okazaki piece synthesis characteristic of the dnaG extract were both apparent in analysis of the dnaB dnaG mutant extract reaction. A hypothetical scheme to explain these results and those of others is presented.

Deletion map of the Escherichia coli K-12 dnaB gene

SummaryTwenty-four dnaB alleles have been ordered by deletion mapping of the Escherichia coli K-12 dnaB gene. The position of the alleles had no linear correlation with the known phenotypes of dnaB mutation: fast shut-off, slow shut-off but immediate change in rate, and GroP. The relevance of the deletion map to future studies of the dnaB protein is discussed.

Complementation of a dnaC initiation defect in vitro

SummaryThe dnaC28 mutant, CT28-3b, is an initiation defective dnaC strain. Extracts of the mutant failed to synthesize DNA in vitro when the strain was incubated at the restrictive temperature for two generation times prior to preparation of the extract. Addition of a complementing extract from a Col-E1::dnaC+ hybrid plasmid containing strain or of partially purified dnaC protein resulted in substantial synthesis. Hybridization of the DNA made by these in vitro complementation extracts showed that a significant portion of this DNA was from the region near the chromosomal origin of replication.