Plasmid ColE1 DNA replication in Escherichia coli strains temperature-sensitive for DNA replication

Mutants of the dnaA, dnaC, dnaD, polC, dnaF and dnaG gene loci were tested for their capacity for colicinogenic plasmid E1 (ColE1) replication at a non-permissive temperature. It was found that ColE1 replication was independent of the dnaA gene function and dependent on dnaC, D, F and G. ColE1 replication in the polC mutant E486 continued for several hours but at a greatly reduced rate. No effect was found of the dnaG mutation on thymine-deprivation-induced "priming" of ColE1 replication at the non-permissive temperature. The mutants also were tested for aberrant replication intermediates of plasmid DNA as well as a temperature sensitive supercoiled DNA-protein relaxation complex. RNA-containing supercoils were found to accumulate in a poIC mutant also blocked for protein synthesis.     

DNA synthesis in an Escherichia coli dna B dnaC mutant.

Summary An Escherichia coli K12 dnaB dnaC mutant was constructed by P1 transduction of the dnaC allele into a dnaB recipient strain dnaB dnaC transductants were discriminated from dnaB mutants by their inability to grow at 40° C after lysogenization with phage P1bac. The dnaB dnaC mutant character was verified by 1. P1 transduction, and 2. by in vitro complementation with dnaB and dnaC wild type protein fractions.DNA synthesis was studied in strains containing dnaB, dnaC, or dnaB dnaC alleles in an otherwise uniform genetic background with the dnaB character either unsuppressed or suppressed by P1bac prophage. Degradation at 42° C of [³H]-thymidine pulselabeled DNA in dnaB and dnaB dnaC mutants is suppressed by P1bac. However, unlike the dnaC mutant, the P1bac lysogen of the dnaB dnaC mutant exhibits an abrupt cessation of DNA synthesis and less residual cell divisions at 42° C indicating an inhibition of DNA chain elongation rather than a defect in DNA initiation. It is suggested that denaturation of the dnaB protein affects the dnaC function.     

Characterization of RNA synthesis in an Escherichia coli mutant with a temperature-sensitive lesion in stable RNA synthesis

Previous experiments with Escherichia coli strain 2S142 have shown that the synthesis of stable RNA is preferentially blocked at the restrictive temperature. In this paper, we have examined the capacity of this mutant strain to synthesize RNA in vitro. Growth of the strain for as short a period as 10 min at 42 degrees C resulted in a 40 to 60% loss of RNA synthetic capacity and a fourfold decrease in percent rRNA synthesized in toluenized cell preparations. The time course for the loss and recovery of this RNA synthetic capacity correlated very well with the changes in RNA synthesis observed in vivo. We found no difference in temperature sensitivity of the purified RNA polymerase from the mutant and the parental strains. Moreover, there was no detectable alteration in the amount of enzyme, specific activity of the enzyme, or electrophoretic mobility of the subunits when the mutant strain was grown at 42 degrees C. The capacity for rRNA synthesis was also measured with the Zubay in vitro system (Reiness et al., Proc. Natl. Acad. Sci. 72:2881-2885, 1975). Supernatant fractions (S-30) prepared from cells grown at 30 degrees C were capable of up to 31.2% rRNA synthesis, using phi 80d3 DNA as template. S-30 fractions from cells grown at 42 degrees C synthesized 8.6% rRNA. The bottom one-third of the S-100 fraction and the ribosomal salt wash from 30 degrees C cells contained one or more factors which partially restored preferential rRNA synthesis in S-30 fractions from cells grown at 42 degrees C. Preliminary evidence suggests that the factor(s) is protein in nature.     

Studies on bacteriophage T7 DNA synthesis in vitro. II. Reconstitution of the T7 replication system using purified proteins.

Summary DNA synthesis in vitro using intact duplex T7 DNA as template is dependent on a novel group of three phage T7-induced proteins: DNA-priming protein (activity which complements a cell extract lacking the T7 gene 4-protein), T7 DNA polymerase (gene 5-protein plus host factor), and T7 DNA-binding protein. The reaction requires, in addition to the four deoxyribonucleoside triphosphates, all four ribonucleoside triphosphates and is inhibited by low concentrations of actinomycin D. Evidence is presented that the priming protein serves as a novel RNA polymerase to form a priming segment which is subsequently extended by T7 DNA polymerase. T7 RNA polymerase (gene 1-protein) can only partially substitute for the DNA-priming protein. At 30°C, deoxyribonucleotide incorporation proceeds for more than 2 hours and the amount of newly synthesized DNA can exceed the amount of template DNA by 10-fold. The products of synthesis are not covalently attached to the template and sediment as short (12S) DNA chains in alkaline sucrose gradients. Sealing of these fragments into DNA of higher molecular weight requires the presence of E. coli DNA polymerase I and T7 ligase. Examination of the products in the electron microscope reveals many large, forked molecules and a few eye-shaped structures resembling the early replicative intermediates normally observed in vivo.     

A novel Escherichia coli mutant capable of DNA replication in the absence of protein synthesis.

An Escherichia coli mutant capable of continued DNA synthesis in the presence of chloramphenicol has been isolated by an autoradiographic technique. The DNA synthesis represents semiconservative replication of E. coli DNA. It can occur in the presence of chloramphenicol or in the absence of essential amino acids, but not in the presence of an RNA synthesis inhibitor, rifampin. The mutant, termed constitutive stable DNA replication (Sdr^c) mutant, appears to grow normally at 37 °C with a slightly slower growth rate than that of the parental strain. DNA replication in the mutant occurs at a reduced rate after 60 minutes in the absence of protein synthesis and continues linearly for several hours thereafter. This distinct slowdown in the DNA replication rate is due to a reduced rate of DNA synthesis in all the cells in the population. Constitutive stable DNA replication appears to require the dnaA and dnaC gene products. The sdr^c mutation has been mapped near the pro-lac region of the E. coli chromosome. The mutation is recessive. Autoradiographic experiments have ruled out the possibility of multiple initiations during a cell cycle. The implication of the above findings is discussed in terms of the regulation of chromosome replication in E. coli.     

DNA synthesis in P1 infected E. coli mutants temperature-sensitive in DNA replication

Summary P1 DNA is synthesized in the E. coli ts dna mutants 165/70 (elongation defect) and 252 (initiation defect) at elevated temperatures. In strain 165/70, P1 infection at 41°C leads to phage production accompanied by a transient recovery of bacterial DNA synthesis. No phages are produced byt P1 DNA is still synthesized in strain 252 if infected after host DNA replication has come to a halt at 42°C.     

Accumulation of bacteriophage T7 head-related particles in an Escherichia coli mutant.

We have analyzed the structure of the late cytoplasmic RNAs made after infection with wild-type simian virus 40 and a set of viable mutants, four of which have deletions and one an insertion within the nucleotide sequence specifying the leader segment of the 16S and 19S mRNA's. The principal findings are: (i) simian virus 40 16S and 19S mRNA's made during infections with wild-type virnds and possibly in the nucleotide sequence comprising the "leader" segments. (II) "Spliced" 16S and 19S mRNA's are made during infections with each of the mutants although, in some cases, the ratio of 19S to 16S mRNA species is reduced. (iii) The deletion or insertion of nucleotides within the DNA segment defined by map position 0.70 to 0.75 causes striking alterations in the types of leader structures in the late mRNAs. (iv) Many of the late RNA leader segments produced after infection with the mutants appear to be multiply spliced, i.e., instead of the major 200- to 205-nucleotide-long leader segment present in wild-type 16S mRNA, the RNAs produced by several of the deletion mutants have leaders with whort discontiguous segments.     

A temperature-sensitive mutant of Escherichia coli with an altered RNA polymerase beta' subunit.

$\text{Penicillium charlesii}$ extracts contain UDP-galactose:NAD⁺ 2-hexosyl oxidoreductase (1). ADP-ribose also serves as a substrate resulting in formation of NADH and an oxidized ADP-ribose derivative. Treatment of the oxidized product with NaBH₄ followed by hydrolysis at pH 2 and 100° releases xylose as well as ribose. We conclude that ADP-D-$\text{glycero}$-D-$\text{glycero}$-3-pentosulose (ADP-3-ketoribose) is the product derived from ADP-ribose.