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D R Helinski

Publications and source records attributed to D R Helinski.

At least 163 records · Page 9Linked to original sources
PubMed1974

Effect of inhibitors of ribonucleic acid and protein synthesis on the cyclic adenosine monophosphate stimulation of plasmid ColE1 replication.

Addition of cyclic adenosine 3'-5'-monophosphate (c-AMP) to growing Escherichia coli cells, colicinogenic for the plasmid ColE1, results in a fourfold stimulation in the rate of synthesis of the plasmid deoxyribonucleic acid (DNA). The stimulation is transient (30 min) and is succeeded by a brief period (30 min) of cessation of plasmid DNA replication. The stimulation of ColE1 DNA replication also occurs in chloramphenicol-treated cells. Rifampin inhibits ColE1 DNA replication in the presence or absence of c-AMP. Employing thymine starvation conditions to stop ColE1 DNA synthesis, it was found that c-AMP, added during the period of thymine starvation, effected a stimulation in the amount of subsequent replication which took place when replicating conditions were restored. The stimulatory effect of c-AMP under these conditions was not prevented by chloramphenicol but was completely eliminated when rifampin was present. Under these conditions, when rifampin was added after the effect of c-AMP was allowed to occur, subsequent replication of the plasmid could take place, but only one round of replication occurred. A model to account for the c-AMP effects is presented.

Carbon Radioisotopes↗
PubMed1974

Properties of the relaxation complexes of supercoiled deoxyribonucleic acid and protein of the R plasmids R64, R28K, and R6K.

The presence of supercoiled deoxyribonucleic acid in the form of a relaxation complex is described for the antibiotic resistance plasmids R64, R28K, and R6K. The properties of these relaxation complexes indicate that they consist of a covalently closed circular deoxyribonucleic acid molecule associated with an activable, single strand-specific endonuclease.

Ampicillin↗
PubMed1973

Temperature-sensitive mutants for the replication of plasmids in Escherichia coli. II. Properties of host and plasmid mutations.

Host mutations in Escherichia coli K12 selected for the temperature-sensitive replication of the bacterial plasmid colicinogenic factor E(1) (ColE(1)) exhibit a pleiotropic effect with respect to the effect of the mutation on other extra-chromosomal elements. The mutations also vary with respect to the time of incubation of the cells at 43 degrees C required for complete cessation of ColE(1) DNA synthesis. While the synthesis of the bacterial chromosome appears unaffected, supercoiled ColE(1) DNA replication stops immediately in some mutants and gradually decreases during several generations of cell growth before stopping in others. Mutations isolated in the ColE(1) plasmid resulted in only a gradual cessation of ColE(1) DNA synthesis over several generations of cell growth at 43 degrees C. Conjugal transfer of the ColE(1) and ColV factors occurs normally in the host mutants when the transfer is carried out at the permissive temperature; however, the presence of a group I mutation in the donor cell prohibited conjugal transfer of either plasmid DNA at 43 degrees C to a normal recipient cell. Similarly, the presence of this mutation in the recipient prevented the establishment of ColE(1) or ColV in the mutant recipient cell upon conjugation with a normal donor at 43 degrees C. Various host ColE(1) replication mutants carrying either ColE(1) or ColE(2) were also defective in the mitomycin C-induced production of colicin E(1) or colicin E(2) at 43 degrees C. The majority of the host mutations examined exhibited a temperature sensitivity to growth in deoxycholate in addition to the inhibition of plasmid DNA replication, suggesting a membrane alteration in these mutants when grown at the restrictive temperature.

Coliphages↗
PubMed1973

Temperature-sensitive mutants for the replication of plasmids in Escherichia coli. I. Isolation and specificity of host and plasmid mutations.

Temperature-sensitive mutants of Escherichia coli defective in the replication of the plasmid colicinogenic factor E1 (ColE(1)) were isolated following mutagenesis of E. coli K12 strain carrying the ColE(1) factor. Following the mutagenic treatment an enrichment procedure utilizing the replacement of thymine with bromouracil in the ColE(1) DNA duplicated at the restrictive temperature was used. The mutants isolated following this enrichment step were the result of a mutation event either in the host chromosome or in the ColE(1) plasmid. The host mutants fell into three phenotypic classes based on the effect each mutation had on the maintenance of a variety of other extrachromosomal DNA elements. Phenotypic class I mutations affected all E. coli plasmids, both the I and F sex factor types as well as the ColE(1) factor. Phenotypic class II mutations affected the maintenance of the ColE(1) and the F sex factor type plasmids and not the I type, while phenotypic class III mutations affected only ColE(1) replication. None of these mutations was found to have a significant effect on the replication of the E. coli chromosome. The plasmid-linked mutations fell into two phenotypic classes on the basis of the ability of the Flac episome to complement the mutation in the ColE(1) plasmid.

Animals↗
PubMed1973

Stimulation by cyclic adenosine monophosphate of plasmid deoxyribonucleic acid replication and catabolite repression of the plasmid deoxyribonucleic acid-protein relaxation complex.

Colicinogenic factors ColE1 and ColE2 are bacterial plasmids that exist in Escherichia coli as supercoiled deoxyribonucleic acid (DNA) and as strand-specific, relaxation complexes of supercoiled DNA and protein. Newly replicated ColE1 DNA becomes complexed with protein after the replication event. This association of DNA and protein can take place under conditions in which DNA or protein synthesis is arrested. The addition of cyclic adenosine monophosphate (c-AMP) to normal cells growing in glucose medium results in a six- to tenfold stimulation in the rate of synthesis of the protein component(s) of the complex and a three- to fivefold stimulation in the rate of ColE1 DNA replication. Employing mutants deficient in catabolite gene activator protein or adenylate cyclase, it was shown that synthesis of both the plasmid-determined protein colicin E1 and the protein component(s) of the ColE1 relaxation complex is mediated through the c-AMP-catabolite gene activator protein system. Addition of c-AMP to ColE2-containing cells results in the stimulation of synthesis of ColE2 DNA and relaxation protein(s) as well as in the production of a protein component of the ColE2 relaxation complex that renders it sensitive to induced relaxation by heat treatment. In the case of ColE2, synthesis of the relaxation protein(s) is not dependent upon catabolite gene activator protein.

Bacterial Proteins↗
PubMed1973

Temperature-sensitive mutants for the replication of plasmids in Escherichia coli: requirement for deoxyribonucleic acid polymerase I in the replication of the plasmid ColE 1 .

An Escherichia coli mutant (polA1), defective in deoxyribonucleic acid (DNA) polymerase I, (EC 2.7.7.7) is unable to maintain colicinogenic factor E1 (ColE1), whereas several sex factor plasmids are maintained normally in this strain. polA1 mutant strains containing these sex factor plasmids do not exhibit a readily detectable plasmid-induced polymerase activity. A series of E. coli mutants that are temperature sensitive for ColE1 maintenance, but able to maintain other plasmids, were isolated and shown to fall into two phenotypic groups. Mutants in one group are defective specifically in ColE1 maintenance at 43 C, but exhibit normal DNA polymerase I activity. Mutations in the second group map in the polA gene of E. coli, and bacteria carrying these mutations are sensitive to methylmethanesulfonate (MMS). Revertants that were selected either for MMS resistance or the ability to maintain ColE1 were normal for both properties. The DNA polymerase I enzyme of two of these mutants shows a pronounced temperature sensitivity when compared to the wild-type enzyme. An examination of the role of DNA polymerase I in ColE1 maintenance indicates that it is essential for normal replication of the plasmid. In addition, the presence of a functional DNA polymerase I in both the donor and recipient cell is required for the ColV-promoted conjugal transfer of ColE1 and establishment of the plasmid in the recipient cell.

Centrifugation, Density Gradient↗
PubMed1972

Isolation of supercoiled colicinogenic factor E 1 DNA sensitive to ribonuclease and alkali.

The synthesis of the covalently-closed, circular DNA form of colicinogenic factor E(1) (ColE(1)) continues in Escherichia coli cells after the addition of chloramphenicol. A large portion of the purified supercoiled ColE(1) DNA molecules made in the presence of chloramphenicol are converted to the open circular DNA form after treatment with alkali (pH 13), RNase A, or RNase H. These treatments do not significantly affect the covalently-closed form of ColE(1) DNA isolated from normally growing E. coli cells. The open circular product resulting from treatment of supercoiled ColE(1) DNA with RNase A possesses a single break in one strand of the circular duplex. The site sensitive to RNase A occurs with equal probability in either of the complementary strands. Both synthesis of ColE(1) DNA and the formation of supercoiled ColE(1) DNA sensitive to RNase A or alkali are prevented by the inhibitor of RNA synthesis, rifampicin. These results indicate that covalently-closed ColE(1) DNA containing one or more ribonucleotides accumulates during ColE(1) replication in the presence of chloramphenicol. It is proposed that this incorporated RNA served as a primer during the initiation of synthesis of ColE(1) DNA and that its removal from the circular DNA is inhibited in cells incubated in the presence of chloramphenicol.

Carbon Isotopes↗
PubMed1972

Effect of growth conditions on the formation of the relaxation complex of supercoiled ColE1 deoxyribonucleic acid and protein in Escherichia coli.

Colicinogenic factor E1 (ColE1) is present in Escherichia coli strain JC411 (ColE1) cells to the extent of about 24 copies per cell. This number does not appear to vary in situations which give rise to twofold differences in the amount of chromosomal deoxyribonucleic acid (DNA) present per cell. If cells are grown in the absence of glucose, approximately 80% of the ColE1 molecules can be isolated as strand-specific DNA-protein relaxation complexes. When glucose is present in the medium, only about 30% of the plasmid molecules can be isolated as relaxation complexes. Medium shift experiments in which glucose was removed from the medium indicate that within 15 min after the shift the majority (>60%) of the plasmid can be isolated as relaxation complex. This rapid shift to the complexed state is accompanied by a two- to threefold increase in the rate of plasmid replication. The burst of replication and the shift to the complexed state are both inhibited by the presence of chloramphenicol. Inhibition of protein synthesis in log cultures by the addition of chloramphenicol or amino acid starvation allows ColE1 DNA to continue replicating long after chromosomal replication has ceased. Under these conditions, noncomplexed plasmid DNA accumulates while the amount of DNA that can be isolated in the complexed state remains constant at the level that existed prior to treatment. In the presence of chloramphenicol, there appears to be a random dissociation and association of ColE1 DNA and "relaxation protein" during or between rounds of replication.

Bacterial Proteins↗
PubMed1971

Strand-specific supercoiled DNA-protein relaxation complexes: comparison of the complexes of bacterial plasmids ColE1 and ColE2.

Certain bacterial plasmids can be isolated as unique complexes of supercoiled circular DNA and protein. These complexes are distinguished by the conversion of the supercoiled DNA to the relaxed or open-circular DNA form upon treatment with ionic detergents, proteases, or alkali. This report demonstrates that the open-circular DNA resulting from the pronase-induced relaxation of the complexes of colicinogenic factors E(1) (ColE(1)) and E(2) (ColE(2)) possesses a strand-specific break. In each case this break is found in the heavy strand of the DNA as defined by CsCl centrifugation in the presence of poly(U,G). In addition, the ColE(1) and ColE(2) complexes exhibit certain properties that are plasmid specific. Heat treatment, and to a lesser extent pronase treatment, inactivates the ColE(2) complex, making it insensitive to agents that formerly were capable of inducing relaxation (conversion of the DNA to the open-circular form). In contrast, the ColE(1) complex is not inactivated by these treatments. The potential role of these strand-specific relaxation complexes in DNA replication is discussed.

Bacterial Proteins↗
PubMed1971

Circular DNA.

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Binding Sites↗