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Biomedical subjects

D R Helinski

Publications and source records attributed to D R Helinski.

At least 127 records · Page 7Linked to original sources
PubMed1979

Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans.

pRK212.2, a derivative of the broad host range plasmid RK2, contains two EcoRI cleavage fragments, A and B, neither of which can replicate by itself in Escherichia coli. Fragment A (41.7 kilobases), but not fragment B (14.4 kilobases), can be cloned by insertion into the unrelated plasmids mini-F and ColE1. Fragment B contains the origin of replication and the ampicillin-resistance determinant of RK2. Transformation of E. coli cells containing the mini-F-fragment A hybrid plasmid with fragment B DNA results in the recircularization and replication of fragment B as a nonmobilizable plasmid (pRK2067) with the copy number and incompatibility properties of RK2. Fragment B cannot be cloned in the absence of fragment A because the latter fragment suppresses a function, specified by fragment B, that results in loss of host cell viability. A small segment (2.4 kilobases) of fragment B that contains the RK2 origin of replication but no longer affects host cell growth in the absence of fragment A had been cloned previously by insertion into a ColE1 plasmid. This hybrid plasmid, designated pRK256, will replicate in E. coli polA mutants only when a fragment A-bearing helper plasmid is present. These results demonstrate that the potentially lethal function specified by fragment B of RK2 is not necessary for replication and that at least one trans-acting function is directly involved in RK2 replication.

Coliphages↗
PubMed1979

Bidirectional replication of the mini-ColE1 plasmid pVH51.

Replicating molecules of the min-ColE1 plasmid pVH51 have been examined by electron microscopy after cleavage with the restriction endonuclease EcoRI. Replication apparently starts at a unique site indistinguishable from the origin of replication used by the parental plasmid ColE1. In contrast to ColE1, the structure of the majority of the replication intermediates was consistent with a bidirectional mode of replication. A minor portion of the molecules appeared to replicate unidirectionally in either direction from the same origin.

DNA Replication↗
PubMed1979

Bacterial plasmids: autonomous replication and vehicles for gene cloning.

The use of recombinant DNA techniques in the analysis of the structure and replication of bacterial plasmids has provided much information on the properties of these genetic elements and has led to the construction of plasmid elements that are potentially very useful as gene cloning vehicles in Escherichia coli and other gram-negative bacteria. The genetic and molecular properties of plasmids mini-F, ColE1, and RK2 are described with particular emphasis on the origin and direction of replication and the identification of genetic regions essential for maintenance of these elements in the extra-chromosomal state. Low molecular weight derivatives of each of these plasmids have been obtained and a restriction enzyme map determined for these various derivatives. A hybrid DNA molecule consisting of a low molecular weight derivative of ColE1 joined to a segment of bacteriophage DNA has been constructed and shown to be capable of existing either as a plasmid element or packaged as an infectious viral particle. Finally, several of the low molecular weight derivatives of these plasmids described have certain advantages as vehicles for the cloning of DNA including derivatives of he broad host range plasmid RK2 that may be useful for gene cloning in gram-negative bacteria distantly related to E. coli.

Chromosomes, Bacterial↗
PubMed1978

Altered DNA-protein relaxation complex in a replication mutant of plasmid ColE1.

Approximately 200,000 clones of Escherichia coli carrying mutagen-treated colicinogenic plasmid E1 (ColE1) were examined for irreversible loss of the plasmid at 43 degrees. Thirty of these clones that appeared to be most defective in plasmid DNA replication at the non-permissive temperature were selected for the study of: (a) the kinetics of plasmid and chromosomal DNA replication during a temperature shift in either the presence or absence of chloramphenicol; (b) the temperature stability of the plasmid DNA-protein relaxation complex; and (c) the temperature effect on F-promoted conjugal transfer. Two mutant plasmids, pJC307 and pJC301, showed defects in their relaxation complex. The relaxation complex of pJC307 exhibited an altered temperature stability in vitro. Reversion to temperature resistant replication resulted in four out of five cases in a concomitant change in the temperature stability of the relaxation complex. Conjugal mobility of this mutant was not markedly reduced at the permissive or non-permissive temperature. Plasmid pJC301 could not be isolated in the form of a relaxation complex and it was very poorly mobilized in an F'-promoted conjugation. These results indicate that the ColE1 plasmid codes for at least one of the proteins of the relaxation complex and that the relaxation complex is involved in ColE1 DNA replication. In addition, the properties of the mutant plasmid pJC301 are consistent with a role for the complex in the mobilization of ColE1 during conjugation.

Colicins↗
PubMed1978

Replication of antibiotic resistance plasmid R6K DNA in vitro.

A soluble extract prepared from cells of an Escherichia coli strain carrying the antibiotic resistance plasmid R6K is capable of carrying out the complete process of R6K DNA replication. DNA synthesis in vitro is dependent on the four deoxyribo- and ribonucleotide triphosphates and is sensitive to rifampin and streptolydigin, inhibitors of DNA-dependent RNA polymerase. The incorporation of deoxyribonucleotides into R6K DNA also is sensitive to actinomycin D, novobiocin, arabinofuranosyl-CTP, and N-ethylmaleimide. Kinetics of synthesis are linear for 60 to 120 min. Replication proceeds semiconservatively and supercoiled closed-circular DNA molecules are synthesized. Analysis by alkaline sucrose gradient centrifugation indicated that the early R6K DNA products contain DNA fragments of approximately 18 S in size, corresponding to the length between the R6K alpha origin of replication and the terminus of replication observed in vivo. Addition of exogenous supercoiled R6K DNA is inhibitory to the in vitro system, whereas the addition of R6K DNA in the form of relaxation complex stimulates R6K DNA synthesis to a small extent.

Anti-Bacterial Agents↗
PubMed1978

Trans-complementation-dependent replication of a low molecular weight origin fragment from plasmid R6K.

A non-self-replicating segment (1370 base pairs) of plasmid R6K was cloned in E. coli and shown to trans-complement temperature-sensitive replication mutants of this plasmid. This segment contains the gene which codes for a protein required for initiation of replication of the plasmid, and was used as a helper in a functional assay for an origin of replication in R6K derivatives. A 420 bp fragment, derived from R6K DNA, was shown to carry a functional origin since it was capable of replicating as a plasmid in E. coli cells carrying the helper segment either on the host chromosome or on a plasmid Col E1 derivative. The copy number of the origin fragment in cells carrying the helper segment on the chromosome is essentially the same as the copy number of R6K. A model for the positive regulation of plasmid R6K replication is presented.

Chromosome Mapping↗
PubMed1978

Requirement of a plasmid-encoded protein for replication in vitro of plasmid R6K.

Conditions are described for the replication of exogeneous R6K DNA in an in vitro system prepared from Escherichia coli cells. Replication of plasmid DNA in this system is semiconservative and sensitive to actinomycin D, novobiocin, arabinofuranosyl-CTP,N-ethylmaleimide, and inhibitors of DNA-dependent RNA polymerase. An ammonium sulfate fraction prepared from cells carrying the R6K plasmid is required for replication. A direct role in replication for a plasmid-encoded protein, designated pi, in this fraction is indicated by the inactivity of this fraction when prepared from cells carrying a temperature-sensitive mutant plasmid and the thermolability of this fraction when prepared from cells carrying a partial revertant of the mutant plasmid. This plasmid-encoded protein is necessary for the initiation of R6K DNA replication and functions before or during the formation of nascent RNA in the initiation process. The results of titration assays of this protein using various template DNAs suggest that the protein interacts with the plasmid DNA at the region essential for DNA replication.

Anti-Bacterial Agents↗
PubMed1978

Construction of a novel plasmid-phage hybrid: use of the hybrid to demonstrate ColE1 DNA replication in vivo in the absence of a ColE1-specified protein.

A hybrid bacteriophage, P420, was constructed in vitro; it contains part of bacteriophage P4 and a 3.6-kilobase derivative of plasmid ColE1. In Escherichia coli, the plasmid-phage hybrid can exist as a stable plasmid or can be packaged into infective bacteriophage particles. Replication of P420, directed by the ColE1 replicon, was found to occur after P420 phage infection of E. coli cells that had been incubated in the presence of chloramphenicol. Replication began without a lag period and resulted in the synthesis of covalently closed circles of P420 DNA. Like ColE1 DNA replication but unlike that of P4, replication was dependent on DNA polymerase I and was sensitive to rifampicin. The presence of a resident ColE1 plasmid in the infected cells resulted in an inhibition of the replication of the incoming P420 DNA. These results indicate that ColE1 does not require a plasmid-coded protein to replicate its DNA in vivo and demonstrate the utility of P4 bacteriophage for coupling bacteriophage properties to a plasmid replicon.

Bacterial Proteins↗
PubMed1977

Unidirectional replication of the P-group plasmid RK2.

The mode of replication of the broad host-range plasmid RK2 has been determined from examination of molecular replicative forms cleaved with the restriction endonucleases EcoRI and Hind III. Replication is unidirectional, and proceeds from a unique origin. The location of the origin and other evidence suggests that genes involved in plasmid maintenance are not tightly clustered.

DNA Replication↗
PubMed1977

Physical and genetic studies with restriction endonucleases on the broad host-range plasmid RK2.

The cleavage map of the plasmid RK2 was determined for the five restriction endonucleases EcoRI, HindIII, BamH-I, SalI and HpaI. DNA has been inserted into several of these sites and cloned in Escherichia coli. Efforts to obtain derivatives of RK2 reduced in size by restriction endonuclease digestion of the plasmid were not successful and indicated that genes required for the maintenance of this plasmid in E. coli are not tightly clustered. An RK2 derivative possessing an internal molecular rearrangement was obtained by transformation with restriction endonuclease digests of the plasmid.

DNA Replication↗
PubMed1977

Purification and characterization of covalently closed replicative intermediates of ColEl DNA from Escherichia coli.

Pulse-labeled ColEl DNA molecules, undergoing replication in Escherichia coli cells either in the absence or presence of chloramphenicol, were extracted and purified by neutral sucrose density gradient sedimentation and equilibrium centrifugation in an ethidium bromide-cesium chloride gradient. In the dye-buoyant density gradient, the replicating molecules were found in regions between the supercoiled and open-circular nonreplicating plasmid DNA, as well as in the open-circular region. In a neutral sucrose gradient, peaks of pulse label were found in the region of 26 to 38 S as well as at the 23 and 17 S positions corresponding to the positions of supercoiled and open-circular ColEl DNA. In alkaline sucrose gradient, nascent ColEl DNA was found to sediment as discrete peaks corresponding to 5-6, 7-9, and 14-16 S, indicating that at least one growing strand of the replicating molecule is produced discontinuously. In the electron microscope, many of the molecules appeared as partially supercoiled structures containing two open-circular branches of equal length, of less than 20% to more than 90% replicated. Branched open-circular molecules were not observed to any significant extent without prior treatment to induce single-strand scissions. The parental strands of the replicating molecules were determined to be covalently closed, but the superhelical density of the DNA was shown to be progressively decreased as replication proceeded.

Centrifugation, Density Gradient↗
PubMed1977

Gene cloning and containment properties of plasmid Col E1 and its derivatives.

Colicinogenic plasmid E1 (Col E1) and Col E1 derivatives offer advantages as plasmid cloning vehicles with regard to both utility and biological containment. The Col E1 derivative pCR1 does not alter those essential characteristics of the enfeebled Escherichia coli strain x1776 that make this strain particularly useful as a host-vehicle system for recombinant DNA research.

Bile Acids and Salts↗