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D R Helinski

Publications and source records attributed to D R Helinski.

At least 145 records · Page 8Linked to original sources
PubMed1977

Bidirection replication from a unique origin in a mini-F plasmid.

Replicating molecules of the mini F-kanamycin resistance plasmid, pML31, derived from F'lac, have been isolated from Escherichia coli as covalently-closed circular DNA molecules. These molecules were examined in the electron microscope after digestion with either EcoRI or BamHI restriction endonuclease. The structure of the majority of the molecules replicating was consistent with a bidirectional mode of replication starting at a unique origin on the F-fragment. This origin is located approximately 2.3 kilobases from one of the EcoRI sites. Orientation of the F-fragment relative to the physical map of F showed the position of the origin to be at 42.6 kilobases. A small proportion of molecules appeared to be replicating unidirectionally in either direction from this origin. Termination of replication of pML31 apparently occurs in the fragment containing the locus for kanamycin resistance in a unique region opposite the origin in the circular DNA molecule.

DNA Replication↗
PubMed1976

Small circular DNA in Drosophila melanogaster.

Covalently closed small circular DNA isolated from Drosophila melanogaster is described. The small circular DNA is found in blastema stage eggs and in Schneider's cell culture line 2 and a cloned subline of line 2. It is heterogeneous in size, although the size distributions and mean sizes differ for each source. The small circular DNA from Schneider's line 2 cells ranges from 0.09-7.3 mum, with a mean contour length of 1.1 mum. This DNA has a buoyant density of 1.703 g/cc and appears to be present predominantly in the nuclear fraction of detergent-disrupted cells. the restriction enzyme EcoRl cleaves approximately 40% of the small circular DNA with a bias toward the larger size classes. Both logarithmic and stationary phase cells contain approximately 3-40 average sized small circular DNA molecules per cell, representing a maximum of 0.03% of the total cellular DNA. Exposure to cycloheximide or puromycin for 14 hr results in a 30 fold increase in the number of small circles per cell, but reduces the mean length of the circular DNA to 0.3 mum. The drug-amplified DNA has a buoyant density in the range of 1.698-1.703 g/cc. No amplification was seen in cells treated with either inhibitor for 3.5 hr. Ethidium bromide, cytosine arabinoside, beta-ecdysone, and insulin all had no significant effect on the amount per cell of either small circular DNA or mitochondrial DNA.

Animals↗
PubMed1976

Generation in vitro of deletions in the broad host range plasmid RK2 using phage Mu insertions and a restriction endonuclease.

Several non-lethal deletions of the broad host range plasmid RK2 (molecular weight of 37.6 . 10(6) have been produced in vitro. The method employed relied on the single HindIII restriction nuclease site in RK2 and the ability of phage Mu to insert and thereby add new HindIII restriction sites at various positions in the plasmid. The deleted plasmids have in each case lost kanamycin (Km) resistance, and in two cases are defective in self-transmissibility. The method used to reduce the size of the RK2 plasmid also results in the cloning of each of the two ends of the Mu phage DNA on the plasmid derivatives.

Ampicillin↗
PubMed1976

Characterization of a mini-ColC1 plasmid.

An in vitro constructed plasmid, pVH15, consisting of the entire genome of the plasmid ColE1, the tryptophan operon of Escherichia coli, and regions of the bacteriophage PHI80pt190, spontaneously gave rise in E. coli to a mini-ColE1 plasmid consisting of approximately one-half of the ColE1 genome and a small segment of phi80pt190 DNA. This mini-ColE1 plasmid, designated pVH51, has a molecular weight of approximately 2.1 X 10(6) and possesses a single EcoRI restriction site. Heteroduplex analyses showed that about 90% of the pVH51 plasmid hybridizes to about 50% of the ColE1 plasmid. Phenotypically, pVH51 did not produce colicin E1 but conferred immunity to this colicin. The number of mini-ColE1 plasmid molecules per cell was maintained at a four- to fivefold higher level than normal ColE1. A mini-ColE1 hybrid plasmid, designated pML21 and consisting of pVH51 and the kan fragment of plasmid pSC105 inserted at the EcoRI restriction site of mini-ColE1, was maintained at a lower copy number level than pVH51. As in the case of normal ColE1, both pVH51 and pML21 continued to replicate in the presence of chloramphenicol. The promotion of conjugal transfer of pVH51 and pML21 by a self-transmissible plasmid was greatly reduced compared with normal ColE1.

Chloramphenicol↗
PubMed1976

Method for the isolation of the replication region of a bacterial replicon: construction of a mini-F'kn plasmid.

A purified fragment of deoxyribonucleic acid (DNA) that determines resistance to kanamycin and is incapable of self-replication was used to select a self-replicating fragment from an EcoRI endonuclease digest of the sex factor F'lac. This F'lac fragment, exhibiting a molecular weight of 6 X 10(6), carries the genes essential for maintenance of the F replicon in Escherichia coli cells. The constructed mini-F'km plasmid also retains the incompatibility properties of the parent F'lac plasmid. Large amounts of the kanamycin resistance fragment of a molecular weight of 4.5 X 10(6) with an EcoRI-cleaved, self-replicating derivative of colicinogenic plasmid E1 that has a molecular weight of 2.2 X 10(6), The recombinant plasmid is able to replicate extensively in E. coli in medium containing chloramphenicol, and, therefore, large quantities of this plasmid DNA can be obtained. The substantial difference in size between the two fragments in the recombinant plasmid greatly facilitates their separation by preparative agarose gel electrophoresis.

Chloramphenicol↗
PubMed1976

Plasmid DNA replication.

Recent studies have provided some insight to the overall characteristics of plasmid replication in bacteria. The ColE 1 and R6K plasmids replicate via a covalently-closed circular intermediate. Replication is initiated at a fixed origin and is unidirectional in the case of ColE 1 and bidirectional for R6K. In the case of the plasmid R6K. the bidirectional replication is asymmetric and sequential, proceeding from a fixed origin to a fixed terminus located approximately 20% of the R6K genome from the origin. RNA serves as a primer for plasmid DNA replication. The base composition and sequence of the 5'-terminus of the RNA segments in ColE 1 DNA have been determined. Finally, the properties of plasmid relaxation complexes and the possible role of these complexes in plasmid DNA replication are discussed.

Chloramphenicol↗
PubMed1975

Molecular vehicle properties of the broad host range plasmid RK2.

The pladmid RK2 is stably maintained in a broad range of gram-negative bacteria. The RK2 DNA has a single Eco RI restriction site. The insertion of a DNA fragment into this site does not interfere with either plasmid maintenance or self-transmissibility. Because RK2 has a broad host range, it should be useful for the construction in vitro of hybrid plasmid molecules capable of being established by conjugal transfer or transformation into many genera of gram-negative organisms.

Conjugation, Genetic↗
PubMed1975

Relaxation complexes of plasmid DNA and protein. I. Strand-specific association of protein and DNA in the relaxed complexes of plasmids ColE1 and ColE2.

The ColE1 and ColE2 relaxation complexes of supercoiled DNA and protein were purified from Escherichia coli cells. Protein remains associated with the open circular DNA of these complexes after induction of relaxation with sodium dodecyl sulfate. The protein is associated specifically with the strand that possesses a site-specific break in the Co1E1 and ColE2 relaxed complexes. This protein remains associated with the DNA after centrifugation of the relaxed complex in a neutral or alkaline (pH 12.5) cesium chloride gradient or treatment with 8 M urea, 2 M NaSCN, 2M LiCl, 0.2 M sodium acetate, pH 4.6, and 70% formamide at 60 degrees.

Acetates↗
PubMed1975

Relaxation complexes of plasmid DNA and protein. II. Characterization of the proteins associated with the unrelaxed and relaxed complexes of plasmid ColE1.

The proteins of the DNA-protein relaxation complex of plasmid ColE1 were labeled with [3H]leucine by growth of ColE1 containing Escherichia coli cells in the presence of this radioactive labeled amino acid. Three major [3H]leucine-labeled proteins are found associated with the supercoiled DNA in the ColE1 relaxation complex. The molecular weights of these proteins, determined by sodium dodecyl sulfate-acrylamide gel electrophoresis, are 60,000, 16,000, and 11,000, respectively. Induction of relaxation of the supercoiled DNA by treatment of the complex with sodium dodecyl sulfate results in a dissociation of the two smaller proteins from the DNA. The 60,000 protein, however, remains associated specifically with the nicked strand of the open circular DNA. The strand-specific association of this protein with the relaxed DNA resists heat denaturation of the DNA, sedimentation through an alkaline (pH 12.5) sucrose gradient, and centrifugation to equilibrium in an alkaline (pH 12.5) CsCl gradient.

Bacterial Proteins↗
PubMed1975

Relaxation complexes of poasmid DNA and protein. III. Association of protein with the 5' terminus of the broken DNA strand in the relaxed complex of plasmid ColE1.

The location of the protein in the open circular DNA form of the ColE1 DNA-protein relaxation complex, induced by treatment with sodium dodecyl sulfate, has been studied using several enzymes of DNA metabolism. Escherichia coli exonucleases I and III are able to degrade extensively the nicked strand of the relaxed complex from the 3' end. DNA polymerase I can initiate synthesis using the relaxed complex as template-primer and specifically extends the 3' end of the nicked strand. The 5' end of the sodium dodecyl sulfate-relaxed complex, however, is blocked to the 5'-3' hydrolitic action T7 exonuclease. This block remains after trypsin treatment of the sodium dodecyl sulfate-relaxed complex but is removed by Pronase treatment. T4 DNA ligase is unable to seal either the sodium dodecyl sulfate-relaxed complex or the Pronase-treated relaxed complex even after pretreatment of the relaxed complex with T4 DNA polymerase and polynucleotide kinase. However, pretreatment with DNA polymerase I and the four deoxyribonucleoside triphosphates facilitates ligase closure of the Pronase-treated relaxed complex but not the sodium dodecyl sulfate-relaxed complex. These studies indicate that the protein in the relaxed ColE1 complex is located at or near the 5' end of the nicked strand.

Bacterial Proteins↗
PubMed1975

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 Replication↗
PubMed1975

Bidirectional replication of plasmid R6K DNA in Escherichia coli; correspondence between origin of replication and position of single-strand break in relaxed complex.

Replicating molecules of plasmid R6K DNA have been purified as covalently closed circular DNA forms and analyzed in the electron microscopy after cleavage with the EcoRI restriction endonuclease. It has been determined that in most cases replication proceeds bidirectionally from an origin whose position is indistinguishable from the site of the single-strand break (nick) in the open circular DNA form of the relaxation complex of R6K DNA and protein. Evidence is presented for the existence of a unique replication terminus asymmetrically placed approximately 20% of genome size from the origin. The positions of the replication forks in a majority of the molecules indicate that replication proceeds sequentially from the fixed origin first in one direction to the terminus and then progresses from the origin in the other direction.

DNA Replication↗
PubMed1974

Relaxation complexes of plasmids ColE1 and ColE2: unique site of the nick in the open circular DNA of the relaxed complexes.

The product of the induced relaxation of supercoiled DNA-protein relaxation complexes of colicinogenic factors E1 (ColE1) and E2 (ColE2) is an open circular DNA molecule with a strand-specific nick. Cleavage of the open circular DNA of each relaxed complex with the EcoRI restriction endonuclease demonstrates that the single-strand break is at a unique position. The site of the single-strand break in the relaxed ColE1 complex is approximately the same distance from the EcoRI cleavage site as the origin of ColE1 DNA replication.

Binding Sites↗
PubMed1974

Plasmid ColEl as a molecular vehicle for cloning and amplification of DNA.

DNA fragments obtained from EcoRI endonuclease digestion of bacteriophage varphi80pt190 (trp(+)) and the plasmid ColE1 were covalently joined with polynucleotide ligase. Transformation of Escherichia coli trp(-) strains to tryptophan independence with the recombined DNA selected for reconstituted ColE1 plasmids containing the tryptophan operon and the varphi80 immunity region. Similarly, an EcoRI endonuclease generated fragment of plasmid pSC105 DNA containing the genetic determinant of kanamycin resistance was inserted into the ColE1 plasmid and recovered in E. coli. The plasmids containing the trp operon (ColE1-trp) and the kanamycin resistance gene were maintained under logarithmic growth conditions at a level of 25-30 copies per cell and accumulate to the extent of several hundred copies per cell in the presence of chloramphenicol. Cells carrying the ColE1-trp plasmid determined the production of highly elevated levels of trp operon-specific mRNA and tryptophan biosynthetic enzymes.

Chloramphenicol↗