Col El Research Articles

Theory for Radii and Second Virial Coefficients. 1. Highly Charged Polyelectrolytes

We develop a theory for the dependence of the radii of gyration and second virial coefficients of highly charged polyelectrolytes on molecular weight and ionic strength. The theory is based on using (i) the Flory-Fisk 1 distribution of chain conformations to describe the elastic entropy and (ii) a charged rod model of local intrachain repulsions based on the full Poisson-Boltzmann equation. To describe the overlap between two approaching chains, we go beyond the rigid sphere model of Orofino and Flory 2 and assume the chains can change coil size and can deform as elastic ellipsoids to avoid unfavorable interchain repulsions. Added salt weakens electrostatic repulsions, leading to reduced radius of gyration and second virial coefficients. Without adjustable parameters this approach gives reasonable agreement with data on T7 DNA, Col El DNA, and poly(styrenesulfonate)s over two decades of ionic strength.

Identification of the plasmid-encoded immunity protein for colicin El in the inner membrane of Escherichia coli

A set of plasmids containing portions of the Col El plasmid were transformed into recA − cells. These cells, after UV irradiation, only incorporate labelled amino acids into plasmid-encoded proteins. UV-irradiated cells label a 14.5 kDa band if they are phenotypically immune to colicin E1, and do not contain this band if they are sensitive to colicin E1. We conclude that the 14.5 kDa protein is the colicin E1 immunity protein. When the inner and outer membranes of these cells are fractionated, the labelled band appears in the inner membrane. The immunity protein must be an intrinsic inner membrane protein, confirming the predictions made by hydrophobicity calculations from primary sequence data. Maxicell Col El plasmid Immunity protein Hydrophobicity calculation

H1 histone, polylysine and spermine facilitate nucleosome assembly in vitro

Nucleosome formation has been studied in a system containing relaxed Col El DNA, core histones and an extract of Drosophila embryos. The formation of nucleosomes was established by the introduction of supercoils into DNA. The degree of DNA supercoiling was shown to be higher if nucleosomes were assem-bled in the presence of the H1 histone, polylysine ( M r 20000) or spermine. These agents do not stimulate relaxation and are the more effective the earlier they are added to the reaction. Thus, the H1 histone, polylysine and spermine facilitate nucleosome assembly in vitro.

Genetic toxicity of dopamine

The genetic toxicity of dopamine was studied in a battery of test systems including DNA single-strand break analysis in cultured human skin fibroblasts, the Salmonella/mammalian-microsome mutagenicity test, sister-chromatid exchange analysis in human lymphocytes, the mouse-lymphoma forward mutation assay, the sex-linked recessive lethal test in Drosophila melanogaster and the micronucleus test in mouse and rat. Dopamine at concentrations of 50–300 μg/ml induced DNA strand breaks in human fibroblasts. It also gave a positive response in the mouse-lymphoma forward mutation assay, where a dose-dependent increase in the frequency of mutant cells was observed in the presence of dopamine, 94–750μ/ml. All other tests showed no response to dopamine. The dopamine-induced DNA strand breaks in human fibroblasts were inhibited by superoxide dismutase or dithiothreitol. Furthermore, dopamine caused nicking of circular Col El DNA and bound to calf thymus DNA in vitro. It is suggested that this genetic activity of dopamine in vitro relates to oxidation of dopamine and the generation of reactive oxygen radicals, semiquinones and quinones. It is unlikely that similar reactions would occur and cause genotoxic activity of dopamine in vivo.

A Col E1 hybrid plasmid containing Escherichia coli genes complementing d-xylose negative mutants of Escherichia coli and Salmonella typhimurium.

The Clarke and Carbon bank of Col El - Escherichia coli DNa hybrid plasmids was screened for complementation of d-xylose negative mutants of E. coli. Of several obtained, the smallest, pRM10, was chosen for detailed study. Its size was 16 kilobases (kb) and that of the insert was 9.7 kg. By transformation or F'-mediated conjugation this plasmid complemented mutants of E. coli defective in either D-xylose isomerase or D-xylulose kinase activity, or both. The activity of D-xylulose kinase in E. coli transformants which bear an intact chromosomal gene for this enzyme was greater than that for the host, due to a gene dosage effect. The plasmid also complemented D-xylose negative mutants of Salmonella typhimurium by F'-mediated conjugation between E. coli and S. typhimurium. Salmonella typhimurium mutants complemented were those for D-xylose isomerase and for D-xylulose kinase in addition to pleiotropic D-xylose mutants which were defective in a regulatory gene of the D-xylose operon. In addition, the plasmid complemented the glyS mutation in E. coli and S. typhimurium. The glyS mutant of E. coli was temperature sensitive, indicating that the plasmid carried the structural gene for glycine synthetase. The glyS mutation in E. coli maps at 79 min, as do the xyl genes. The behaviour of the plasmid is consistent with the existence of a d-xylose operon in E. coli. The data also suggest that the plasmid carries three of the genes of this operon, specifically those for D-xylose isomerase, D-xylulose kinase, and a regulatory gene.

The nucleotide sequence of the bacteriocin promoters of plasmids Clo DF13 and Co1 E1: role of lexA repressor and cAMP in the regulation of promoter activity.

Treatment of cells, harbouring the bacteriocinogenic plasmic Clo DF13 with mitomycin-C, which induces the cellular SOS response, results in a significantly increased transcription of the operon encoding the bacteriocin cloacin DF13, the immunity protein and the lysis protein H. The nucleotide sequences of the promoter regions and N-terminal parts of the bacteriocin genes of Clo DF13, Col E1 and the pMB1 derivative pBR324 have been determined. A comparison of these sequences with those of corresponding regions of the lexA, recA and uvrB genes revealed that the promoter regions of the bacteriocin genes studied contain binding sites for the lexA protein, which is the repressor of the E. coli DNA-repair system. Using both, a thermosensitive lexA host strain and a host with pACYC184 into which the lexA gene had been cloned, we were able to demonstrate, that in vivo the lexA protein is involved in the regulation of bacteriocin synthesis. From the data presented, we conclude that bacteriocin synthesis is controlled at least by the lexA repressor. It has been reported that also catabolite repression might play an essential role in the control of bacteriocin synthesis. Computer analysis of the DNA sequence data indicated that the promoter regions of both, the cloacin DF13 and colicin E1 genes contain potential binding sites for the cyclic AMP-cyclic AMP Receptor Protein complex.

Effect of ionic strength on the pKa of ligands bound to DNA

AbstractThe pKa of 3,8‐diamino‐6‐phenyl‐phenanthridine (DAPP), a nonquaternary analog of ethidium bromide, has been determined spectrophotometrically as a function of sodium ion concentration both free in solution and complexed to DNA. Unwinding angle determinations with this compound were determined with Col El DNA using ethidium bromide as a standard. The unwinding angle for DAPP was 24 ± 2° relative to 26° for ethidium, and this suggests that DAPP binds in a manner quite similar to ethidium and with no significant outside bound DAPP under these experimental conditions. Isobestic behavior was obtained on spectrophotometric pH titration above pH 5 as long as the ratio of DNA‐phosphate to ligand was between 100 and 300 and the DNA phosphate concentration was approximately 0.01M or greater. The loss of isosbestic behavior which occurred below pH 5 is probably due to titration of the 8 amino group of the ligand complexed to DNA. To circumvent this problem, pKa values and the extinction coefficient of the acidic species were both determined by a computer program using experimental data obtained above pH 5. The pKa of the free compound has only a minor dependence on ionic strength, while the pKa of the ligand bound to DNA in an intercalated complex depends strongly on the sodium ion concentration. The pKa of the DAPP‐DNA complex is a linear function of –log[Na+] as predicted by the ion‐condensation theory of polyelectrolytes. It was determined that DAPP is essentially completely bound to DNA under the conditions of these experiments by (1) determination of apparent pKa values as a function of total DNA concentration, (2) calculation of binding constants for the neutral species of DAPP, and (3) spectral analysis of the protonated and neutral species of DAPP bound to DNA relative to DAPP free in solution. These results support the ion‐condensation theory; provide an independent method for measuring ψ*, the average number of counterions associated per phosphate of DNA in the intercalated conformation; and illustrate that there are no specific pH effects or absolute pKa values for ligands bound to DNA, but only ionic‐strength‐dependent results.

Preparative separation of the complementary strands of DNA restriction fragments by alkaline RPC-5 chromatography.

High pressure reversed phase chromatography (RPC-5) at pH 12 was used for preparative separation of the complementary strands of the smaller DNA fragments which are generated by the Hae III restriction endonuclease digestion of Col El DNA. A single high pressure RPC-5 chromatographic step at neutral pH served to purify duplex fragments 70, 172, 250 and 440 base pairs long; each of these yielded two elution peaks upon chromatography under alkaline denaturing conditions.

Construction of Chimeric Phages and Plasmids Containing the Origin of Replication of Bacteriophage Lambda

Segments of the replication control region of bacteriophage lambda (lambda) and lambda mutants defective in replication were attached in vitro to the phi80 phage vector Charon 3 and to the plasmid vector mini Col El (pVH51). The chimeric phages and plasmids have been used to localize the origin of lambda DNA replication and to facilitate a structural analysis of the lambda replicator.

Involvement of DNA gyrase in bacteriophage T7 DNA replication.

NOVOBIOCIN and the related antibiotic coumermycin A1 (referred to as coumermycin) inhibit the supercoiling of circular double-stranded DNA catalysed by Escherichia coli DNA gyrase1. They also inhibit the replication of chromosomal DNA in E. coli cells2, and of circular double-stranded DNA in cell-free systems from E. coli3–6. The activity of DNA gyrase obtained from a coumermycin-resistant (cour) mutant and the replication of colicin El plasmid (Col El) DNA in a cell extract of the mutant are resistant to both drugs7. These results show that DNA gyrase is an essential component of these systems for the replication of the circular double-stranded DNA. It is interesting to examine whether DNA gyrase also has a role in replication of linear double-stranded DNA. The DNA of E. coli bacteriophage T7 is linear and double stranded8. Linear replicating molecules have been isolated from infected cells and it has been claimed that the phage DNA replicates as a linear form, at least in the first round of replication9. We show here that the replication of phage T7 DNA, including the first round, is inhibited by coumermycin.

Plasmid Detection and Sizing in Single Colony Lysates

A simple and contained procedure for the rapid assay of the presence and size of plasmids similar to Col El is described. Bacteria are picked from an agar plate with a toothpick, lysed with dodecyl sulfate and heat, and placed directly on an agarose gel for electrophoresis.

DNA double-strand breaks and alkali-labile bonds produced by bleomycin.

Both in linear T2 DNA, analyyzed by velocity sedimentation, and in supercoiled Col EL DNA, analyzed by gel electrophoresis, the number of double-strand breaks produced by bleomycin was directly propotional to the number of single-strand breaks and was far greater than the number expected from random coincidence of single-strand breaks, suggesting that the bleomycin-induced double-strand breaks occur as an independent event. In Col EL DNA, at least twice as many single-strand breaks were found under alkaline assay conditions as were found under neutral conditions, showing the production of alkaline-labile bonds by bleomycin.

Construction of a hybrid col El plasmid carrying the gene for bacteriophage repressor

A biologically active hybrid DNA molecule was constructed from plasmid Col E1 and the Eco R1 fragment of lambda DNA containing the gene for lambda repressor. The presence of this gene in the hybrid molecule was demonstrated genetically. The hybrid plasmid contains two closely located targets for restriction endonuclease Hind 111 in the integrated fragment. Thus, the plasmid may be used as a vector not only for Eco R1 fragments but also for Hind 111 fragments.

Mutants of Salmonella typhimurium deficient in DNA polymerase I: further characterization and genetic analysis.

Tests with a plasmid-borne ochre suppressor (sup-812) and a chromosomal amber suppressor (supD501) revealed that one of three mutants of S. typhimurium deficient in DNA polymerase I was an amber mutant. Assays performed on crude extracts established that derivatives of this mutant (designated polA3) carrying ochre and amber suppressors had about 13 to 20% respectively of the enzyme activity found in the wild-type parent. The unsuppressed mutant showed less than 1% of the wild-type level of activity. Other properties of the polA3 mutant that were also partially or in some cases completely reversed by the sup-812 and supD501 suppressors included: u.v. sensitivity, methyl methanesulphonate (MMS) sensitivity, reduced ability to effect host-cell reactivation of u.v.-irradiated or MMS-treated bacteriophages, inability to maintain the (Col El) plasmid, and reduced ability to plate the phage mutant P22 c2 hpi-308. Mapping by P1-mediated transduction showed that all three polA mutations lie between metE and rha on the S. typhimurium chromosome, and that the polA mutation is cotransduced with metE at a frequency of 20% and with rha at a frequency of 8%.

Sequences spanning the EcoRI substrate site

Substrate recognition by the EcoRI restriction endonuclease was investigated by analysis of the nucleotide sequences at the sites of enzymatic cleavage in various DNA molecules. 5'-end labeling and homochromatographic fingerprinting led to the determination of a 17-base-pair sequence spanning the EcoRI site of simian virus 40 DNA and a 15-base-pair sequence overlapping the EcoRI site of Col El plasmid DNA. Three other DNAs were similarly tested, although extended sequences were not determined in these cases. The EcoRI site was shown to be symmetric double-stranded equivalent of -N-G-A-A-T-T-C-N-.