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J Inselburg

Publications and source records attributed to J Inselburg.

At least 55 records · Page 3Linked to original sources
PubMed1981

Selection and characterization of ColE1 plasmid mutants that exhibit altered stability and replication.

This report describes a method for isolating mutants of plasmid ColE1 that exhibit unstable maintenance and altered replication characteristics. It also describes the initial characterization of four mutants isolated by that method. A chimeric plasmid, pHSG124, containing a ColE1 derivative and a temperature-sensitive replication derivative of pSC101 was mutagenized in vitro, using hydroxylamine. By adjusting the growth conditions of transformants containing the mutagenized chimeric deoxyribonucleic acid, it was possible to rapidly screen colonies and identify those that had a high probability of carrying ColE1 mutants that exhibit unstable maintenance. Of those mutants, some exhibited altered copy number or accumulated catenated structures. Evidence is presented which suggests that the mutations in three of the mutants are probably located in the HaeII A fragment of ColE1.

Bacteriocin Plasmids↗
PubMed1980

Functional relationship between parts of the replication region of plasmid ColE1.

The inhibition of plasmid ColE1 replication caused by a deletion of the ColE1 plasmid replication origin has been previously reported (T. Hashimoto-Gotoh and J. Inselburg, J. Bacteriol. 139:597-619). Evidence is presented showing that restoration of the deleted nucleotide sequence in the precise relationship it normally has to the rest of the replication region is essential for restoration of ColE1 replication capability to the deletion mutant.

Bacteriocin Plasmids↗
PubMed1979

Studies of colicin induction with an imm- col+ mutant of the plasmid colicin E1.

A mutant of a derivative of the colicin E1 plasmid has been isolated that does not confer immunity to colicin E1 on its host (imm-) although it is still capable of producing colicin (col+). Cells carrying the col+, imm- plasmid are capable of forming colonies and grow best in liquid culture in the presence of trypsin. The induction of colicin synthesis by ultraviolet light has been analysed using this mutant plasmid. The results suggest that a) the expression of the col+ gene may be delayed for many generations after the inducing stimulus, b) although induced cells are usually killed they can reproduce and c) the capacity to produce colicin can be propagated and segregated into the progeny of an induced cell.

Bacteriocin Plasmids↗
PubMed1979

A complementation analysis of mobilization deficient mutants of the plasmid ColE1.

Hydroxylamine was used to induce mutants of the ColE1 derived plasmid pML2 that are inefficiently mobilized (Mob-) during conjugation by an Hfr donor. The ability of those mutants to be complemented by deletion mutants and Tn3 insertion mutants of ColE1 was examined. Three complementation groups were identified and localized on the ColE1 genetic map (Mob1, Mob2, and Mob3). One hydroxylamine mutant was not complemented by any mobilization deficient mutant but was complemented by mobilizable ColE1 mutants. Two hydroxylamine mutants were not complemented by any ColE1 derivatives. A mutant that had its relaxation nick site deleted had a markedly reduced mobilizability. The relationship between DNA relaxation nick site deleted had a markedly reduced mobilizability. THe relationship between DNA relaxation, replication and mobilization is considered.

Colicins↗
PubMed1979

Isolation and characterization of replication-deficient mutants of ColE1 plasmids.

Replication-defective mutants of plasmid ColE1 were isolated from a chimeric plasmid formed by ligating a temperature-sensitive replication derivative of pSC101, pHSG1, with a ColE1-Tn3-containing plasmid. The replication-defective ColE1 mutants isolated were all spontaneous deletion mutants that had lost the ColE1 replication origin and regions adjacent to it. The extent of a deletion was determined by analyzing restriction endonuclease-generated deoxyribonucleic acid fragments of the ColE1 plasmid component of the chimeras by both agarose and polyacrylamide gel electrophoresis. None of the chimeras containing the replication-defective ColE1 mutants was able to replicate in the presence of chloramphenicol. The expression of ColE1 incompatibility was either markedly reduced or not detectable in the replication mutants isolated.

Chloramphenicol↗
PubMed1979

ColE1 plasmid incompatibility: localization and analysis of mutations affecting incompatibility.

Deletion mutants of plasmid ColE1 that involve the replication origin and adjacent regions of the plasmid have been studied to determine the mechanism by which those mutations affect the expression of plasmid incompatibility. It was observed that (i) a region of ColE1 that is involved in the expression of plasmid incompatibility lies between base pairs -185 and -684; (ii) the integrity of at least part of the region of ColE1 DNA between base pairs -185 and -572 is essential for the expression of ColE1 incompatibility; (iii) the expression of incompatibility is independent of the ability of the ColE1 genome to replicate autonomously; (iv) plasmid incompatibility is affected by plasmid copy number; and (v) ColE1 plasmid-mediated DNA replication of the lambda phage-ColE1 chimera lambda imm434 Oam29 Pam3 ColE1 is inhibited by ColE1-incompatible but not by ColE1-compatible plasmids.

Colicins↗
PubMed1978

Proteins synthesized in minicells containing plasmid ColE1 and its mutants.

Minicells containing the ColE1 plasmid and derivatives of it that contain a Tn3 insertion and deletions of the plasmid were shown to synthesize a variety of polypeptides: (i) a 56,000-dalton polypeptide was found that is colicin E1. (ii) A 15,000-dalton polypeptide is involved in plasmid DNA relaxation. (iii) A 14,000-dalton polypeptide may be the colicin immunity protein. (iv) A correlation between the location of various Tn3 insertions and the proteins synthesized from the plasmid DNA was found that suggests the locations of promoter sites in the plasmid and directions of transcription in two regions of the plasmid. (v) Polypeptides associated with the presence of Tn3 were identifiable. (vi) The nature and extent of gene expression of deletion mutants only generally correlates with the extent of the deletions.

Bacterial Proteins↗
PubMed1977

Isolation, mapping, and examination of effects of TnA insertions in ColE1 plasmids.

Twelve TnA insertions of ColE1 deoxyribonucleic acid have been isolated and mapped by electron microscopic studies of heteroduplex molecules. Insertions only blocking the production of active colicin clustered in one region of the map, whereas insertions only blocking the expression of deoxyribonucleic acid nicking activity associated with the plasmid "relaxation complex" clustered in another region of the map. The location of one insertion that blocks both the expression of colicin immunity and the production of active colicin suggests that the expression of both characteristics are coordinately controlled.

Ampicillin↗
PubMed1977

Isolation and genetic analysis of deletion mutants of colicin E1 plasmids carrying a TnA insertion.

Deletions of colicin E1 (colE1) plasmid deoxyribonucleic acid (DNA) carrying the TnA transposon have been isolated. All except two were generated by nuclease digestion of plasmid DNA from its EcoRI-sensitive site. A plasmid containing about 16% of the ColE1 DNA (6.5 X 10(5) daltons) was generated that also contained the part of the TnA transposon conferring ampicillin resistance. The extents of different deletions were determined by analysis of restriction endonuclease fragments generated by the restriction endonucleases HaeII, BamHI, and HincII.

Ampicillin↗
PubMed1977

Studies of colicin E1 plasmid functions by analysis of deletions and TnA insertions of the plasmid.

The further identification of regions of the colicin E1 plasmid that affect plasmid functions has been achieved by studying deletions and TnA insertions of the plasmid. Colicin production, colicin immunity, relaxation of plasmid deoxyribonucleic acid, and plasmid incompatibility functions have been examined. A strong correlation has been observed between the ability of colicin E1 plasmid deoxyribonucleic acid to be relaxed and the ability of that plasmid to be transferred by conjugation.

Colicins↗
PubMed1975

Replicative intermediates of colicin E1 plasmid DNA in minicells.

Pulse-labeled colicin E1 plasmid (Col E1) DNA in minicells was examined to characterize replicating molecules. Replication of Col E1 DNA principally occurred in covalently closed molecules and involved the synthesis of variable length single-stranded DNA fragments that were dissociable from the template DNA and ultimately incorporated into the completely replicated molecules. RNA was found to be associated with some of these newly synthesized fragments. Replicating molecules containing different size displacement loops were observed by electron microscopy.

Centrifugation, Density Gradient↗
PubMed1975

Mapping of colicin E2 and colicin E3 plasmid deoxyribonucleic acid EcoR-1-sensitive sites.

Colicin plasmids E2 and E3 (Col E2 and Col E3) deoxyribonucleic acid (DNA) has been shown to contain, respectively, two and three EcoR1 restriction endonuclease-sensitive sites. This was determined by measuring the DNA fragments generated after EcoR1 endonuclease treatment by agarose gel electrophoresis and electron microscopy. The structure of heteroduplex Col E2-col E3 DNA molecules formed from EcoR1-generated fragments permitted a localization of the EcoR1-sensitive sites on the plasmid chromosomes.

Binding Sites↗
PubMed1975

Discontinuous replication of colicin E1 plasmid deoxyribonucleic acid.

The plasmid pML 21, which was found to contain approximately 49% of the Col E1 genome was used to determine the template origin of single-stranded deoxyribonucleic acid (DNA) fragments (4 to 32% of the Col E1 units length) associated with Col E1 dna replicative intermediates. The results of DNA hybridization competition experiments indicate that the single-stranded fragments derive from the full length of the Col E1 DNA template as expected for Okazaki fragments and the plasmid pML 21 contains the replication origin of Col E1 DNA.

Colicins↗
PubMed1974

Replication of colicin E1 plasmid DNA in minicells from a unique replication initiation site.

Replicating DNA molecules of the colicin E1 plasmid isolated from minicells are cleaved at a single site by R1 restriction endonuclease (EcoR1). Electron microscopic measurements of the replicating molecules treated with the endonuclease indicate that (a) replication is initiated at a site between 14% and 20% of the distance from the EcoR1 endonuclease cleavage site; and (b) extensive replication of most molecules occurs in one direction from the initiation site, although a limited amount of replication in the opposite direction may occur. Single-stranded regions at one or both replication forks, involving one or both DNA strands, can be frequently found in replicating molecules.

Colicins↗
PubMed1974

Isolation and characterization of mutants of colicin plasmids E1 and E2 after Mu bacteriophage infection.

Cells colicinogenic for the colicin plasmids E1 or E2 (Col E1 and Col E2, respectively) were selected for a loss of colicin production after infection with bacteriophage Mu. Extrachromosomal deoxyribonucleic acid that was larger than the original colicin plasmids was found in such cells. A small insertion mutant in Col E1 deoxyribonucleic acid affecting active colicin production without affecting either expression of colicin immunity or Col E1 deoxyribonucleic acid replication was found. Cells carrying this Col E1 plasmid mutant do not exhibit the lethal event associated with colicin E1 induction, suggesting that synthesis of active colicin is required for killing during induction. The altered Col E2 plasmid, containing an insertion at least as large as phage Mu, was maintained unstably in the mutants examined.

Carbon Radioisotopes↗
PubMed1974

Incompatibility exhibited by colicin plasmids E1, E2, and E3 in Escherichia coli.

Escherichia coli strains were made multiply colicinogenic for the colicin plasmids E1, E2, or E3 (Col E1, Col E2, or Col E3, respectively) by both a deoxyribonucleic acid transformation system and bacterial conjugation. The multiply colicinogenic bacteria constructed exhibited an immunity to the colicins produced by all the plasmids they carried and also produced colicins corresponding to all the plasmids they carried. An incompatibility was observed among the plasmids. In doubly colicinogenic cells where the presence of two plasmids was established, Col E2 was lost more frequently than Col E3. In triply colicinogenic cells, Col E1, Col E2, and Col E3 were lost, with Col E3 being lost least frequently. A significant reduction in the acquisition of a conjugationally transferred Col E1 plasmid by cells colicinogenic for Col E1 was demonstrated.

Colicins↗