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R J Sheehy

Publications and source records attributed to R J Sheehy.

12 recordsLinked to original sources
PubMed1983

Membrane-bound fractions of R6K plasmid DNA in Escherichia coli.

The intracellular location of plasmid DNA has been of interest in an effort to understand the maintenance of these molecules. We have employed a simple procedure which enables us to isolate from exponentially grown cells on sucrose gradients membrane-complexed forms of R6K plasmid DNA. Electron micrographs identified the complexing of membrane fractions to circular forms of R6K DNA. Biochemical studies of the complexed R6K molecules showed the presence of membrane-specific proteins and suggested that complexing of R6K DNA was primarily with inner membrane fractions of Escherichia coli.

Bacterial Proteins↗
PubMed1982

Effects of genetically different strains of mice on the microsomal activation of 2-aminofluorene.

Five strains of mice were examined for microsomal cytochrome P450 (Cyt P450) activity and ability of the microsomal preparation to activate 2-aminofluorene (2-AF) to its mutagenic form. Each strain was assayed under normal and induced conditions. Group A strains known to be easily induced to carcinogenesis by chemicals (C57BL/6J and BALB/cJ) showed higher levels of Cyt P450 and increased mutagenesis activity of the metabolized 2-AF than Group B strains which are known to be high spontaneous tumor producers (CE/J, A/HeJ, C3H/HeJ). Induction of the hepatic microsomes with Aroclor 1254 increased the difference between the two groups.

Animals↗
PubMed1982

Survey of the extrachromosomal gene pool of Clostridium difficile.

Pseudomembranous colitis, a severe diarrheal disease, has been linked to the administration of antibiotics and to two toxins produced by Clostridium difficile. Eighty-two strains of C. difficile isolated from humans and hamsters were assayed for the presence of plasmid DNA. Agarose gel electrophoresis of Sarkosyl-lysed cells indicated that 18% of the strains contained from one to four plasmids. The plasmid DNA in these strains ranged in molecular weight from 2.7 X 10(6) to 60 X 10(6). Strains with and without plasmids were examined for the cytopathogenic effect of the toxins on MRC-5 cells. No correlation was observed between plasmid content and cytopathogenic effect. The results of in vitro antibiotic susceptibility testing with plasmid-containing strains revealed that 33% of the strains tested exhibited growth with four or more of the antimicrobial agents used.

Anti-Bacterial Agents↗
PubMed1981

Effects of rifampicin on the relationship of R6K replicating forms to the folded chromosomal complex of Escherichia coli.

An examination of the relationship of R6K plasmic DNA to the folded chromosome has shown that replicating forms of this plasmid, when compared to the non-replicating forms, were preferentially associated to the apparently mediated by RNA molecules. In this report we show that inhibition of RNA synthesis with rifampicin prior to pulse-labeling cells harboring R6K plasmid DNA resulted in the release of the replicating forms. Analyses of the single-stranded length of rifampicin-released nascent molecules indicate that continuous replication of R6K plasmid DNA to unit length was not affected by the presence of rifampicin. Thus, it appears that complexing was not required for continued synthesis of this plasmid. Further, the inhibition of protein synthesis does not appear to alter the complexing frequency of R6K plasmid DNA to the folded structure. These results suggest that active RNA synthesis is required for maintaining the association of the replicating forms to their replicational site(s).

Chromosomes, Bacterial↗
PubMed1978

Relationship of R6K replicating forms to the folded chromosome of Escherichia coli.

An examination of the relationship of both nonreplicating and replicating forms of R6K plasmid DNA to the Escherichia coli folded chromosome showed that both forms cosediment with the chromosome in neutral sucrose gradients. Approximately 20% of the nonreplicatin molecules was found as freely sedimenting forms when the folded-configuration of the chromosomes was preserved. However, under the same conditions negligible amounts of the replicating forms were found as freely sedimenting molecules. Thus, it is concluded that the replicating forms, when compared with nonreplicating molecules, are preferentially associated with the folded chromosomal structure. Exposure of the folded chromosomal structure to RNase resulted in an unfolding of the chromosome and a concomitant increase in the amount of freely sedimenting replicating and nonreplicating forms of R6K DNA. Analyses of the single-stranded length of RNase-released nascent molecules suggest that they replicate in continuous association with the folded chromsome complex. Nonenzymatic unfolding of the chromosomes by progressively lowering the sodium ion concentration during lysis resulted in a progressive increase in the release of nonreplicating molecules. Replicating molecules wer not released by unfolding the chromosome in this fashion.

Centrifugation, Density Gradient↗
PubMed1975

DNA degradation in minicells of Escherichia coli K-12. II. Effect of recA1 and recB21 mutations on DNA degradation in minicells and detection of exonuclease V activity.

The properties of minicell producing mutants of Escherichia coli deficient in gentic recombination were examined. Experiments were designed to test recombinant formation in conjugal crosses, survival following UV-irradiation in cells, and the state of DNA metabolism in minicells. The REC- phenotypes are unaffected by min+/- genotypes in whole cells. In contrast to minicells produced by rec+ parental cells, minicells from a recB21 strain have limited capacity to degrade linear, Hfr transfereed DNA. The lack of a functional recA gene product, presumably involved in inhibiting the recBC nuclease action(s), permits unrestricted Hfr DNA breakdown in minicells produced by a recA1 strain. This results in an increase in TCA soluble products and in the formation of small DNA molecules that sediment near the top of an alkaline sucrose gradient. Unlike the linear DNA, circular duplex DNA from plasmids R 64-11 or lambdadv, segregated into the minicells, is resistant to breakdown. By using in vitro criteria, and [32P]-labelled linear DNA from bacteriophage T7 for substrate, we found that the ATP-dependent exonuclease of the recBC complex (exo V) is present in rec+ and recA- minicells, and is lacking in the recB21 mutant. In fact, the absence of a functional exo V in recBC- minicells results in isolation of larger than average Hfr DNA from minicells. We suggest that recombination (REC) enzymes segregate into the polar minicells at the time of minicell biogenesis. This system should be useful for studies on DNA metabolism and functions of the recBC and recA gene products.

DNA, Bacterial↗
PubMed1973

Cryptic plasmids in a minicell-producing strain of Salmonella typhimurium.

A minicell-producing strain of Salmonella typhimurium contains two cryptic plasmids. One has a molecular weight of 2.6 x 10(6) to 2.8 x 10(6), is present in multiple copies per cell, and segregates into minicells. The other has a molecular weight of 130 x 10(6), is present in few copies per cell, and probably does not segregate into minicells.

Bromides↗
PubMed1973

Molecular nature of R-factor deoxyribonucleic acid isolated from Salmonella typhimurium minicells.

In earlier reports it was shown that a variety of extrachromosomal elements harbored in the Escherichia coli minicell producer segregate into the minicells. We show in this report that the fi(+) R factor R-100-1 (derepressed derivative of R-100) similarly segregates into minicells produced by a Salmonella typhimurium strain. Four distinct classes of covalently closed circular deoxyribonucleic acid molecules are found in minicells derived from the R(+)Salmonella minicell producer. The sum of the average molecular weights or contour lengths of the circular molecules in two of the classes is equal to the average molecular weight or contour length of those in a third class. The data suggest that R-100-1 dissociates into resistance determinants (i.e., genes that specify the molecules that confer resistance) and the resistance transfer factor (i.e., genes responsible for the transferability of the R factor to a recipient). In contrast, only one molecular species is found in minicells derived from the R(+) (R-100-1) Escherichia coli minicell producer. The fourth size class consists of small covalently closed circles (minicircles), which were originally found in the R(-)Salmonella minicell producer and are shown in this report to be enhanced in number in R(+)Salmonella minicells.

Bromides↗
PubMed1972

Molecular studies on entry exclusion in Escherichia coli minicells.

Minicells produced by abnormal cell division in a strain of Escherichia coli (K-12) have been employed here to investigate the phenomenon of "entry exclusion." When purified minicells from strains containing F' or R factors, or both, are mated with radioactive thymidine-labeled Hfr or R(+) donors, the recipient minicells can be conveniently separated from normal-sized donors following mating, and the products of conjugation can be analyzed in the absence of donors and of further growth of the recipients. Transmissible plasmids or episomes are transferred less efficiently to purified minicells derived from strains carrying similar or related elements than to strains without them. Measurement of deoxyribonucleic acid (DNA) degradation and determination of weight-average molecular weights following transfer indicate that degradation of transferred DNA or transfer of smaller pieces cannot account for the comparative reduction in transfer to entry-excluding recipients. Therefore, we conclude that entry exclusion operates to prevent the physical entry of DNA into recipients expressing the exclusion phenotype. The R-produced repressor (product of the drd(+) gene), which represses fertility (i.e., ability to act as donor), reduces exclusion mediated by R or F factor, or both, in matings between strains carrying homologous elements. Furthermore, the data suggest that the presence of the F pilus or F-like R pilus on recipient cells ensures maximum expression of the exclusion phenotype but is not essential for its expression. In contrast to previous suggestions, we found no evidence for a reduction of entry exclusion attributable to the DNA temperature-sensitive chromosomal mutation dnaB(TS).

Centrifugation, Density Gradient↗