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Biomedical subjects

R W Murray

Publications and source records attributed to R W Murray.

51 records · Page 3Linked to original sources
PubMed1986

Sequence-specific interaction between the replication initiator protein of plasmid pT181 and its origin of replication.

The replication of the pT181 plasmid is dependent on the plasmid-encoded initiator protein RepC. We have previously shown that RepC protein has sequence-specific endonuclease and topoisomerase-like activities. In this paper we demonstrate that this initiator protein has sequence-specific DNA-binding properties. Based on filter binding of plasmid restriction fragments, RepC protein specifically recognizes only the pT181 origin region. Using DNase I and neocarzinostatin "footprinting" techniques, we show that RepC protein specifically binds to a 32-base-pair sequence within the origin that is part of the initiator cistron. Using dimethyl sulfate as a chemical probe, we have identified the purine residues that interact with the initiator protein. The features of the DNA region that interacts with RepC protein include sequences with the potential to form Z DNA and/or hairpin structures. The specific DNA-protein interaction at the origin may be critical in the initiation of pT181 DNA replication by RepC protein in association with other host initiation proteins.

Bacterial Proteins↗
PubMed1985

Purification of pT181-encoded repC protein required for the initiation of plasmid replication.

The plasmid pT181 of Staphylococcus aureus consists of 4437 base pairs and encodes resistance to tetracycline. Initiation of pT181 replication specifically requires the plasmid-encoded repC protein. An in vitro system has been shown to carry out semiconservative replication of pT181 and its derivative plasmids (Khan, S A., Carleton, S. M., and Novick, R. P. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 4902-4906). We have used this replication assay to isolate repC protein, which was purified to near homogeneity. The repC gene was cloned into the pKJB825 plasmid that contains the phage lambda temperature-sensitive repressor gene, cI857, and the rightward promoter, PR. Upon temperature induction, Escherichia coli clones containing the recombinant plasmid overproduced repC protein, which was purified in significant quantities. The molecular weight of repC protein under denaturing conditions is 38,000, which is consistent with the size predicted from the DNA sequence data. Presence of repC protein was absolutely essential for the initiation of replication of pT181 and its derivatives in vitro.

Bacterial Proteins↗
PubMed1985

The replication initiator protein of plasmid pT181 has sequence-specific endonuclease and topoisomerase-like activities.

Initiation of pT181 DNA replication specifically requires the plasmid-encoded RepC protein. Here we demonstrate that highly purified RepC protein has sequence-specific endonuclease and topoisomerase-like activities. A maximum sequence of 127 base pairs containing the pT181 origin of replication is required for nicking-closing by RepC protein. RepC introduces a single strand break within the pT181 origin. The nick site has been shown by DNA sequencing to lie between nucleotides 70 and 71 in the bottom strand of the DNA within the origin sequence. This nick site probably corresponds to the start site of pT181 replication. The results presented here suggest that, unlike most other plasmids, pT181 replicates by a rolling circle mechanism.

Bacterial Proteins↗
PubMed1984

Production of singlet delta oxygen by atmospheric pollutants.

Singlet delta oxygen (1O2) was generated at atmospheric pressure using heterogeneous gas-phase photosensitization. Polycyclic aromatic hydrocarbons (PAH), known constituents of the atmosphere, were found to be efficient sensitizers in the production of singlet delta oxygen under simulated environmental conditions. A novel mechanism involving singlet delta oxygen is proposed in which PAH on particle surfaces are self-activated to form direct-acting mutagens.

Air Pollutants↗
PubMedDate not supplied

Gamma glutamyl transpeptidase as a sensitive marker of fetal alcohol exposure.

Changes in gamma-glutamyl transpeptidase (gamma-GTP) activity have been shown to occur concomitantly with ethanol use. Alterations in gamma-GTP isozymal forms have also been demonstrated to accompany various developmental stages, diseases, and drug exposures. In view of these properties, electrophoretic examination of gamma-GTP isozymal forms was performed on sera from neonatal rats exposed to ethanol during gestation. The purpose of this study was to see whether or not a form of the enzyme specific for prenatal ethanol exposure could be demonstrated. Pregnant Wistar rats were pair-fed on diets containing either ethanol (1, 4, 5, or 6.7% v/v) or diets without ethanol which were isocalorically equivalent. Sera collected from one day old neonates were separated into gamma-GTP fractions using polyacrylamide gradient gel electrophoresis. The zymograms were specifically stained for gamma-GTP activity and desitometrically scanned. All alcohol groups had 4 bands of gamma-GTP activity shared in common with the control groups. However, in addition to these bands the sera from the neonates exposed to ethanol in utero were found to have a unique band of gamma-GTP activity. This unique band was present in all zymograms of sera from ethanol-exposed neonates and absent in all control neonates. The possibility therefore exists that gamma-GTP serum isozymes may be useful as a diagnostic index available for the assessment of Fetal alcohol exposure in man.

Animals↗