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Biomedical subjects

E Orr

Publications and source records attributed to E Orr.

At least 55 records · Page 3Linked to original sources
PubMed1982

DNA primase of plasmid ColIb is involved in conjugal DnA synthesis in donor and recipient bacteria.

The sog gene of the IncI alpha group plasmid ColIb is known to encode a DNA primase that can substitute for defective host primase in dnaG mutants of Escherichia coli during discontinuous DNA replication. The biological significance of this enzyme was investigated by using sog mutants, constructed from a derivative of ColIb by in vivo recombination of previously defined mutations in a cloned sog gene. The resultant Sog- plasmids failed to specify detectable primase activity and were unable to suppress a dnaG lesion. These mutants were maintained stably in E. coli, implying that the enzyme is not involved in vegetative replication of ColIb. However, the Sog- plasmids were partially transfer deficient in E. coli and Salmonella typhimurium matings, consistent with the hypothesis that the normal physiological role of this enzyme is in conjugation. This was confirmed by measurements of conjugal DNA synthesis. Studies of recipient cells have indicated that plasmid primase is required to initiate efficient synthesis of DNA complementary to the transferred strand, with the protein being supplied by the donor parent and probably transmitted between the mating cells. Primase specified by the dnaG gene of the recipient can substitute partially for the mutant enzyme, thus providing an explanation for the partial transfer proficiency of the mutant plasmids. Conjugal DNA synthesis in dnaB donor cells was deficient in the absence of plasmid primase, implying that the enzyme also initiates synthesis of DNA to replace the transferred material.

Bacteriocin Plasmids↗
PubMed1981

Identification of gene products programmed by restriction endonuclease DNA fragments using an E. coli in vitro system.

DNA restriction enzyme fragments have been used to programme the synthesis of polypeptides in an in vitro system without apparent loss in fidelity compared with supercoiled templates. The system is extremely sensitive, less than 1 microgram of DNA can be used to direct the synthesis of 35S-labelled polypeptides of sufficiently high specific activity such that products can be identified by SDS-PAGE after a few hours autoradiography. The ability to analyse fragments can be used to readily assign specific proteins to small regions of the coding template, to identify cloned gene products distinct from those of the vector, and to identify cloned genes expressed from their own promoters. The in vitro system can be used successfully with bacterial DNA from other species and efficient extracts can be prepared from any E. coli K-12 strain, which should greatly facilitate the purification of factors controlling the expression of specific genes by complementation assay.

Cloning, Molecular↗
PubMed1981

DNA gyrase: affinity chromatography on novobiocin-Sepharose and catalytic properties.

Novobiocin-Sepharose was prepared by coupling of novobiocin to Epoxy-activated Sepharose 6B and used as an affinity adsorbent. Four novobiocin-binding proteins were isolated from crude extracts of Escherichia coli with molecular weights of 105, 92, 85 and 40 kdal. The two larger proteins were identified as the A subunit (gyrA protein) and the B subunit (gyrB protein) of DNA gyrase topoisomerase II). By this method the two gyrase components can be easily separated and purified in high yield. Although both proteins are involved in the ATP-dependent supercoiling of relaxed plasmid DNA, only the gyrB protein is required for catalyzing the cleavage of ATP. The gyrB protein ATPase activity is competitively inhibited by novobiocin and related coumarin antibiotics. ATP hydrolysis is unaffected by the addition of either gyrA protein or DNA but stimulated in the presence of both.

Adenosine Triphosphatases↗
PubMed1981

An Escherichia coli mutant thermosensitive in the B subunit of DNA gyrase: effect on the structure and replication of the colicin E1 plasmid in vitro.

An E. coli strain which carries a mutation conferring clorobiocin resistance and temperature sensitivity for growth has recently been described and evidence has been presented suggesting that the mutation is located in the gyrB gene (Orr et al. 1979). The replication of the ColE1 plasmid was analysed in cell-free extracts from this thermosensitive strain. These extracts were totally deficient in the replication of exogenous plasmid DNA and were unable to maintain the superhelical structure of the plasmid DNA. Both defects could be fully complemented by addition of purified gyrB protein.

Bacterial Proteins↗
PubMed1980

Chloramphenicol releases a block in initiation of chromosome replication in a dnaA strain of Escherichia coli K12.

DNA-DNA hybridisation experiments show that chloramphenicol induces a burst of initiation from the oriC region of a dnaA46 mutant of Escherichia coli at 36.5 degrees C but not from the isogenic dnaA+ strain. Following this stimulation of initiation is in parallel with the induced stimulation of RNA synthesis caused by chloramphenicol in the same strain. This is consistent with the hypothesis that the stimulation of initiation in the dnaA mutant is the result of the stimulation of the synthesis of an RNA species.

Chloramphenicol↗
PubMed1980

Inhibition of deoxyribonucleic acid gyrase: effects on nucleic acid synthesis and cell division in Escherichia coli K-12.

Mutants of Escherichia coli resistant to the antibiotic clorobiocin are also coumermycin resistant, and the mutation to resistance in at least one mutant was mapped near gyrB. We conclude, therefore, that clorobiocin inhibits deoxyribonucleic acid gyrase, and the drug was used to probe the role of this enzyme in vivo. Deozyribonucleic acid synthesis was preferentially inhibited but not completely blocked by the antibiotic. Transcription and cell division were also markedly affected. However, unlike other inhibitors of deoxyribonucleic acid synthesis, clorobiocin failed to induce the synthesis of protein X, the recA gene product. In mutants resistant to clorobiocin the replication velocity was unaffected, but initiation of deoxyribonucleic acid synthesis appeared to be delayed. We conclude that deoxyribonucleic acid gyrase, and hence the supercoiled structure of the chromosome, is important for transcription, normal initiation of deoxyribonucleic acid replication, and cell division. The possible role of deoxyribonucleic acid gyrase in the elongation of replication forks is also discussed.

Bacterial Proteins↗
PubMed1979

Isolation and characterisation of a strain carrying a conditional lethal mutation in the cou gene of Escherichia coli K12.

A strain which carries a mutation conferring clorobiocin resistance and temperature sensitivity for growth was isolated from Escherichia coli K12. Genetic mapping and the molecular weight of the gene product suggest that the mutation is in the cou gene, specifying a sub-unit of DNA gyrase. Nuclear organisation and segregation and placement of septa are grossly abnormal in the mutant at 42 degrees C. RNA synthesis and initiation of DNA replication are also affected at the restrictive temperature but the rate of DNA chain elongation continues almost undisturbed.

Chromosome Mapping↗
PubMed1978

Diameter of cells of a thermosensitive dnaA mutant of Escherichia coli cultivated at intermediate temperatures.

Strains of Escherichia coli K-12 carrying the dnaA46 mutation exhibited a progressively decreasing DNA concentration and a progressively increasing cell size as the temperature was raised from 31 to 37 degrees C. Above 37 degrees C, steady-state exponential growth could not be maintained. The increase in average cell size with increasing growth temperature was due to an increase in cell length. There was no change in cell width. This seems to rule out the hypothesis that the increase in cell width in thy strains cultivated on low concentrations of thymine is due to the decrease in DNA concentration which also occurs under these conditions.

DNA, Bacterial↗
PubMed1976

Determination of the execution points of mutations in the nuclear replication cycle of Aspergillus nidulans.

Cultures of nuclear replication cycle mutants of Aspergillus nidulans were transferred to the nonpermissive temperature, and the fraction of nuclei still able to reach mitosis was determined. For the determinations, benomyl [methyl-1(butylcarbomoyl)benzimidazolecarbamate] was added to trap nuclei in mitosis, and these were detected by staining with aceto-orcein. The assumptions and controls required to relate the experimentally determined fractions to the points where a mutation blocks the nuclear cycle are discussed. Nine genetically distinct mutants were tested. Two of these were blocked early in the cycle, two in the middle, and five close to, or during, mitosis.

Aspergillus nidulans↗
PubMed1975

Hypothalamic 24-hour rhythms in histamine, histidine, decarboxylase and histamine-N-methyltransferase.

Twenty-four hour rhythms of histamine (H), its biosynthetic enzyme, L-histidine decarboxylase (EC 4.1.1.22, HD) and its inactivation enzyme, histamine N-methyltransferase (EC 2.1.1.8, HMT) were assayed in the 63-day-old male rat hypothalamus using a modified single isotopeenzyme microassay. H was found to have a 24-h rhythm with a maximum concentration at 11:00 h (lights on 10:00-22:00 h) and a minimum at 23:00 h. In vitro HD and HMT activities were also found to have 24-h rhythms with peak activites in both occurring at 04:00 h. HMT also had a broad secondary peak throughout the day, whereas HD activity during the day was dominated by a mid-light trough at 16:00 h. These rhythms are considered significant for evaluation of the control and physiological role of histamine in the mammalian hypothalamus.

Animals↗
PubMed1975

The matron.

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Administrative Personnel↗
PubMedDate not supplied

The omnipotent suppressor SUP45 affects nucleic acid metabolism and mitochondrial structure.

Yeast (Saccharomyces cerevisiae) strains sensitive to a variety of drugs were used to select for novobiocin-resistant mutants that were simultaneously temperature-sensitive. The mutants remained as sensitive as the parent strains to a wide range of drugs other than novobiocin, and did not exhibit any suppression of suppressible auxotrophic markers. At the non-permissive temperature, the mutant cells arrested mainly as unbudded cells, and were instantly defective in DNA and RNA synthesis, but not protein synthesis. The cloned wild-type gene was identified as SUP45, which has been previously implicated in the translation process. Our results suggest that SUP45 may have a function in addition to, or different from, the one that has been assigned to it previously.

Cloning, Molecular↗