Hiroshi Hiasa

Distinct functional contributions of three potential secondary structures in the phage G4 origin of complementary DNA strand synthesis

Three potential secondary structures, stem-loops I, II, and III, are contained in the phage G4 origin of complementary DNA strand synthesis, G4oric, and are believed to be involved in its recognition by dnaG-encoded primase and the synthesis of primer RNA. In a previous publication [Sakai et al., Gene 71 (1988) 323-330], we suggested that base pairing between the loops of stem-loops I, and II, and/or II and III, might play a role in G4oric function. To test this hypothesis, site-directed mutagenesis was used to construct mutants which carried base substitutions in loops I, II and III that destroyed possible interloop base pairing. These mutations, however, did not seriously affect G4oric activity. This indicates that base pairing between the loops is not essential for G4oric functional activity, and also that base substitutions which do not affect the secondary structure of stem-loops I, II and III, do not affect G4oric activity. To complete an analysis of the effects of altering the structure of the G4oric stem-loops, insertions were made into stem-loop III. In contrast to stem-loops I and II, all insertions into stem-loop III destroyed in vivo G4oric activity.

Identification of single-strand initiation signals in the terC region of the Escherichia coli chromosome

On the basis of clear‐plaque formation, we detected initiation signals in the terC region of the Escherichia coli chromosome. At least two single‐strand initiation signals were identified from the terC region. The nucleotide sequences of these two signals were determined. Sequence homologies, variations of the consensus sequence of n′ protein recognition sites, 5′‐GAAGCGG‐3′, were found within these signals. A novel conserved sequence was also found within these signals. Their initiation activities were measured both by the infection growth assay and by the ability to convert the single‐stranded DNA to the duplex replicative form DNA in vivo.

Catalytic Oxidation of D-Glucose at an Enzyme-Modified Electrode with Entrapped Mediator

Biocatalyst electrodes, that is, modified electrodes carrying an immobilized enzyme in which the electrode behaves as a substitute for a chemical electron acceptor or donor in the enzyme reaction can be used in such novel applications as biosensors, bioreactors and biofuel cells1,2. The bioelectrocatalytic oxidation or reduction of substrates at biocatalyst electrodes can be accelerated by the presence of a small molecule which functions as an electron transfer mediator between the electrode and the prosthetic group of the immobilized enzyme1–6. Two types of the enzymemodified electrode with entrapped mediator have been designed7,8,9, a carbon paste electrode and a porous electrode, both modified with enzyme and a reservoir of mediator. In the former type of electrode7,8, where glucose oxidase (GOD) was immobilized on the surface of a carbon paste electrode along with p-benzoquinone (BQ) by coating the enzyme-loaded surface with a semipermeable membrane, BQ was dissolved into the immobilized enzyme layer and retained there effectively to serve as an electron transfer mediator between the carbon paste electrode and the immobilized enzyme. It has been shown8–11 that the kinetics of the bioelectrocatalytic oxidation of D-glucose (Glc) at the membrane-coated GOD-modified carbon paste electrode with mixed-in BQ can be explained by theoretical equations in which the diffusion enzyme reaction of the substrate and mediator in the immobilized-enzyme layer and the diffusion of the substrate (and mediators) in the coating membrane are taken into account8–10.