Hideo Hirokawa

ISL1: a new transposable element in Lactobacillus casei

SummaryThe genome structures of a temperate Lactobacillus phage, ϕFSW, and its virulent mutants, ϕFSVs, were examined by restriction, heteroduplex and nucleotide-sequence analyses. The results showed that two out of three ϕFSVs had the same 1.3 kbp insertion (designated as ISL1) at different positions in the ϕFSW sequence. ISL1 was 1,256 bp long and contained at least two long open reading frames of 279 and 822 bases on one strand. Inverted repeats were found at the termini of the ISL1 which was bracketed by 3 bp direct repeats of the ϕFSW sequence. From this evidence, we concluded that ISL1 was a transposable element in Lactobacillus casei.

Analysis of radiation damage of DNA by atomic force microscopy in comparison with agarose gel electrophoresis studies.

DNA damage induced with ionizing radiation is considered one of the main causes of cell inactivation. Several methods including gel electrophoresis, pulsed-field gel electrophoresis, neutral filter elution method, neutral sedimentation and electron microscopy have been applied to analyze this type of DNA damage. A new method employing an atomic force microscope (AFM) for nanometer-level-structure analysis of DNA damage induced with gamma-irradiation is introduced in this report. Structural changes of plasmid DNA on a molecular size scale of about 3 kbp were visually analyzed by AFM after irradiation with 60Co gamma-rays at doses of 1.9, 5.6, and 8.3 kGy. Three forms of plasmid DNA, closed circular (intact DNA), open circular (DNA with a single strand break) and linear form (DNA with a double strand break) were visualized by dynamic force mode AFM after gamma-irradiation. The torsional feature of the plasmid DNA was visualized better with AFM than with a transmission electron microscope (TEM). All three forms of plasmid DNA were observed in the sample irradiated with gamma-rays at the dose of 1.9 kGy. Open circular and linear forms were observed in the samples irradiated with gamma-rays at doses of 5.6 and 8.3 kGy, though no closed circular form was observed. A shortening of the length of a linear form of DNA irradiated with 5.6 and 8.3 kGy gamma-rays was observed by AFM. Structural changes of DNA after gamma-irradiation were visualized by AFM at nanometer level resolution. In addition, shortening of the length of the linear form of DNA after radiation exposure was observed by AFM.

The replication origin of proplastid DNA in cultured cells of tobacco

SummaryWhen tobacco suspension culture line BY2 cells in stationary phase are transferred into fresh medium, replication of proplastid DNA proceeds for 24 h in the absence of nuclear DNA replication. Replicative intermediates of the proplastid DNA concentrated by benzoylated, naphthoylated DEAE cellulose chromatography, were radioactively labelled and hybridized to several sets of restriction endonuclease fragments of tobacco chloroplast DNA. The intermediates hybridized preferentially to restriction fragments in the two large inverted repeats. Mapping of D-loops and of restriction fragment lengths by electron microscopy permitted the localization of the replication origin, which was close to the 23S rRNA gene in the inverted repeats. The replication origins in both segments of the inverted repeat in tobacco proplastid DNA were active in vivo.

In vitro initiation of bacteriophage Φ29 and M2 DNA replication: Genes required for formation of a complex between the terminal protein and 5′dAMP

SummaryCell-free extracts prepared from Φ29 and M2-infected Bacillus subtilis cells catalyse the formation of complexes between terminal protein and [α-32P]-dAMP in the presence of [α-32P]-dATP, MgCl2, ATP, and phage DNA with terminal protein covalently linked at both the 5′ends. The complex formation does not take place when proteinase K-treated DNA is added or when uninfected extract is used. The Φ29 complex thus formed is smaller than the M2 complex, primarily due to the different molecular weights of the respective terminal proteins.Extracts prepared from cells infected with suppressor-sensitive mutants of genes 2 or 3 of Φ29 or genes G or E of M2 do not support complex formation. When the pair of extracts of Φ29 or M2-infected cells are mixed, however, formation of the complex takes place as a result of in vitro complementation. These results indicate that the complex formation observed in vitro reflects in vivo initiation of phage DNA replication. The product of gene 2 of Φ29 may be the enzyme that catalyses formation of the complex.

Bacteriophage ϕ29 DNA replication in vitro: Participation of the terminal protein and the gene 2 product in elongation

SummaryFrom ϕ29-infected Bacillus subtilis cells, we have isolated a protein fraction which promotes in vitro replication of ϕ29 DNA. This fraction catalyses both initiation and elongation, indicating that it contains the product of gene 3 (tp: terminal protein) and the product of gene 2 (gp2: probably a DNA polymerase), since initiation requires the two products (Blanco et al. 1983; Matsumoto et al. 1983). The fractions isolated from cells infected with temperature-sensitive (ts) mutants of gene 2 and gene 3 were thermolabile in both the initiation and elongation assays. When the pre-initiated material from the ts fractions of each mutant was heat-inactivated and mixed no complementation, restoring the elongation activity, was found. These results indicate: (i) tp and gp2 participate not only in the initiation but also in the elongation of ϕ29 DNA replication, (ii) they probably function in tight physical association with each other.

Aphidicolin-resistant mutants of bacteriophage φ29: Genetic evidence for altered DNA polymerase

Aphidicolin-resistant mutants (Aphr) of Bacillus subtilis bacteriophage phi 29 were isolated after mutagenesis with hydroxylamine. Efficiency of plating (e.o.p.) of the resistant mutants was not reduced at 500 microM aphidicolin, although e.o.p. of wild type phi 29 was less than 10(-5) at the same concentration of aphidicolin. By recombination and complementation analyses, both sites of the mutations, aph-71 and aph-101, of Aphr71 and Aphr101, respectively, were mapped in gene 2 which encodes phi 29 DNA polymerase. The activity of wild type phi 29 DNA polymerase, in a partially purified fraction, was inhibited by aphidicolin. DNA polymerases from Aphr71 and Aphr101, prepared in the same manner as that of wild type, were resistant to the drug. These results indicate that the acquisition of the aphidicolin resistance of Aphr71 and Aphr101 of bacteriophage phi 29 results from a structural alteration of phi 29 DNA polymerase which reduces sensitivity to aphidicolin.

An inhibitory effect of RGD peptide on protein, priming reaction of bacteriophages ϕ29 and M2

SummaryThe amino acid sequence, arginine-glycine-aspartic acid (RGD), found in some cell adhesive proteins, is a recognition signal for the receptor protein. It is interesting that we have found the RGD sequence in terminal protein (TP) of bacteriophages ϕ29 and M2 near an amino acid, the serine residue at 232, covalently linked to the terminal nucleotide of their DNAs. At the initiation of proteinprimed DNA replication, TP is essential for the recognition of replication machinery containing DNA polymerase and primer protein (PP; PP becomes TP upon linking the first nucleotide, and hence the primary structure of TP is the same as that of PP). Synthetic peptide RGD specifically inhibited transfection of ϕ29 and M2. The target of the RGD peptide is shown to be TP by marker rescue experiments, suggesting that a receptor for the RGD sequence exists in TP. Furthermore, the peptide inhibited the in vitro protein-priming reaction of DNA replication. We propose that the RGD sequence of PP and a putative receptor on TP is utilized for the molecular recognition initiating DNA replication.

Physical arrangement of suppressor-sensitive mutations of Bacillus phage M2.

SummaryThe order of the fragments derived from bacteriophage M2 DNA by digesting it with restriction endonucleases Xba1, HindIII, and EcoRI has been determined. The locus of each representative mutation in 13 cistrons of the M2 genome has been determined by transfection/marker rescue with the individual restriction enzyme-digested fragments derived from wild-type M2 DNA.

Primer protein of bacteriophage M2 exposes the RGD receptor site upon linking the first deoxynucleotide

SummaryPrimer protein (PP) of bacteriophages M2 and ϕ29 contains an Arg-Gly-Asp (RGD) sequence. The RGD-mediated protein-protein interaction in protein-primed DNA replication of M2 was studied in vitro using three purified and indispensable components: PP, DNA polymerase (POL) and template DNA linked to terminal protein (TP). PP competed with a synthetic RGD peptide for binding to the template DNA-TP complex (TP-DNA). In addition, POL bound to template TP-DNA only when complexed with PP. These results indicate that the RGD sequence of PP is responsible for the interaction of the PP-POL complex with TP-DNA, which contains the initiation site for the protein priming of DNA synthesis. At the moment when PP converts to TP upon linking the first deoxynucleotide, a conformational change results in exposure of the RGD binding site.

The nucleotide sequences of the heterologous region between the genomes of Bacillus phages M2 and Nf that indicate the two phages are originally identical

It has been suggested that the heterologous population of Bacillus phage M2 is derived from an original clone, which is identical with phage Nf, by the deletion on a particular region of the genome. We have determined the nucleotide sequence of this region of M2 subclones and Nf genomes. The results clearly indicate that the homologous recombination through the short direct repeated sequence is the main cause of the varied deletions found in the genomes of M2 subclones.