Yonosuke Ikeda

Species specificity of the ribosomal RNA cistrons in bacteria

Abstract Hybridization of labelled ribosomal RNA of Bacillus subtilis Marburg strain with DNAs from various microorganisms was studied by means of a DNA-agar method involving ribonuclease digestion. The size and base ratio of the hybridized rRNA portion were analyzed. The B. subtilis rRNA was found to be highly complementary to the DNAs of Bacilli tested: the extent of the analogy seemed to be correlated with the gehetic relatedness and the guanine-cytosine content of the DNA. Appreciable cross-hybridization was also observed between B. subtilis rRNA and DNA from microorganisms of different families, e.g. the extents of hybridization with DNAs from Staphylococcus epidermidis, Alcaligenes faecalis, and Escherichia coli were 55, 33 and 20 %, respectively, of that with the homologous DNA. Competition experiments revealed similar extents of the analogy of rRNA in these bacteria. In addition, the heterologous hybridization between Staphylococcus DNA and labelled rRNA from B. subtilis was reduced by unlabelled rRNA from A. faecalis by the expected amount. The results suggest that analogous base sequences may occur at the same site(s) in the rRNAs from these bacteria. The majority of experiments were carried out with 23-S RNA, but similar results were also obtained with 16-S RNA. These results suggest the conservation of rRNA sites in the DNA of microorganisms.

Ribonuclease-resistant RNA found in cells of Escherichia coli infected with RNA phage

RNA phages were used to infect ultraviolet-irradiated cells of Escherichia coli, and the ribonucleic acid newly formed in the cells was fractionated in a methylated albumin column. Among the three radioactive peaks found, one which appeared overlapping the DNA peak was resistant to ribonuclease. This RNA was purified by re-chromatography after treatment with deoxyribonuclease and ribonuclease. It has a sharp thermal transition with a Tm of 99°C in standard saline citrate, as determined by susceptibility to ribonuclease, and consists of equal molar percentages of adenylic acid and uridylic acid and of guanylic acid and cytidylic acid. The guanine—cytosine content (51·6) is identical with that of phage RNA (51·2). These results suggest that this RNA is of double-stranded form. It is not detectable in non-irradiated cells or non-infected cells.

A specialized transducing phage constructed from Bacillus subtilis phage φ105

Chromosomal DNA of Bacillus subtilis 168 (trpC2) prepared from defective phage P BSX was digested by restriction endonuclease Eco RI and ligated in vitro with DNA fragments of page phi 105C digested by the same endonuclease. The ligated DNA was used to transform a competent culture of B. subtilis (trpC2 lys3 metB10) which was lysogenic for phi 105, and transformants of the auxotroph markers were selected. The bacterial DNA ligated to the phage DNA fragments could be integrated into the prophage genome by transformation. The transformants in toto were treated with mitomycin C and the lysate was used to transduce B. subtilis (trpC2 lys3 metB10). Among metB+ transductants, one clone appeared to be a double lysogen carrying both plaque forming and metB+ transducing phage genomes. The latter defective phage was designated phi 105dmetB. Physical mapping of these phages was carried out by agarose gel electrophoresis of the restriction endonuclease digests and also by electron microscopic analysis of heteroduplex DNA. These results indicate that two adjacent fragments Eco RI-G and E of phi 105 DNA had been substituted with a foreign fragment Eco RI-M in phi 105dmetB DNA. Transformation experiments showed that the metB+ gene resided on the fragment Eco RI-M. This fragment was found to have a BamHI-sensitive site. The transforming activity for the metB marker, however, was not affected by the treatmment with BamHI.

Studies on the conidia of Aspergillus oryzae: VII. Development of protein synthesizing activity during germination☆

Abstract A cell-free protein synthesizing system from Aspergillus oryzae has been established. Amino acid incorporation is dependent on ATP, ribosomes, supernatant, polyuridylic acid (poly U), and a critical concentration of magnesium. Amino acid incorporation in vivo into protein fractions during germination of conidia increases after a lag of 30–40 min. The cell-free extract from dormant conidia has less capacity for protein synthesis; following germination protein synthesizing activity rises to 3–4 times that of the dormant system. Analysis of a “limiting factor” in the protein synthesizing system shows that a “limiting factor” is in the ribosome fraction which has lower phenylalanyl-transfer RNA (tRNA) binding activity than that of vegetative ribosomes.

Inactivation of transforming DNA by ultraviolet irradiation

Abstract Transforming activities of various markers of Bacillus subtilis were inactivated by UV irradiation to different degrees. The results were compared with the studies on the differential inactivation by heat denaturation and on the fractionation on a methylated albumin column, which suggested that these markers might reside on DNA molecules of different base compositions. The markers on GC-rich DNA molecules seemed to be more resistant to UV inactivation and vice versa. This conclusion was supported by comparing the photoreactivable sectors of two markers showing different UV sensitivities. Experimental results also indicated a correlation between the UV resistance and the frequency of transformation. It seems likely that the base composition in the marker region might be an important factor causing the difference in transformation frequency and that the UV irradiation might exaggerate the difference. In contrast, X-rays inactivated all the activities at almost similar rates. Minor differences might have been caused by certain indirect effects.

A mutant of Bacillus subtilis producing ultraviolet-sensitive spores

Abstract In general, bacterial spores are more resistant to ultraviolet (UV) irradiation than the vegetative cells of the same strain ( Zelle and Hollaender, 1955 ) ( Donnellan and Stafford, 1968 ). It is thought that the spores might be protected against UV effects by a special mechanism. This paper deals with isolation of a mutant of Bacillus subtilis producing UV-sensitive spores. This mutant was derived from a mutant producing UV-sensitive vegetative cells. In the new isolate, the spores were almost as sensitive to UV irradiation as the vegetative cells. Spore-specific protection or repair mechanism is discussed.

Isolation of double-helical regions rich in guanine-cytosine base pairing from bacteriophage f1 DNA

Abstract Bacteriophage f1 DNA was digested with single-strand specific nuclease S1 at 30° for 5 hours. About 1.9% of the treated DNA was resistant to S1 under the conditions and the fraction was termed core fraction. The core fraction was characterized by high (G+C) content and exhibited reversible thermal denaturation. It was considered on these bases that the f1 DNA might be looped out at the GC-rich regions.

Bacteriophage ϕ1 as a gene-cloning vector in Bacillus subtilis

SummaryWe attempted to use Bacillus subtilis phage ϕ1 as a gene-cloning vector since the ϕ1 genome was found to have few cleavage sites upon digestion with several kinds of restriction endonucleases. A ϕ1 stock supplied by J. Ito (University of Arizona, Tucson, USA) consisted of two phages, ϕ1E1 and ϕ1E2, having one and two EcoRI-cleavage sites in their genomes respectively. From the latter isolate a deletion mutant ϕ1E2Δ1 was induced to increase the size range of DNA segments to be cloned. It was demonstrated, by in vitro recombination experiments with phage ρ11 DNA, that ϕ1E2Δ1 can be used for cloning EcoRI fragments of various sizes. We analyzed the DNAs of ten ϕ1 clones isolated from independent transfectants and found that six of them carried ρ11 DNA fragments inserted at either of the two EcoRI-cleavage sites. Some of the hybrid phage DNAs were found to be cleaved with BamHI and HaeIII endonucleases at the ρ11 DNA portion, whereas the parental ϕ1E2Δ1 DNA was insensitive to any of these enzymes. These hybrid phages would therefore be useful vectors for cloning foreign DNA fragments generated by cleavage with BamHI or HaeIII endonucleases.

A new site-specific endonuclease from Streptomyces lavendulae (SlaI).

A new restriction-like endonuclease, SlaI, was found and partially purified from Streptomyces lavendulae ATCC8664. This endonuclease cleaved bacteriophage lambda DNA at only one site, and cytosine-substituted bacteriophage T4 DNA at 16 sites. The recognition sequence was determined by using SlaI fragments of cytosine-substituted bacteriophage T4 DNA. The hexanucleotide recognized by SlaI endonuclease was (Formula: see text), with the sites of cleavage as indicated by the arrows. Therefore, SlaI endonuclease was an isoschizomer of XhoI endonuclease.

Inhibition of RNA phage growth by phenetyl alcohol

Abstract Whereas several papers have been published in concern to the inhibitory effect of phenetyl alcohol (PA) on DNA synthesis ( Berrah and Konetzka, 1962 ; Konetzka and Berrah, 1962 ), possibility that PA might act in otherwise has not been ruled out ( Slepecky, 1963 ). This communication concerns the inhibitory effect of PA on the growth of RNA phages. The phages used are β ( Nonoyama, Yuki, and Ikeda, 1963 ) and MS-2 (kindly supplied by Dr. A. J. Clark).