Nobuto Irino

Isolation and genetic characterization of a thymineless death-resistant mutant of Escherichia coli K12: Identification of a new mutation (recQ1) that blocks the RecF recombination pathway

SummaryAn Escherichia coli K12 mutant resistant to thymineless death (TLD) was isolated, and its genetic analysis led us to identify a new mutation (recQ1) located between corA and metE on the standard linkage map. The mutation was found to result in increased sensitivity to ultraviolet light and deficiency in conjugational recombination when placed in the recBC sbcB background, indicating that it blocked the RecF pathway of recobbination. It seemed likely that this mutation is also capable of causing partial resistance to TLD, but we reserve the possibility of a separate mutation closely linked to recQ1 giving rise to this phenotype. The original mutant was shown to carry an additional mutation probably in the vicinity of the uhp locus, which was also required for the full TLD resistance of the mutant to be expressed.

The recQ gene of Escherichia coli K12: molecular cloning and isolation of insertion mutants

SummaryThe recQ gene of Escherichia coli K12 was subcloned from plasmid pKO1 (Oeda et al. 1981) by monitoring the capacity of the resulting recombinant plasmids partially to reverse the increased ultraviolet (UV) sensitivity of a recF143 recQ1 double mutant. We were able to trace this complementation activity to a 3.4 kilobase (kb) SalI-PvuII fragment. Furthermore, analysis of the Tn3 insertion mutations that abolished the complementation revealed the exclusive localisation of such insertions in the same 3.4 kb segment. This segment was situated about 4 kb clockwise from corA on the chromosome, a result consistent with the transductional data previously reported. In addition, a comparison of our restriction endonuclease cleavage map with the published data has placed recQ between pldA and pldB. When relocated to the recQ site on the chromosome, the recQ::Tn3 mutations conferred partial resistance to thymineless death (TLD) or, in the case of a recBC sbcB background, recombination deficiency and increased UV sensitivity. This has provided the firm evidence that both the TLD resistance and the deficiency in the RecF recombination pathway result from loss of the functional recQ gene. We also identified the recQ gene product as a 74 kilodalton polypeptide by using the maxicell technique.

Thymine starvation-induced structural changes in Escherichia coli DNA Detection by pulsed field gel electrophoresis and evidence for involvement of homologous recombination

Effect of thymine starvation on Escherichia coli DNA was investigated by using pulsed field gel electrophoresis combined with cell lysis in agarose gel. Post-lysis treatment with restriction enzymes generating relatively large fragments (NheI, SpeI or XbaI) revealed peculiar electrophoretic profiles specific for thymine-starved cells. Thus, a substantial portion of the DNA remained in the origin of electrophoresis (non-migrating DNA), and the amounts of the migrating fragments correspondingly decreased in an inverse relation to the map distance between the origin of replication (oriC) and each fragment. The formation of non-migrating DNA seems to depend upon the presence of replicated portions of the chromosome (sister duplexes), as judged by the effect of a preincubation at the non-permissive temperature in a dnaA(Ts) mutant. Electron microscopy showed that the non-migrating fraction of DNA was enriched with such structures as single-stranded tails or gaps and branchings with single-stranded arms. It was also found that the appearance of non-migrating DNA was highly dependent on the functional recA gene and moderately on certain RecF-family genes. These results strongly suggest that homologous recombination between sister duplexes is involved in the formation of the peculiar structures found in non-migrating DNA. A possible causal relationship between the formation of non-migrating DNA and viability loss (thymineless death) is also discussed.

The recQ gene of Escherichia coli K12: primary structure and evidence for SOS regulation

SummaryA 2,695 bp chromosomal segment of Escherichia coli K12 containing the recQ gene was sequenced. Analysis of the sequence revealed an open reading frame thought to represent recQ, with a clockwise direction of transcription relative to the standard genetic map of E. coli K12 and having a coding capacity for a protein of Mr 68,350. The—10 region of the presumptive recQ promoter overlapped the putative terminator for the upstream gene pldA, and was immediately followed by a 15 bp stretch of DNA bearing a strong resemblance to the reported sequences of LexA repressor binding sites. This latter finding suggested the possibility of SOS regulation of recQ gene expression, which was substantiated by experiments with recQ-lacZ fusions.

Constituents of regenerated and shoot cultured root tissue of Rehmannia glutinosa

Abstract Callus formation from leaf segments of Rehmannia glutinosa var. purpurea was obtained after the addition of 1 mg/1 2,4-D. When the calli were subcultured embryogenesis occurred. Mature somatic embryos developed normal shoots when transferred to 1/2 MS medium containing 1 mg/1 GA BAP. Root differentiation were cultured in a MS liquid medium supplemented with 1 mg/1 IBA. Root tissues produced two iridoid glycosides, melittoside and rehmannioside D. A caffeoyl glycoside, acetoside, and ethyl-β- d -glucose were detected in the regenerated shoots.

recA+ gene-dependent regulation of a urvD::lacZ fusion in Escherichia coli K12

SummaryThe expression of the Escherichia coli uvrD gene was studied with a uvrD::Mud(Aprlac) insertion mutant. The results indicate that it is inducible by DNA damaging agents in a recA+ gene-dependent manner.

Melanin biosynthesis inhibitory activity of a compound isolated from young green barley (Hordeum vulgare L.) in B16 melanoma cells

In the course to find compounds that inhibit melanin biosynthesis (i.e., whitening agents), we evaluated the effects of the methanol-soluble fraction (i.e., the water-soluble portion of methanol extracts-CHP20P-MeOH eluted fraction) from young green barley leaves on melanin production in B16 melanoma cells. Activity-guided fractionation led to an isolate called tricin (compound 1) as an inhibitory compound of melanin production in B16 melanoma cells. Furthermore, tricin analogs such as tricetin, tricetin trimethyl ether, luteolin, and apigenin were used for analyzing the structure–activity relationships (SAR) of 5,7-dihydroxyflavones studies. Tricin demonstrated stronger inhibitory activity compared to three other compounds. The results suggest that a hydroxyl group at the C-4′ position and methoxy groups at the C-3′,5′ positions of the tricin skeleton may have important roles in this inhibitory activity in B16 melanoma cells. Our results suggest that tricin inhibits melanin biosynthesis with higher efficacy than arbutin, and it could be used as a whitening agent.