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An intracellular 3-hydroxybutyrate (3HB)-oligomer hydrolase (PhaZ[2.sub.Reu]) of Ralstonia eutropha was purified from Escherichia coli harboring a plasmid containing phaZ[2.sub.Reu]. The purified enzyme hydrolyzed linear and cyclic 3HB-oligomers. Although it did not degrade crystalline poly(3-hydroxybutyrate) (PHB), the purified enzyme degraded artificial amorphous PHB at a rate similar to that of the previously identified intracellular PHB (iPHB) depolymerase (PhaZ[1.sub.Reu]). The enzyme appeared to be an endo-type hydrolase, since it actively hydrolyzed cyclic 3HB-oligomers. However, it degraded various linear 3HB-oligomers and amorphous PHB in the fashion of an exo-type hydrolase, releasing one monomer unit at a time. PhaZ2 was found to bind to PHB inclusion bodies and as a soluble enzyme to cell-free supernatant fractions in R. eutropha; in contrast, PhaZ1 bound exclusively to the inclusion bodies. When R. eutropha H16 was cultivated in a nutrient-rich medium, the transient deposition of PHB was observed: the content of PHB was maximized in the log growth phase (12 h, ca. 14% PHB of dry cell weight) and decreased to a very low level in the stationary phase (ca. 1% of dry cell weight). In each phaZ1-null mutant and phaZ2-null mutant, the PHB content in the cell increased to ca. 5% in the stationary phase. A double mutant lacking both phaZ1 and phaZ2 showed increased PHB content in the log phase (ca. 20%) and also an elevated PHB level (ca. 8%) in the stationary phase. These results indicate that PhaZ2 is a novel iPHB depolymerase, which participates in the mobilization of PHB in R. eutropha along with PhaZ1.

Abstract Background Heavy-ion mutagenesis is recognised as a powerful technology to generate new mutants, especially in higher plants. Heavy-ion beams show high linear energy transfer (LET) and thus more effectively induce DNA double-strand breaks than other mutagenic techniques. Previously, we determined the most effective heavy-ion LET (LETmax: 30.0 keV μm-1) for Arabidopsis mutagenesis by analysing the effect of LET on mutation induction. However, the molecular structure of mutated DNA induced by heavy ions with LETmax remains unclear. Knowledge of the structure of mutated DNA will contribute to the effective exploitation of heavy-ion beam mutagenesis. Results Dry Arabidopsis thaliana seeds were irradiated with carbon (C) ions with LETmax at a dose of 400 Gy and with LET of 22.5 keV μm-1 at doses of 250 Gy or 450 Gy. The effects on mutation frequency and alteration of DNA structure were compared. To characterise the structure of mutated DNA, we screened the well-characterised mutants elongated hypocotyls (hy) and glabrous (gl) and identified mutated DNA among the resulting mutants by high-resolution melting curve, PCR and sequencing analyses. The mutation frequency induced by C ions with LETmax was two-fold higher than that with 22.5 keV μm-1 and similar to the mutation frequency previously induced by ethyl methane sulfonate. We identified the structure of 22 mutated DNAs. Over 80% of the mutations caused by C ions with both LETs were base substitutions or deletions/insertions of less than 100 bp. The other mutations involved large rearrangements. Conclusions The C ions with LETmax showed high mutation efficiency and predominantly induced base substitutions or small deletions/insertions, most of which were null mutations. These small alterations can be determined by single-nucleotide polymorphism (SNP) detection systems. Therefore, C ions with LETmax might be useful as a highly efficient reverse genetic system in conjunction with SNP detection systems, and will be beneficial for forward genetics and plant breeding.

Abstract Background Plant promoter architecture is important for understanding regulation and evolution of the promoters, but our current knowledge about plant promoter structure, especially with respect to the core promoter, is insufficient. Several promoter elements including TATA box, and several types of transcriptional regulatory elements have been found to show local distribution within promoters, and this feature has been successfully utilized for extraction of promoter constituents from human genome. Results LDSS (Local Distribution of Short Sequences) profiles of short sequences along the plant promoter have been analyzed in silico, and hundreds of hexamer and octamer sequences have been identified as having localized distributions within promoters of Arabidopsis thaliana and rice. Based on their localization patterns, the identified sequences could be classified into three groups, pyrimidine patch (Y Patch), TATA box, and REG (Regulatory Element Group). Sequences of the TATA box group are consistent with the ones reported in previous studies. The REG group includes more than 200 sequences, and half of them correspond to known cis-elements. The other REG subgroups, together with about a hundred uncategorized sequences, are suggested to be novel cis-regulatory elements. Comparison of LDSS-positive sequences between Arabidopsis and rice has revealed moderate conservation of elements and common promoter architecture. In addition, a dimer motif named the YR Rule (C/T A/G) has been identified at the transcription start site (-1/+1). This rule also fits both Arabidopsis and rice promoters. Conclusion LDSS was successfully applied to plant genomes and hundreds of putative promoter elements have been extracted as LDSS-positive octamers. Identified promoter architecture of monocot and dicot are well conserved, but there are moderate variations in the utilized sequences.

Abstract Background Describing adverse health effects and identifying vulnerable populations during and after a disaster are important aspects of any disaster relief operation. This study aimed to describe the mortality and related risk factors which affected the displaced population over a period of two and a half months after the 2004 Indian Ocean tsunami in an eastern coastal district of Sri Lanka. Methods A cross-sectional household survey was conducted in 13 evacuation camps for internally displaced persons (IDP). Information on all pre-tsunami family members was collected from householders, and all deaths which occurred during the recall period (77 to 80 days starting from the day of the tsunami) were recorded. The distribution of mortality and associated risk factors were analysed. Logistic regression modelling using the generalized estimating equations method was applied in multivariate analysis. Results Overall mortality rate out of 3,533 individuals from 859 households was 12.9% (446 deaths and 11 missing persons). The majority of the deaths occurred during and immediately after the disaster. A higher mortality was observed among females (17.5% vs. 8.2% for males, p < 0.001), children and the elderly (31.8%, 23.7% and 15.3% for children aged less than 5 years, children aged 5 to 9 years and adults over 50 years, respectively, compared with 7.4% for adults aged 20 to 29 years, p < 0.001). Other risk factors, such as being indoors at the time of the tsunami (13.8% vs. 5.9% outdoors, p < 0.001), the house destruction level (4.6%, 5.5% and 14.2% in increasing order of destruction, p < 0.001) and fishing as an occupation (15.4% vs. 11.2% for other occupations, p < 0.001) were also significantly associated with increased mortality. These correlations remained significant after adjusting for the confounding effects by multivariate analysis. Conclusion A significantly high mortality was observed in women and children among the displaced population in the eastern coastal district of Sri Lanka who were examined by us. Reconstruction activities should take into consideration these changes in population structure.