LET dependence of lethality in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> irradiated by heavy ions

To clarify the effect of heavy ions in plants, dry seeds of Arabidopsis were irradiated with carbon, neon, and argon ions with various linear energy transfer (LET) values. The relative biological effectiveness (RBE) for lethality peaked at LET values over 350 keV/microns for neon and argon ions. This LET giving the peak RBE was higher than the LET of 100-200 keV/microns which was reported to have a maximum RBE for other types of cells, such as mammalian cells. Furthermore, sterility showed a higher RBE at an LET of 354 keV/microns with neon ions than that at an LET of 113 keV/microns with carbon ions. Lethality and sterility are both considered to be caused by damage to DNA. The results indicate that the LET having a maximum of RBE for lethality is higher in Arabidopsis seeds than in other unicellular systems. The most likely explanation for this shift of LET is that the DNA in dry seeds has a different chemical environment and/or hydration state than the DNA in cells in culture.     

Changes in biomass,productivity and decomposition along topographical gradients under different geological conditions in tropical lower montane forests on Mount Kinabalu,Borneo

We have examined how the structure and function of a forest ecosystem change with topography (lower-slope versus ridge) and how the changes are modified by nutrient availability depending on geological substrate (Quaternary and Tertiary sedimentary rocks and ultrabasic rock) in the tropical montane rain forests of Mt. Kinabalu (Borneo) where climate is humid and aseasonal. Reflecting the difference in site age and parent rock, the pool size of soluble-P and inorganic-N in topsoil decreased from Quaternary sedimentary >Tertiary sedimentary >ultrabasic rock on the lower-slope, and they decreased from the lower-slope to the ridge on all substrates. Forest structural attributes [stature, above-ground biomass, and leaf area index (LAI)] decreased in the order of Quaternary sedimentary >Tertiary sedimentary >ultrabasic rock in association with soil nutrients on the lower-slopes, and decreased upslope consistently on each of the three substrates. Functional attributes [above-ground net primary productivity (ANPP) and decomposition rate] demonstrated similar patterns to structure. ANPP significantly correlated with LAI among the six sites, while net assimilation rate (ANPP divided by LAI assuming an even productivity between above vs below-ground system) was nearly constant. Therefore, ANPP could be explained primarily by LAI. Topographical change in LAI could be explained by leaf mass per area (LMA) combined with stand-level leaf biomass. LMA increased upslope on all substrates in association with the decrease in individual leaf area. Stand-level leaf biomass decreased upslope on all substrates but the Tertiary sedimentary rock. Our study demonstrated that topography and geological substrates interactively affected forest structure and processes. The effect of topography on forest structure and processes was greater on nutrient-rich substrates than on poor substrates, and the effect of geological substrate was greater on lower-slopes than on ridges.     

A kaiC-interacting sensory histidine kinase, SasA, necessary to sustain robust circadian oscillation in cyanobacteria

Both regulated expression of the clock genes kaiA, kaiB, and kaiC and interactions among the Kai proteins are proposed to be important for circadian function in the cyanobacterium Synechococcus sp. strain PCC 7942. We have identified the histidine kinase SasA as a KaiC-interacting protein. SasA contains a KaiB-like sensory domain, which appears sufficient for interaction with KaiC. Disruption of the sasA gene lowered kaiBC expression and dramatically reduced amplitude of the kai expression rhythms while shortening the period. Accordingly, sasA disruption attenuated circadian expression patterns of all tested genes, some of which became arrhythmic. Continuous sasA overexpression eliminated circadian rhythms, whereas temporal overexpression changed the phase of kaiBC expression rhythm. Thus, SasA is a close associate of the cyanobacterial clock that is necessary to sustain robust circadian rhythms.     

Soil phosphorus fractionation and phosphorus-use efficiencies of tropical rainforests along altitudinal gradients of Mount Kinabalu, Borneo

We studied soil phosphorus (P) fractionation and P-use efficiencies (PUEs) of rainforests along altitudinal gradients (700–3100 m) on two types of parental rocks (sedimentary versus ultrabasic) on Mount Kinabalu, Borneo. Sedimentary rocks were known to contain more quartz (which does not adsorb P) than ultrabasic rocks. The pool (top 30 cm) of total P was always greater on sedimentary (ranging from 34.9 to 72.6 g m^–2) than on ultrabasic (9.0–29.2 g m^–2) rocks at comparable altitudes. Accordingly, the pools of organic P and labile inorganic P were always greater on sedimentary than on ultrabasic rocks. The pool of primary mineral, calcium P increased upslope from 1.7 to 4.3 g m^–2 on sedimentary rock, suggesting that the altitudinal sequence of the sites reflected a decreasing magnitude of soil weathering upslope. The pool of calcium P on ultrabasic rock did not vary consistently with altitude (1.2–2.8 g m^–2), probably reflecting the greater between-site variability of primary mineral P in parent rocks. When all sites were compared, the pool of most labile, bicarbonate-extracted inorganic P increased (ranging from 0.02 to 1.85 g m^–2) with increasing calcium P. Calcium P was therefore considered to be an important P source to the biota on Kinabalu. Gross patterns in the variation of PUE (indexed as the reciprocal of the P concentration in litter) were best explained by the pool size of actively cycling P (total P minus occluded inorganic P). PUE, however, demonstrated distinct altitudinal patterns to generate an intricate conrol of P use pattern by soil P pools and altitude. Received: 2 August 1998 / Accepted: 28 November 1999     

Monitoring of the refolding process for immobilized firefly luciferase with a biosensor based on surface plasmon resonance

In order to examine the possibility of the use of a surface plasmon resonance (SPR) sensor for real-time monitoring of the process of refolding of immobilized proteins, the refolding of firefly luciferase immobilized on a carboxymethyldextran matrix layer was analyzed. The SPR signal of the immobilized luciferase decreased after unfolding induced by GdnCl and increased gradually in the refolding buffer, while there was no signal change in the reference surface lacking the immobilized protein. The decrease in the SPR signal on unfolding was consistent with the difference between the refractive indices of the native and unfolded protein solutions. The effects of blocking of the excess NHS-groups of the matrix layer on the refolding yield were examined by means of an SPR sensor. The results were consistent with those obtained with the enzymatic activity assay, indicating that the changes in the SPR signal reflected the real-time conformational changes of the immobilized protein. Hence, an SPR biosensor might be used for monitoring of the process of refolding of immobilized proteins and as a novel tool for optimization of the refolding conditions. This is the first demonstration that SPR signal changes reflect the conformational changes of an immobilized protein upon unfolding and refolding.     

Design of the linkers which effectively separate domains of a bifunctional fusion protein

With the aim of separating the domains of a bifunctional fusion protein, the ability of several lengths of helix-forming peptides to separate two weakly interacting beta-can domains was compared with that of flexible linkers or of a three alpha-helices bundle domain. We introduced helix-forming peptide linkers A(EAAAK)nA (n = 2-5) between two green fluorescent protein variants, EBFP and EGFP, and investigated their spectral properties. The fluorescence resonance energy transfer from EBFP to EGFP decreased as the length of the linkers increased. The circular dichroism spectra analysis suggested that the linkers form an alpha-helix and the alpha-helical contents increased as the length of the linkers increased. The results clearly suggested the ability of the helical linkers to control the distance and reduce the interference between the domains. This 'linker engineering' may open a way to the rational design of linkers which maximize the multiple functions of fusion proteins or de novo multi-domain proteins.     

Global gene expression profiling and cluster analysis in Xenopus laevis

We have undertaken a large-scale microarray gene expression analysis using cDNAs corresponding to 21,000 Xenopus laevis ESTs. mRNAs from 37 samples, including embryos and adult organs, were profiled. Cluster analysis of embryos of different stages was carried out and revealed expected affinities between gastrulae and neurulae, as well as between advanced neurulae and tadpoles, while egg and feeding larvae were clearly separated. Cluster analysis of adult organs showed some unexpected tissue-relatedness, e.g. kidney is more related to endodermal than to mesodermal tissues and the brain is separated from other neuroectodermal derivatives. Cluster analysis of genes revealed major phases of co-ordinate gene expression between egg and adult stages. During the maternal-early embryonic phase, genes maintaining a rapidly dividing cell state are predominantly expressed (cell cycle regulators, chromatin proteins). Genes involved in protein biosynthesis are progressively induced from mid-embryogenesis onwards. The larval-adult phase is characterised by expression of genes involved in metabolism and terminal differentiation. Thirteen potential synexpression groups were identified, which encompass components of diverse molecular processes or supra-molecular structures, including chromatin, RNA processing and nucleolar function, cell cycle, respiratory chain/Krebs cycle, protein biosynthesis, endoplasmic reticulum, vesicle transport, synaptic vesicle, microtubule, intermediate filament, epithelial proteins and collagen. Data filtering identified genes with potential stage-, region- and organ-specific expression. The dataset was assembled in the iChip microarray database, , which allows user-defined queries. The study provides insights into the higher order of vertebrate gene expression, identifies synexpression groups and marker genes, and makes predictions for the biological role of numerous uncharacterized genes.