We describe a highly efficient alkali cation method and library transducing vectors for cloning mammalian cDNAs by trans-complementation of fission yeast Schizosaccharomyces pombe mutants. cDNA libraries constructed with the pcD or pcD2 vector are transduced into yeast by cotransfection with a linearized vector, which allows an enhanced homologous recombination between the yeast vector and the library plasmid leading to the efficient formation of concatemers containing pcD molecules. The transformation frequencies obtained by the method are 10(6) colonies per 10(8) cells transfected with 2 micrograms of library and 1 microgram of vector, 50-60% of which contain pcD molecules. The high-efficiency alkali cation method circumvents many of the shortcomings of the spheroplast method generally used for Schiz. pombe transfection. The vectors are maximized for the efficiency of library transduction and minimized for the rearrangements of pcD molecules during propagation in yeast. This system allows rapid screening of multi-million cDNA clone libraries for rare cDNAs in a routine scale of experiments. Using this system, various mammalian cDNAs that are extremely difficult, time-consuming, or unclonable to clone by other methods have been cloned.

SummaryA large number of metabolites are found in each plant, most of which have not yet been identified. Development of a methodology is required to deal systematically with unknown metabolites, and to elucidate their biological roles in an integrated 'omics' framework. Here we report the development of a 'metabolite annotation' procedure. The metabolite annotation is a process by which structures and functions are inferred for metabolites. Tomato (Solanum lycopersicum cv. Micro-Tom) was used as a model for this study using LC-FTICR-MS. Collected mass spectral features, together with predicted molecular formulae and putative structures, were provided as metabolite annotations for 869 metabolites. Comparison with public databases suggests that 494 metabolites are novel. A grading system was introduced to describe the evidence supporting the annotations. Based on the comprehensive characterization of tomato fruit metabolites, we identified chemical building blocks that are frequently found in tomato fruit tissues, and predicted novel metabolic pathways for flavonoids and glycoalkaloids. These results demonstrate that metabolite annotation facilitates the systematic analysis of unknown metabolites and biological interpretation of their relationships, which provide a basis for integrating metabolite information into the system-level study of plant biology.

Unlike ubiquitin, the ubiquitin-like protein modifier SUMO-1 and its budding yeast homologue Smt3p have been shown to be more important for posttranslational protein modification than for protein degradation. Here we describe the identification of the SUMO-1 homologue of fission yeast, which we show to be required for a number of nuclear events including the control of telomere length and chromosome segregation. A disruption of the pmt3 ؉ gene, the Schizosaccharomyces pombe homologue of SMT3, was not lethal, but mutant cells carrying the disrupted gene grew more slowly. The pmt3⌬ cells showed various phenotypes such as aberrant mitosis, sensitivity to various reagents, and high-frequency loss of minichromosomes. Interestingly, we found that pmt3 ؉ is required for telomere length maintenance. Loss of Pmt3p function caused a striking increase in telomere length. When Pmt3p synthesis was restored, the telomeres became gradually shorter. This is the first demonstration of involvement of one of the Smt3p/SUMO-1 family proteins in telomere length maintenance. Fusion of Pmt3p to green fluorescent protein (GFP) showed that Pmt3p was predominantly localized as intense spots in the nucleus. One of the spots was shown to correspond to the spindle pole body (SPB). During prometaphase-and metaphase, the bright GFP signals at the SPB disappeared. These observations suggest that Pmt3p is required for kinetochore and/or SPB functions involved in chromosome segregation. The multiple functions of Pmt3p described here suggest that several nuclear proteins are regulated by Pmt3p conjugation.Ubiquitin is a small (76-residue), abundant protein conserved in all eukaryotic cells. It exists in several cellular compartments, such as the cytosol, nucleus, and cell surface. It is well known that ubiquitin regulates the function and stability of target proteins through its posttranslational conjugation to target proteins. Before conjugation to target proteins, ubiquitin must be processed by a C-terminal hydrolase. The first step of the ubiquitin conjugation pathway is the ATP-dependent formation of a thioester bond between the conserved C-terminal glycine of processed ubiquitin and the active-site cysteine residue of an E1 ubiquitin-activating enzyme. The second step is the transfer of activated ubiquitin to the active-site cysteine of an E2 ubiquitin-conjugating enzyme. In the final step, the E2 enzyme may cooperate with an E3 ubiquitin protein ligase to form an isopeptide bond between the C-terminal glycine of ubiquitin and the ε-amino groups of lysine residues of target proteins. Ubiquitin covalently conjugated to target proteins can be removed by a ubiquitin isopeptidase (89).Recently, a number of novel ubiquitin-like proteins were independently discovered in a number of species, suggesting that ubiquitin is part of a family of related proteins involved in the covalent modification of proteins. The first example of such a protein was the 15-kDa interferon-inducible, ubiquitin crossreacting protein UCRP (25). UCRP contains two ubiquitinr...

We identified the phhl ÷ gene that encodes a MAP kinase as the effector of Wisl MAP kinase kinase in fission yeast, which is highly homologous with HOG1 of S. cerevisiae. Heterothalic phhl dsiruptant is phenotypically indistinguishable from wisl deletion mutant, both displaying the same extent of partial sterility and enhanced sensitivity to a variety of stress. In phhl disruptant, nitrogen starvation-induced expression of stell +, a key controller of sexual differentiation, is markedly diminished. Ectopic expression of stell ÷ effectively restores fertility, but not stress resistance, to the phhl disruptant. These data show that stress signal, mediated by a MAP kinase, is required for efficient start of sexual differentiation.

We have isolated a new cell division cycle gene (resl+) required for entry into S phase, as a multicopy dual suppressor of the paut and cdclO mutants of the fission yeast Schizosaccharomyces pombe. The resi+ gene specifies a 72 kDa protein with two copies of the cdclO/SWI6 motif. A disruptant of resl+ grows poorly at 30°C with severe heat-and cold-sensitivities, and completely arrests in G1 at 36°C and 23°C. The arrested disruptant retains a full conjugation ability. In addition to the cdclO/SWI6 motif, Resl and SWI4 proteins share a remarkable homology in their aminoterminal region, whereas CdclO and SWI6 do so in their carboxy-terminal region. Moreover, the amino-terminal region is essential for the function of Resl as it is for the function of SWI4. Furthermore, analogous to the relationship of SWI4 to SWI6, the resl+ gene effectively rescues cdclO mutants, but the cdclO+ gene cannot rescue the resl-phenotype. Thus, striking similarities exist in both structural and functional relationships between Resl and SWI4, and between CdclO and SWI6. In view of the fact that SWI4 and SWI6 form a transcription factor complex and activate promoters containing the SWI4/SWI6 dependent cell-cycle box, Resl might be a putative association partner of CdclO which appears to be involved at least in the activation of promoters containing a MluI cell-cycle box.

A Schizosaccharomyces pombe spindle pole body (SPB) protein interacts in a two-hybrid system with Dlc1, which belongs to the 14-kDa Tctex-1 dynein light chain family. Green fluorescent protein-tagged Dlc1 accumulated at the SPB throughout the life cycle. During meiotic prophase, Dlc1 was present along astral microtubules and microtubule-anchoring sites on the cell cortex, reminiscent of the cytoplasmic dynein heavy chain Dhc1. In a dlc1-null mutant, Dhc1-dependent nuclear movement in meiotic prophase became irregular in its duration and direction. Dhc1 protein was displaced from the cortex anchors and the formation of microtubule bundle(s) that guide nuclear movement was impaired in the mutant. Meiotic recombination in the dlc1 mutant was reduced to levels similar to that in the dhc1 mutant. Dlc1 and Dhc1 also have roles in karyogamy and rDNA relocation during the sexual phase. Strains mutated in both the dlc1 and dhc1 loci displayed more severe defects in recombination, karyogamy, and sporulation than in either single mutant alone, suggesting that Dlc1 is involved in nuclear events that are independent of Dhc1. S. pombe contains a homolog of the 8-kDa dynein light chain, Dlc2. This class of dynein light chain, however, is not essential in either the vegetative or sexual phases.

Intermediate-depth earthquakes in cold subduction zones are observed within the subducting oceanic crust, as well as the mantle. In contrast, intermediate-depth earthquakes in hot subduction zones predominantly occur just below the Mohorovičić discontinuity. These observations have stimulated interest in relationships between blueschist-facies metamorphism and seismicity, particularly through dehydration reactions involving the mineral lawsonite. Here we conducted deformation experiments on lawsonite, while monitoring acoustic emissions, in a Griggs-type deformation apparatus. The temperature was increased above the thermal stability of lawsonite, while the sample was deforming, to test whether the lawsonite dehydration reaction induces unstable fault slip. In contrast to similar tests on antigorite, unstable fault slip (that is, stick-slip) occurred during dehydration reactions in the lawsonite and acoustic emission signals were continuously observed. Microstructural observations indicate that strain is highly localized along the fault (R1 and B shears), and that the fault surface develops slickensides (very smooth fault surfaces polished by frictional sliding). The unloading slope during the unstable slip follows the stiffness of the apparatus at all experimental conditions, regardless of the strain rate and temperature ramping rate. A thermomechanical scaling factor for the experiments is within the range estimated for natural subduction zones, indicating the potential for unstable frictional sliding within natural lawsonite layers.

Slow earthquakes occur at the plate interface in subduction zones. These low-frequency tremors and slow-slip events are often located at about 30 km depth 1-3 , near the boundary between the crust and mantle (Moho) on the overriding plate. Slow earthquakes occur on fault patches with extremely low frictional strength 4-6 . This weakness is generally assumed to result from increased pore-fluid pressures and may be linked to the release of fluids from the descending plate. Here we propose that a contrast in permeability across the Moho results in the accumulation of water and the build-up of pore-fluid pressure at the corner of the mantle wedge that overlies the subducting plate. We use laboratory measurements of permeability to show that gabbroic rock layers in the crust are two orders of magnitude less permeable than serpentinite layers in the underlying hydrated mantle rocks. Inserting our experimental data into a numerical model that simulates pore pressure evolution across the Moho, we show that the porefluid pressure at this boundary can be as high as lithostatic pressure. We suggest that water released from the descending plate is trapped at the corner of the mantle wedge owing to this permeability barrier, and then causes the localized slow earthquakes that are triggered by fault instabilities.The presence of pore fluid has long been recognized as playing an important role in the crustal deformation and earthquake mechanism, because it weakens the frictional strength and can result in fault instability 7,8 . As episodic tremor and slow-slip events found in subduction zones exhibit extremely low effective stresses, the occurrence of these slow earthquakes is attributed to the effect of high pore-fluid pressures on the plate boundaries 4-6 . Low seismicwave velocities and high Poisson's ratios of the source regions also suggest the presence of aqueous fluids that facilitate these slow earthquakes 1-3 . The occurrence of these events is mostly localized at triple junctions between the subducting plate and the island-arc Moho; consequently, a mechanism must be operating to facilitate water accumulation in such localized regions.Water in the subducting plate is released into the overlying mantle wedge by the dehydration of hydrous minerals 9 . These fluids tend to migrate upwards along the plate interface because the hydrated mantle (serpentinite) can develop a strong fabric 10 that results in a significant anisotropy in permeability 11 . At the island-arc Moho, migrating fluids can become trapped at the gabbroic layer, which acts as a cap rock, similar to the structures that trap petroleum resources 12 . Thus, the permeability contrast across the Moho may be the key to water accumulation in the source regions of slow earthquakes (Fig. 1). A similar model of a permeability barrier has also been proposed to account for the anomalous pore pressures in subducted crust [13][14][15] . In this study we Figure 1 | Schematic model of water circulating in a subduction zone.Subducting plates release most of their water in...