UV-induced ubiquitylation of XPC protein mediated by UV-DDB-ubiquitin ligase complex

The xeroderma pigmentosum group C (XPC) protein complex plays a key role in recognizing DNA damage throughout the genome for mammalian nucleotide excision repair (NER). Ultraviolet light (UV)-damaged DNA binding protein (UV-DDB) is another complex that appears to be involved in the recognition of NER-inducing damage, although the precise role it plays and its relationship to XPC remain to be elucidated. Here we show that XPC undergoes reversible ubiquitylation upon UV irradiation of cells and that this depends on the presence of functional UV-DDB activity. XPC and UV-DDB were demonstrated to interact physically, and both are polyubiquitylated by the recombinant UV-DDB-ubiquitin ligase complex. The polyubiquitylation altered the DNA binding properties of XPC and UV-DDB and appeared to be required for cell-free NER of UV-induced (6-4) photoproducts specifically when UV-DDB was bound to the lesion. Our results strongly suggest that ubiquitylation plays a critical role in the transfer of the UV-induced lesion from UV-DDB to XPC.     

Bio-photosensor: Cyanobacterial photosystem I coupled with transistor via molecular wire

We report on the first successful output of electrons directly from photosystem I (PSI) of thermophilic cyanobacteria to the gate of a field-effect transistor (FET) by bypassing electron flow via a newly designed molecular wire, i.e., artificial vitamin K(1), and a gold nanoparticle; in short, this newly manufactured photosensor employs a bio-functional unit as the core of the device. Photo-electrons generated by the irradiation of molecular complexes composed of reconstituted PSI on the gate were found to control the FET. This PSI-bio-photosensor can be used to interpret gradation in images. This PSI-FET system is moreover sufficiently stable for use exceeding a period of 1 year.     

Seed reserve composition in 19 tree species of a tropical deciduous forest in Mexico and its relationship to seed germination and seedling growth

Background and Aims

The size and composition of seed reserves may reflect the ecological strategy and evolutionary history of a species and also temporal variation in resource availability. The seed mass and composition of seed reserves of 19 co-existing tree species were studied, and we examined how they varied among species in relation to germination and seedling growth rates, as well as between two years with contrasting precipitation (652 and 384 mm).

Methods

Seeds were collected from a tropical deciduous forest in the northwest of Mexico (Chamela Biological Station). The seed dry mass, with and without the seed coat, and the concentrations of lipids, nitrogen and non-structural carbohydrates for the seed minus seed coat were determined. The anatomical localization of these reserves was examined using histochemical analysis. The germination capacity, rate and lag time were determined. The correlations among these variables, and their relationship to previously reported seedling relative growth rates, were evaluated with and without phylogenetic consideration.

Key Results

There were interannual differences in seed mass and reserve composition. Seed was significantly heavier after the drier year in five species. Nitrogen concentration was positively correlated with seed coat fraction, and was significantly higher after the drier year in 12 species. The rate and lag time of germination were negatively correlated with each other. These trait correlations were also supported for phylogenetic independent contrasts. Principal component analysis supported these correlations, and indicated a negative association of seedling relative growth rate with seed size, and a positive association of germination rate with nitrogen and lipid concentrations.

Conclusions

Nitrogen concentration tended to be higher after the drier year and, while interannual variations in seed size and reserve composition were not sufficient to affect interspecific correlations among seed and seedling traits, some of the reserves were related to germination variables and seedling relative growth rate.     

Sequencing of three lambda clones from the genome of alkaliphilic Bacillus sp. strain C-125

The nucleotide sequences of three independent fragments (designated no. 3, 4, and 9; each 15–20 kb in size) of the genome of alkaliphilic Bacillus sp. C-125 cloned in a λ phage vector have been determined. Thirteen putative open reading frames (ORFs) were identified in sequenced fragment no. 3 and 11 ORFs were identified in no. 4. Twenty ORFs were also identified in fragment no. 9. All putative ORFs were analyzed in comparison with the BSORF database and non-redundant protein databases. The functions of 5 ORFs in fragment no. 3 and 3 ORFs in fragment no. 4 were suggested by their significant similarities to known proteins in the database. Among the 20 ORFs in fragment no. 9, the functions of 11 ORFs were similarly suggested. Most of the annotated ORFs in the DNA fragments of the genome of alkaliphilic Bacillus sp. C-125 were conserved in the Bacillus subtilis genome. The organization of ORFs in the genome of strain C-125 was found to differ from the order of genes in the chromosome of B. subtilis, although some gene clusters (ydh, yqi, yer, and yts) were conserved as operon units the same as in B. subtilis. Received: April 17, 1998 / Accepted: June 23, 1998     

Characterization of two genes encoding putative cysteine synthase required for cysteine biosynthesis in Schizosaccharomyces pombe

Cysteine synthase catalyzes the formation of cysteine from O-acetylserine, and is the key enzyme for de novo cysteine biosynthesis in Schizosaccharomyces pombe. An examination of the S. pombe database revealed that two gene products are predicted to encode proteins homologous to eukaryotic cysteine synthases. Disruption of one of these candidates, cys1a+ (SPBC36.04), caused an obvious cysteine auxotrophy, while disruption of cys1b+ (SPAC3A12.17c) had no effect on the growth phenotype. Furthermore, overexpression of cys1b+ did not complement the cysteine auxotrophic phenotype of cys1a mutant cells. These results indicated that cys1a+, not cys1b+, primarily functions in the biosynthesis of cysteine in S. pombe cells. We constructed a bacterial-S. pombe shuttle vector containing cys1a+ as a selective marker gene. The combination of the cysteine auxotroph and new vector could be useful for the expression of a heterologous protein.     

A crucial role for matrix metalloproteinase 2 in osteocytic canalicular formation and bone metabolism

Extracellular matrix production and degradation by bone cells are critical steps in bone metabolism. Mutations of the gene encoding MMP-2, an extracellular matrix-degrading enzyme, are associated with a human genetic disorder characterized by subcutaneous nodules, arthropathy, and focal osteolysis. It is not known how the loss of MMP-2 function results in the pathology. Here, we show that Mmp2(-/-) mice exhibited opposing bone phenotypes caused by an impaired osteocytic canalicular network. Mmp2(-/-) mice showed decreased bone mineral density in the limb and trunk bones but increased bone volume in the calvariae. In the long bones, there was moderate disruption of the osteocytic networks and reduced bone density throughout life, whereas osteoblast and osteoclast function was normal. In contrast, aged but not young Mmp2(-/-) mice had calvarial sclerosis with osteocyte death. Severe disruption of the osteocytic networks preceded osteocyte loss in Mmp2(-/-) calvariae. Successful transplantation of wild-type periosteum restored the osteocytic canalicular networks in the Mmp2(-/-) calvariae, suggesting local roles of MMP-2 in determining bone phenotypes. Our results indicate that MMP-2 plays a crucial role in forming and maintaining the osteocytic canalicular network, and we propose that osteocytic network formation is a determinant of bone remodeling and mineralization.     

Ethanol-inducible gene expression using gld1 + promoter in the fission yeast Schizosaccharomyces pombe

In the fission yeast Schizosaccharomyces pombe, the gld1 ⁺ gene encoding glycerol dehydrogenase is repressed by glucose and induced by ethanol and 1-propanol. The promoter region of gld1 ⁺ was cloned into a multicopy vector designated as pEG1 for evaluation as an ethanol-inducible expression vector using EGFP as a model heterologous protein. Expression of EGFP was repressed in the presence of high glucose and induced in the presence of ethanol, low-glucose, and 1-propanol in the absence of glucose. Addition of ethanol to cells harboring pEG1–EGFP was found to be the most effective means for inducing EGFP production. Protein yields were found to increase in proportion to ethanol concentration. As a further test of effectiveness, secreted recombinant human growth hormone was produced using the pEG1 expression vector in medium containing glycerol and ethanol. The pEG1 gene expression system is an effective tool for the production of heterologous proteins under glucose-limiting conditions, including medium containing glycerol as a carbon source.     

Excitation-energy distribution in green algae. The existence of two independent light-driven control mechanisms

Three distinct states can be identified for cells of the green alga Chlorella vulgaris; State 1 and State 2 obtained by preillumination in far-red and red light, respectively, and the dark state obtained by dark-adaptation. Addition of the inhibitor DCMU to algal cells leads to an initial rapid increase in chlorophyll-a fluorescence reflecting the closure of Photosystem II traps. This, in the case of dark and state-2-adapted algae is followed by a slow light-dependent increase to a fluorescence yield typical of State-1-adapted cells. Measurements of low temperature (77 K) emission spectra indicate that the low fluorescence yields of dark and State-2-adapted algae reflect similar balances in excitation-energy distribution between the two photosystems. In both cases, the balance favours PS I and the slow fluorescence increase seen in the poisoned algae reflects a redressing of this balance in favour of PS II. The low fluorescence yield of State-2-adapted algae is thought to be associated with the phosphorylation of chlorophyll a/b light-harvesting protein (Biochim. Biophys. Acta (1983) 724, 94–103). Measurements of the uncoupler and ATPase sensitivity of the light-dependent increases seen in DCMU-poisoned cells indicate that the low fluorescence yield of dark-adapted algae is of different origin. Evidence is presented showing that the light-driven changes in excitation-energy distribution seen in green algae involve two distinct processes; a low-intensity, wavelenght-independent change reflecting simple light/dark changes and a higher intensity, wavelength-dependent change reflecting State 1/State 2 adaptation. The former changes appear to be associated with changes in the local ionic environment within the algal chloroplast, whilst the latter appear to reflect changes in the phosphorylation state of chlorophyll a/b light-harvesting protein.