Tapetum-Specific Expression of the Gene for an Endo-{beta}-1,3-glucanase Causes Male Sterility in Transgenic Tobacco

A cDNA for a pathogenesis-related endo-ß-1,3-glucanaseisolated from soybean, was fused to an anther tapetum-specificpromoter (Osg6B promoter) isolated from rice and the resultingchimeric gene was introduced into tobacco. The Osg6B promoterbecame active in the anther tapetum during formation of tetradsand the tapetal glucanase activity in the transgenic plantscaused in a significant reduction in the number of fertile pollengrains. Most of the pollen grains were aberrant in shape, lackedgerminal apertures and aggregate of the pollen grains. Granulesof ß-1,3-glucan, which have not previously been reported,were often observed to adhere to the surface of the pollen grains.Further observations revealed that the callose wall was almostabsent in the pollen tetrads of transgenic plants. In wild-typeplants, by contrast, the tetrads were surrounded by callosethat was degraded soon after the tetrad stage to release freemicrospores. Thus, the introduced gene for endo-ß-1,3-endoglucanaseunder the control of the Osg6B promoter caused digestion ofthe callose wall at the beginning of the tetrad stage, a timethat was just a little earlier than the time at which endogenousglucanase activity normal appears. These results demonstratethat premature dissolution of the callose wall in pollen tetradscauses male sterility and suggest that the time at which tapetallyproduced glucanase is activate is critical for the normal developmentof microspores. (Received September 29, 1994; Accepted January 30, 1995)     

Autophagy is activated for cell survival after endoplasmic reticulum stress

Eukaryotic cells deal with accumulation of unfolded proteins in the endoplasmic reticulum (ER) by the unfolded protein response, involving the induction of molecular chaperones, translational attenuation, and ER-associated degradation, to prevent cell death. Here, we found that the autophagy system is activated as a novel signaling pathway in response to ER stress. Treatment of SK-N-SH neuroblastoma cells with ER stressors markedly induced the formation of autophagosomes, which were recognized at the ultrastructural level. The formation of green fluorescent protein (GFP)-LC3-labeled structures (GFP-LC3 “dots”), representing autophagosomes, was extensively induced in cells exposed to ER stress with conversion from LC3-I to LC3-II. In IRE1-deficient cells or cells treated with c-Jun N-terminal kinase (JNK) inhibitor, the autophagy induced by ER stress was inhibited, indicating that the IRE1-JNK pathway is required for autophagy activation after ER stress. In contrast, PERK-deficient cells and ATF6 knockdown cells showed that autophagy was induced after ER stress in a manner similar to the wild-type cells. Disturbance of autophagy rendered cells vulnerable to ER stress, suggesting that autophagy plays important roles in cell survival after ER stress.     

Generation of three-dimensional retinal organoids expressing rhodopsin and S- and M-cone opsins from mouse stem cells

Purpose

Three-dimensional retinal organoids can be differentiated from embryonic stem cells/induced pluripotent stem cells (ES/iPS cells) under defined medium conditions. We modified the serum-free floating culture of embryoid body-like aggregates with quick reaggregation (SFEBq) culture procedure to obtain retinal organoids expressing more rod photoreceptors and S- and M-cone opsins.

Interaction between elongation factors 1beta and 1gamma from Bombyx mori silk gland

Elongation factor 1 (EF-1) from the silk gland of Bombyx mori consists of four subunits: alpha (51 kDa), beta (26 kDa), gamma (49 kDa), and delta (33 kDa). The EF-1alpha subunit catalyzes the binding of aminoacyl-tRNA to the ribosome concomitant with the hydrolysis of GTP. The EF-1alpha-bound GDP is then exchanged for GTP by the EF-1betagammadelta complex. To facilitate analysis of the roles of the individual EF-1beta, gamma, and delta subunits in GDP/GTP exchange on EF-1alpha, we cloned the cDNAs for these subunits and expressed them in Escherichia coli. EF-1beta, EF-1gamma, and the carboxyl-terminal half of EF-1delta were expressed, purified, and examined for protein:protein interactions by gel filtration chromatography and by a quartz-crystal microbalance method. An 80-kDa species containing EF-1beta and gamma subunits in a 1:1 molar ratio was detected by gel filtration. A higher molecular weight species containing an excess of EF-1gamma relative to EF-1beta was also detected. The amino-terminal region of EF-1beta (amino acid residues 1-129) was sufficient for binding to EF-1gamma. The carboxyl-terminal half of EF-1delta did not appear to form a complex with EF-1gamma.     

Detection of von Willebrand factor-cleaving protease (ADAMTS-13) in human platelets

The hemostatic activity of von Willebrand factor (vWF) is strongly dependent on its multimeric structure, with the highest activity in 'unusually large' multimers secreted from endothelial cells. The multimeric structure is regulated by a plasma protease, vWF-cleaving protease (vWF-CP, or ADAMTS-13). ADAMTS-13 mRNA is variably expressed in liver and other tissues. Because 15-25% of circulating vWF is stored in platelets, the presence and function of ADAMTS-13 in platelets are important issues. Here we report ADAMTS-13 expression in human platelets. Western blot analysis and flow cytometric analysis on permeabilized platelets revealed the presence of ADAMTS-13 protein. Real-time PCR demonstrated that ADAMTS-13 mRNA is present in platelets of six healthy volunteers, with little quantitative difference. The presence of ADAMTS-13 in platelets may imply the functional role of this enzyme in the local regulation of platelet function at the site of vascular injury or thrombus formation, and provide a useful tool for the analysis of structure and function of this enzyme.     

Genetic characterization of Raffaelea quercivora isolates collected from areas of oak wilt in Japan

This study was undertaken to improve understanding of the phylogenetic position of pathogenic fungi implicated in the oak wilt in Japan. Sequences were obtained from three regions of partial nuclear ribosomal DNA of 25 isolates of Raffaelea quercivora including an ex-type strain, all of which were collected from seven areas of disease outbreak. All the isolates formed one clear clade with high bootstrap values, distinctly delimited from the closest species, R. montetyi. These results indicate that the R. quercivora is phylogenetically a well-defined taxon.     

Analysis of spatiotemporal regulation of aromatase in the brain using transgenic mice

Brain aromatase is widely distributed in the vertebrates, from fish to mammals, and plays important roles in functional reproductive behavior through production of estrogen as a neurosteroid. It is expressed only in the nerve cells of specific brain regions with a transient peak in the neonatal period when sexual behavior becomes organized, and therefore provides a good model system to study regulatory mechanism of cell-specific, brain region-specific, and developmental stage-specific expression.

To elucidate spatiotemporal regulation of brain aromatase, we prepared transgenic mice carrying a reporter gene under the promoter of brain-specific exon 1f of the mouse aromatase gene. The reporter transgene carrying a 6.5 kb upstream region of the brain-specific promoter accurately reproduced the spatiotemporal expression patterns of aromatase in mouse brain, whereas transgenes carrying smaller fragments of the promoter showed ambiguous or inconsistent expression patterns.

The binding sites of Aro-AI, Aro-AII, and Aro-B for nuclear factors were also identified in the proximal region of the exon 1f brain-specific promoter. Introduction of a mutation into the Aro-AII site in the reporter transgene carrying −6.5 kb promoter region of exon 1f caused complete alteration of the spatiotemporal expression pattern of the reporter gene in the transgenic mice.

These results indicate that the −6.5 kb promoter region of exon 1f is the minimal essential element for brain-specific regulation, with both proximal and distal promoter regions required for accurate spatiotemporal expression of aromatase in the mouse brain.