Atsushi Higashitani

Effects of High Temperature on the Development of Pollen Mother Cells and Microspores in Barley Hordeum vulgare L.

The development of the inflorescence, microspores and anthesis were well synchronized among individuals or in the panicles of barley under controlled environmental conditions. To study the effects of high-temperature stress on the development of pollen mother cells (PMCs) and microspores, the plants were subjected to high temperature treatment at different stages of reproductive growth. When plants were exposed to high temperature for five days at the early differentiation stage of the panicle, pollen grains had apparently normal exine but no or little cytoplasm. At the pre-meiotic stage of PMCs, high temperature caused subsequent development of short anthers possessing no pollen grains. When plants were exposed to high temperature during meiosis of PMCs, all pollen grains possessed exine and were swollen but showed little starch accumulation. In these plants treated at high temperature, the panicles at the heading stage had a normal appearance, but their seeds were virtually sterile. These results indicated that there are at least three stages of reproductive growth hypersensitive to high temperature, which resulted in abnormal terminal phenotypes different from one another.

Caenorhabditis elegans RBX1 is essential for meiosis, mitotic chromosomal condensation and segregation, and cytokinesis

Background:u2002 The RING‐H2 finger protein RBX1 (ROC1/HRT1) is a common subunit of SKP1‐CDC53/CUL1‐F‐box (SCF), other cullins and von Hippel‐Lindau (VHL) tumour suppressor E3 ubiquitin ligase complexes. RBX1 protein sequences are highly conserved in various species, including yeasts, Drosophila melanogaster, mice and humans. In Saccharo‐myces cerevisiae, RBX1 is essential for the G1/S transition.

Cell cycle arrest and apoptosis in Caenorhabditis elegans germline cells following heavy-ion microbeam irradiation

Purpose: To investigate positional effects of radiation with an energetic heavy-ion microbeam on germline cells using an experimental model metazoan Caenorhabditis elegans. Materials and methods: The germline cells were irradiated with raster-scanned broad beam or collimated microbeam of 220 MeV 12C5+ particles delivered from the azimuthally varying field (AVF) cyclotron, and subsequently observed for cell cycle arrest and apoptosis. Results: Whole-body irradiation with the broad beam at the L4 larval stage arrested germ cell proliferation. When the tip region of the gonad arm was irradiated locally with the microbeam at the L4 stage, the same arrest was observed. When the microbeams were used to irradiate the pachytene region of the gonad arm, at a young gravid stage, radiation-induced apoptosis occurred in the gonad. In contrast, arrest and apoptosis were not induced in the non-irradiated neighboring region or the opposite gonad. Similar results were confirmed in the c-abl-1 (mammalian ortholog of cellular counterpart of Abelson murine leukemia virus) mutant that is hypersensitive to radiation-induced apoptosis. Conclusion: These results indicate that the microbeam irradiation is useful in characterizing tissue-specific, local biological response to radiation in organisms. DNA damage-induced cell cycle arrest and apoptosis were observed in locally irradiated regions, but there was little, if any, ‘bystander effect’ in the nematode.

Caenorhabditis elegans Chk2-like gene is essential for meiosis but dispensable for DNA repair

A Chk2‐like gene was identified in the genome of Caenorhabditis elegans. The putative gene product, termed Ce‐chk‐2 consists of 450 amino acid residues, and shows good homology with the Chk2/Cds1 gene family. The results of RNA‐mediated interference (RNAi) indicated that the F1 generation from dsRNA injected animals grew to adulthood, but approximately 95% of their eggs (F2) died during early embryogenesis. Among the few surviving progeny, males (XO animals) arose at an abnormally high frequency (30%). In addition, 12 univalents were observed in full grown oocytes of the F1, while six bivalents were normally observed in wild‐type oocytes. Ce‐chk‐2 gene expression increased in the adult stage, and their expression level decreased in the glp‐4 mutant, which is defective in germ line proliferation. The radiation sensitivity of F1 embryos carrying Ce‐chk‐2 RNAi was not significantly affected.

Checkpoint and physiological apoptosis in germ cells proceeds normally in spaceflown Caenorhabditis elegans.

It is important for human life in space to study the effects of environmental factors during spaceflight on a number of physiological phenomena. Apoptosis plays important roles in development and tissue homeostasis in metazoans. In this study, we have analyzed apoptotic activity in germ cells of the nematode C. elegans, following spacefight. Comparison of the number of cell corpses in wild type or ced-1 mutants, grown under either ground or spaceflight conditions, showed that both pachytene-checkpoint apoptosis and physiological apoptosis in germ cells occurred normally under spaceflight conditions. In addition, the expression levels of the checkpoint and apoptosis related genes are comparable between spaceflight and ground conditions. This is the first report documenting the occurrence of checkpoint apoptosis in the space environment and suggests that metazoans, including humans, would be able to eliminate cells that have failed to repair DNA lesions introduced by cosmic radiation during spaceflight.

Characterization of Ce-atl-1, an ATM-like gene from Caenorhabditis elegans

Abstract An ATM-like gene was identified in the genome of Caenorhabditis elegans. The putative product of the gene, termed Ce-atl-1 (C. elegans ATM-like 1) consists of 2514 amino acid residues. The C-terminal sequence, which contains a PI-3 kinase-like domain, showed good homology with the products of the gene MEC1/ESR1 from budding yeast, the rad3+ gene of fission yeast and mammalian ATR (ataxia-telangiectasia and rad3+ related) genes. The results of RNA-mediated interference indicated that the major phenotype associated with repression of Ce-atl-1 was lethality (approximately 50–80%) during early embryogenesis. Among the surviving progeny, males (XO animals) arose at a high frequency (2–30%). In addition, 5% of oocyte chromosomes demonstrated aneuploidy due to a defect in pre-meiotic chromosomal segregation. Gene expression analyses indicated that Ce-atl-1 mRNA was expressed in all larval stages and that its level increased about fivefold in the adult stage. The adult expression level was decreased in the glp-4 mutant, which is defective in germ line proliferation. Ce-atl-1 was strongly expressed in both the mitotic and meiotic cells of adult gonads. In summary, Ce-atl-1 appears to be important for early embryogenesis, and loss of its function results in a defect in chromosome segregation, similar to what has been observed for AT-related proteins.

The effect of high strength static magnetic fields and ionizing radiation on gene expression and DNA damage in Caenorhabditis elegans.

Magnetic resonance imaging with high static magnetic fields (SMFs) has become widely used for medical imaging purposes because SMFs cause fewer genotoxic side effects than ionizing radiation (IR). However, the effect of exposure to high SMFs on global transcription is little understood. We demonstrate that genes involved in motor activity, actin binding, cell adhesion, and cuticles are transiently and specifically induced following exposure to 3 or 5 T SMF in the experimental model metazoan Caenorhabditis elegans. In addition, transient induction of hsp12 family genes was observed after SMF exposure. The small-heat shock protein gene hsp16 was also induced but to a much lesser extent, and the LacZ-stained population of hsp-16.1::lacZ transgenic worms did not significantly increase after exposure to SMFs with or without a second stressor, mild heat shock. Several genes encoding apoptotic cell-death activators and secreted surface proteins were upregulated after IR, but were not induced by SMFs. Real-time quantitative RT-PCR analyses for 12 of these genes confirmed these expression differences between worms exposed to SMFs and IR. In contrast to IR, exposure to high SMFs did not induce DNA double-strand breaks or germline cell apoptosis during meiosis. These results suggest that the response of C. elegans to high SMFs is unique and capable of adjustment during long exposure, and that this treatment may be less hazardous than other therapeutic tools.

Caenorhabditis elegans Ce-rdh-1/rad-51 functions after double-strand break formation of meiotic recombination.

During meiotic prophase 1, homologous recombination is accompanied by dynamic chromosomal changes. The Ce-rdh-1/rad-51 gene is the only bacterial recA-like gene in the nematode C. elegans genome. Upon depletion of Ce-rdh-1/rad-51 using the RNA interference method, abnormal ‘kinked’ chromosomes can be observed in mature oocytes at diakinesis, whereas synapsis between homologous chromosomes during the pachytene stage is normal. Following fertilization, Ce-rdh-1/rad-51-depleted embryos die early in embryogenesis, and their nuclei exhibit abnormal chromosome fragments and bridges. From epistasis analyses with Ce-spo-11 defective mutant and ionizing radiation, it is indicated that Ce-rdh-1/rad-51 functions after double-strand break (DSB) formation of meiotic recombination. Under the Ce-chk-2 defective condition, whose meiotic synapsis and meiotic recombination between homologous chromosomes are completely inhibited, the Ce-rdh-1/rad51 is normally expressed in the gonadal cells. Moreover, it seems that exogenous DSBs in the Ce-chk-2 defective nuclei at the pachytene stage can be repaired between sister chromatids in a Ce-rdh-1/rad-51-dependent manner. These results indicate that Ce-rdh-1/rad51 functions after both endogenous and exogenous DSB formation during meiosis, but not as ‘pairing centers’ for meiotic synapsis.

C. elegans RBX-2-CUL-5- and RBX-1-CUL-2-based complexes are redundant for oogenesis and activation of the MAP kinase MPK-1

Cul5‐based complex is a member of ECS (Elongin B/C‐Cul2/Cul5‐SOCS‐box protein) ubiquitin ligase family. The cellular function of the Cul5‐based complex is poorly understood. In this study, we found that oocyte septum formation and egg production did not occur in either cul‐5‐ or rbx‐2‐depleted cul‐2 homozygotes, although control cul‐2 homozygotes laid approximately 50 eggs. These phenotypes are reminiscent of those caused by the MAP kinase mpk‐1 depletion. In fact, activation of MPK‐1 was significantly inhibited in cul‐5‐depleted cul‐2 mutant and cul‐2‐depleted cul‐5 mutant. Yeast two‐hybrid analysis and RNAi‐knockdown experiments suggest that oocyte maturation from pachytene exit and MPK‐1 activation are redundantly controlled by the RBX‐2‐CUL‐5‐ and RBX‐1‐CUL‐2‐based complexes.

Caenorhabditis elegans Elongin BC complex is essential for cell proliferation and chromosome condensation and segregation during mitosis and meiotic division II.

The ubiquitin-mediated protein degradation system is involved in a wide variety of cellular functions. The RING-H2 finger protein RBX1 is a common subunit of Cullin-based ubiquitin ligases. Caenorhabditis elegans RBX1 and CUL2 are essential for regulating chromosome condensation and segregation during mitosis and meiosis and are also critical for cell proliferation. Here, we demonstrate that Elongin B (ELB1) and C (ELC1) form a stable complex, and that depletion of either gene product by RNA-mediated interference (RNAi) causes pronounced defects in the second meiotic division. Embryos and adults that escape meiotic arrest have several irregular phenotypes. These include defects in mitotic chromosomal condensation and segregation, pronuclear rotation, and germ cell proliferation, abnormal cortical protrusion, and accumulation of the cyclin-dependent kinase inhibitor CKI1. All these defects are consistent with those found after depletion of CUL2. In addition, direct interaction between ELC1 and CUL2 is revealed by bacterial two-hybrid analysis. Thus, the RBX1/CUL2/ELC1/ELB1 complex acts as an E3 ubiquitin ligase in C. elegans and is essential for diverse functions relevant to chromosomal dynamics and cell cycle control.