|Budget Amount *help
¥1,300,000 (Direct Cost : ¥1,300,000)
Fiscal Year 1991 : ¥200,000 (Direct Cost : ¥200,000)
Fiscal Year 1990 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1989 : ¥600,000 (Direct Cost : ¥600,000)
1. To investigate origin of the cytoplasmic and nuclear nucleoloids, detail examinations have been carried out under electron microscopy in Lilium male reproductive cells by applying ribonucleoprotein (RNP) preferential staining technique. The nucleolus-derived material (nucleolar material) completely dispersed in the cytoplasm during prophase-metaphase was found to first form minute amorphous masses in late anaphase. Then they fused each other to form large nucleoloids in the cytoplasm in early telophase. Coincidentally, part of nucleolar material began to accumulate on the surface of chromosomes and form nuclear nucleoloids.
2. Estimation of RNA content of the nucleolar-derived structures (cytoplasmic and nuclear nucleoloids, nucleolar material on chromosoms)
(1) RNase-gold labeling - In root-tip meristematic cells of Vicia faba, all the nucleolus-derived structures were not significantly labeled with the RNase-gold complex. In Lilium cells, on the other hand, the nuclear nucleoloids
were moolerately labeled and the the labeling on the cytoplasmic nucleoloids was about 50% of that over the nuclear nucleoloids.
(2) In situ hybridization using rDNA probe. - Lilium cytoplasmic nucleoloids were heavily labeled with rDNA probe, suggesting that they contain rDNA transcripts.
3. Estimation of DNA by indirect immunolabeling technique. - No DNA molecule has been discerned for all the nucleolar-derived structures in Vicia faba cells by the immunolabeling technique using Anti-DNA monoclonal antibody.
4. Effect of colchicine and cytochalacin B on behavior of the nucleolar material. - When cells of Vici faba were exposed to colchicine, the dispersed nucleolar material formed numerous cytoplasmic nucleoloids and accumulated on the chromosome surface even in metaphase. Cytochalacin B, an inhibitor of F-actin, caused remarkable increase of the cytoplasmic nucleoloids in early telophase. These suggest that cytoskelton plays a role to transport the nucleolar material.
5. Silver impregnation technique applied to serail ultrathin sections has suggested that the nucleclar material transported into the daughter nuclei goes into the nucleolonema.