1 Hokuriku National Agricultural Experiment Station, Joetsu 943-0193, Japan 2 Japan science and Technology Corporation, Saitama 332-0012, Japan 3 Science University of Tokyo, Shinjuku-ku 162-8601, Japan 4 Faculty of Engineering, Graduated School of Osaka University, Suita 565-0871, Japan
A number of studies on nuclear proteins which are stage specific in the cell cycle are carried out in mammals. However, a few studies on plant were carried out because synchronization of plant cells to collect nuclei at specific stage is not easy. Flow cytometry and sorter is one of the most effective tools in cell biology. J. R. Dynlacht et al. (1996) showed that flow cytometry can also be used for separation of the nuclei with high quality in the study of nuclear matrix proteins. We tried to analyze stage specificity on nuclear protein by using this system. Nuclear samples for the analyses were prepared from barley buds of 4 day-old seedling according to K. Arumuganathan et al. (1991) with slight modifications (protein inhibitor was added to the isolation buffer). After isolation of nuclei, the suspension of nuclei stained with propidium dide was analyzed and sorted with Altra flow sorter (Beckman Coulter, USA) equipped with a water-cooled argon ion laser (Coherent, CA) operating at 488 nm. Nuclei were sorted to G1 and G2 phases on the basis of the fluorescent intensity, the ratio of obtained nuclei G1 to G2 is approximately 4 to 1. Their nuclear proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis. The result showed that there are some different spots between G1 phase and G2 phase on the 2-D protein maps. By this method, we can readily and easily detect some differences of nuclear protein between G1 and G2 phase. This result showed that the flow sorting to separate the nuclei is also effective for studies on nuclear proteins at the specific cell cycle in plant.
Plant & Animal Genome VIII Conference Plant & Animal Genome VIII Conference