Studies on Plastid-Nuclei (Nucleoids) in Nicotiana tabacum L. I. Isolation of Proplastid-Nuclei from Cultured Cells and Identification of Proplastid-Nuclear Proteins

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We have developed a method of isolating morphologically intact proplastid-nuclei (nucleoids) in large quantities from Nicotiana tabacum cultured cells (line BY-2) without contamination by mitochondria and cell-nuclei. Fluorescence microscopy using 4',6-diamidino-2-phenylindole (DAPI) revealed that the compact structure of the isolated proplastid-nuclei (pp-nuclei) was disorganized by DNase I, micrococcal nuclease, proteinase K, 2_M NaCl and 2_M KCl, but was not affected by RNase A, suggesting that the pp-nuclei are compactly organized by an electrostatic interaction between the proplastid-DNA (pp-DNA) and some protein(s). Although SDS-polyacrylamide gel electrophoresis showed that the isolated pp-nuclear fraction still contained a number of polypeptides, only four of them (mol wt: 69 kDa, 31 kDa, 30 kDa and 14 kDa) were found to be solubilized by treatments of the pelletable pp-nuclear fraction with DNase I, micrococcal nuclease and 2_M NaCl. Furthermore, when the supernatant of the pp-nuclei treated with DNase I was applied onto a denatured or a native DNA-cellulose affinity column, these four polypeptides were bound to both DNAs and eluted by raising the NaCl concentration. These findings, taken together, show that these proteins are proplastid DNA-binding proteins and strongly suggest that the pp-nuclei are compactly organized by interaction between the pp-DNA and these proteins.
日本植物生理学会の論文