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

We have developed a method of isolating morphologically intactproplastid-nuclei (nucleoids) in large quantities from Nicotianatabacum cultured cells (line BY-2) without contamination bymitochondria 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 KC1, but was not affected byRNase A, suggesting that the pp-nuclei are compactly organizedby an electrostatic interaction between the proplastid- DNA(pp-DNA) and some protein(s). Although SDS-polyacrylamide gelelectrophoresis showed that the isolated pp-nuclear fractionstill contained a number of polypeptides, only four of them(mol wt: 69 kDa, 31 kDa, 30 kDa and 14 kDa) were found to besolubilized by treatments of the pelletable pp-nuclear fractionwith DNase I, micrococcal nuclease and 2 M NaCl. Furthermore,when the supernatant of the pp-nuclei treated with DNase I wasapplied onto a denatured or a native DNA-cellulose affinitycolumn, these four polypeptides were bound to both DNAs andeluted by raising the NaCl concentration. These findings, taken together, show that these proteins areproplastid DNA-binding proteins and strongly suggest that thepp-nuclei are compactly organized by interaction between thepp-DNA and these proteins. ⁴Present address: Department of Biology, Faculty of Science,University of Tokyo, Hongo, Tokyo 113, Japan.⁵Present address: School of Food and Nutritional Sciences, Universityof Shizuoka, Yata, Shizuoka 422, Japan. (Received August 13, 1987; Accepted November 9, 1987)