SIMULTANEOUS ISOLATION OF CELL-NUCLEI, PLASTID-NUCLEI AND MITOCHONDRIAL-NUCLEI FROM CULTURED TOBACCO CELLS - COMPARATIVE-ANALYSIS OF THEIR TRANSCRIPTIONAL ACTIVITIES IN-VITRO

A procedure was established for simultaneously isolating cell-nuclei, plastid-nuclei (plastid-nucleoids) and mitochondrial-nuclei (mitochond rial-nucleoids) from cultured tobacco cells (Nicotiana tabacum L., lin e BY-2), Quantitative Southern hybridization analysis revealed cross-c ontamination to be minimal (less than 1% in most cases). The isolated cell-, plastid- and mitochondrial-nuclei incorporated about 17, 15 and 7-pmoles of UTP, respectively, into RNA per microgram of DNA during a 60 min incubation under appropriate conditions. The isolated cell-, p lastid- and mitochondrial-nuclei exhibited different sensitivities to various transcriptional inhibitors, and synthesized RNAs that are sele ctively hybridizable to their own DNA. The three isolated nuclei also incorporated [gamma-P-32]ATP radioactivity into the DEAE-paper bound f raction: Although most of this incorporation was due to protein phosph orylation, a small fraction of the incorporated radioactivities was re cover-ed as RNA, suggesting that de novo transcription initiation may occur in our assay system. The simultaneous isolation of these three n uclei and the assay of their transcriptional activities in vitro were useful for comparative analysis of their transcription apparatus. This system may also be a useful means of analyzing the transcriptional st ales of cell nuclear-, plastid- and mitochondrial-genomes simultaneous ly. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.