Comparative analysis of DNA synthesis activity in plastid-nuclei and mitochondrial-nuclei simultaneously isolated from cultured tobacco cells

Plastid-nuclei (plastid-nucleoids) and mitochondrial-nuclei (mitochondrial- nucleoids) were simultaneously isolated from cultured tobacco cells (Nicoti ana tabacum L., line BY-2), and their activity synthesizing DNA in vitro wa s examined. The isolated plastid- and mitochondrial-nuclei incorporated abo ut 20 and 50 pmol of dCTP, respectively, into DNA per microgram of template DNA during a 60-min incubation. The DNA synthetic activity of the two orga nelle-nuclei exhibited similar responses to various inhibitors; it was resi stant to aphidicolin and sensitive to N-ethylmaleimide and high concentrati ons of ddCTP, which are all characteristics of gamma-like DNA polymerase. T he responses of the DNA synthetic activity in the two organelle-nuclei to p H and divalent- and monovalent-metal cation concentrations were also simila r. Moreover, an in situ DNA polymerase assay following SDS-polyacrylamide g el electrophoresis revealed that DNA. polymerases with an apparent molecula r mass of 116 kDa were present in both the isolated plastid- and mitochondr ial-nuclei, and that the two 116-kDa DNA polymerases were quite similar in terms of their sensitivity to various inhibitors, optimum assay conditions, and template preferences. These results indicate that DNA replication in p lastids and mitochondria may be conducted by DNA polymerases that have quit e similar characteristics. (C) 1999 Elsevier Science Ireland Ltd. All right s reserved.