In vitro chloroplast DNA synthesis. Part I. In vitro DNA synthesis by chloroplasts isolated from Marchantia polymorpha L. cell suspension cultures.

Abstract

Lysate of chloroplasts prepared from liverwort Marchantia polymorpha L. cell suspension cultures incorporated [3H]-dTTP into acid insoluble materials when DNA was added exogenously as a template. The incorporation was highly dependent on the addition of template DNA, four deoxynucleoside triphosphates and magnesium ions (maximum incorporation at 5mM). Magnesium ions could be replaced by manganese ions. DNA synthesis inhibitors, Nethylmaleimide (NEM) and ethidium bromide (EtBr), strongly inhibited the incorporation. Dideoxythymidine triphosphate (ddTTP), an inhibitor of DNA polymerases β and γ, inhibited the incorporation at the concentration of 50μM (molar ratio of ddTTP/dTTP= 17). On the other hand, the incorporation by the chloroplast lysate was resistant to arabinofuranosyl cytosine triphosphate (araCTP) and aphidicolin as well as the RNA polymerase inhibitors, rifampicin and α-amanitin. The chloroplast lysate highly utilized denatured calf thymus DNA and bacteriophage ΦX174 singlestranded DNA as templates when added exogenously, while a synthetic homopolymer, poly(rA)- oligo(dT)12-18, did not stimulate the incorporation at all. Autoradiographic analysis of DNA synthesized in isolated chloroplasts showed that the chloroplast DNA synthesis took place at several specific sites on the chloroplast DNA from cells of the liverwort, Marchantia polymorpha.