Abstract.-The effect of rifampin, an inhibitor of bacterial DNA-dependent RNA polymerase, was studied in Chlamydomonas reinhardi. It was shown, in vivo and in vitro, that chloroplast-located, but not nuclear, DNA-dependent RNA polymerase is inhibited by this drug. The inhibition of chloroplast RNA polymerase results in the inhibition of chloroplast rRNA synthesis, and thus in the loss of chloroplast ribosomes. The ability to carry out photosynthesis is also lost after prolonged heterotrophic growth in the presence of rifampin, but cell division and chloroplast replication are not affected. It is proposed that chloroplast DNA contains information for chloroplast rRNA, but this DNA does not have the information for chloroplast DNA polymerase. Moreover, the DNA polymerase is not synthesized on chloroplast ribosomes.The nature and amount of information encoded in chloroplast DNA is not known. A possible approach to this problem is to study in vivo the effect of specific inhibitors of chloroplast DNA-dependent RNA polymerase.It was recently shown that antibiotics of the rifamycin group specifically inhibit DNA-dependent RNA polymerase (E.C. 2776) of bacterial, but not mammalian, origin.1-4 The inhibitors act by specifically binding to the enzyme itself, and not to the DNA template.5 The effect of this inhibitor on the DNAdependent RNA polymerase of chloroplasts of Chlamydomonas reinhardi is reported in this paper.Materials and Methods.-UTP, GTP, CTP, and ATP were purchased from Calbiochem, a'2P-ATP from ICN (1 c/mmole), adenine-8-14C from Schwarz (54 mc/mmole), pyruvate kinase from Sigma, ribonuclease (crystallized) and calf thymus DNA from Worthington, and actinomycin D from Merck. Rifampin, 3-(4-methylpiperazinyliminomethyl) rifamycin SV, was a gift from Dow Chemical. Wild-type C. reinhardi (strain 137c, mating type plus) was used in all experiments. The cells for chloroplast isolation and for nucleic acid labeling experiments were grown at 250 on minimal medium6 for 50 hr in a 3-liter Volume. The cultures were agitated by bubbling with 5% CO2 in air. The light intensity was 4000 lux. Cells grown in the presence of rifampin were cultured on an acetate-containing medium.7 Because of the instability of the drug to light, the cells were grown in the dark or under reduced light intensity when experiments were carried out longer than 24 hr.A new method of chloroplast isolation which yields highly purified and intact chloroplasts was developed. Six liters of cells were collected and washed three times with cold buffer I (0.25 M sucrose, 0.05 M Tris-HCl buffer, pH 8.0, 1 X 10-31 I Mg EDTA, and 2 X 10-2 ,M MgSO4) and then twice with cold buffer II (0.5 Al sucrose, 0.05 A!l Tris-HCl buffer, pH 8.0, 2 X 10-2 M MgSO4, 0.25% BSA, 2 X 10-3 M 2-mercaptoethanol). They were then suspended in cold buffer II at the ratio of 1 vol of buffer to 2 vol of pelleted cells and were broken in a French pressure cell at a pressure of 35 kg/cm2. The suspension of broken cells was diluted with 3 vol of cold buffer II and centrifuged in 20-ml vo...
