At high concentration, actinomycin D kills the protozonChlamydomonas reinhardi with exponential kinetics. The rate of killingis dependent upon the temperature of incubation. This dependenceis partially a function of the increased extent of binding of the drug at the higher temperature (33°C), but sensitivity of the cellmust also be stimulated by the higher temperature. While actinomycinD probably kills cells in a reaction which requires binding to DNA,there is no correlation between the lethal event and the inhibitionof macromolecular synthesis or the breakdown of macromolecules.
I have developed a simple model to explain the difference insensitivity of various species of RNA to actinomycin D inhibition.This model predicts that frequently transcribed genes will be muchmore sensitive to the drug than infrequently transcribed genes.
Mutants of Chlamydomonas reinhardi have also been isolated which are both temperature sensitive in growth and resistant to killing by actinomycin D. These mutants, unlike other actinomycin D resistant cell lines, are neither impermeable to the drug nor do they excrete it at an accelerated rate. The mutants are partially temper ature sensitive in their ability to synthesize RNA. In the presence of actinomycin D, however, RNA synthesis is partially protected at the nonpermissive temperature (and, in some cases, at the permissive temperature also) when compared to the inhibition of wild type cells.
Extraction and examination of RNA from these mutants revealsthat actinomycin D inhibits different species of RNA to different extents.
I propose that the mutants have an altered chromosomal constituent,which impedes the binding to the genome. At the nonpermissivetemperature the alteration is postulated to partially interfere withtranscription.
In the course of these experiments it became necessary to determinethe maturation pathways of the ribosomal RNA species of Chlamydomonas reinhardi. Cytoplasmic rRNAs of C. reinhardi are cleaved from asingle precursor of molecular weights 2.4 · 106 to a mature rRNA(0.69 · 106mol.wt) and a 1.4 · 106-mol. wt precursor of a mature 1.3 · 106-mol. wt rRNA. The kinetics of incorporation of radioactive label into the rRNAs suggest that the 0.69 · 106-mol. wt rRNA gene is located closer to the promotor than is the gene for the 1.4 · 106-mol. wt rRNA. The synthesis of cytoplasma rRNAs is extremely sensitive to camptothecin, an inhibitor of nuclear rRNA synthesis, but synthesis ofchloroplast rRNA is quite resistant to the inhibitor. This has allowedus to demonstrate that chloroplast rRNAs are processed from precursorswhich resemble those of blue-green algae. A 1.14 · 106-mol. wt precursoris processed to the 1.07 · 106-mol. wt mature chloroplast rRNA, anda 0.64 · 106-mol. wt precursor is cleaved to a 0.56 · 106-mol. wt species and then to the mature 0.54 · 106-mol. wt rRNA. This studydemonstrates two new ways in which the function of the chloroplast genome resembles those of prokaryotes more than those of the nucleus.
