Specific Inhibitors of the Three RNA Polymerases from the Aquatic Fungus Blastocladiella emersonii

Abstract

Alpha-amanitin, a toxin from the basidiomycete, Amanita phalloides, has been shown to specifically inhibit the DEAE fraction II enzyme (2-5) that is found in the nonnucleolar portion of the nucleus. It has been suggested that the fraction II enzyme functions in the synthesis of DNA-like RNA. The fraction I enzyme's cellular location is in the nucleolus, and it is reported to serve specifically in the synthesis of ribosomal RNA (1, 2, 6). At present there is no evidence or suggested function for the fraction III enzyme, which is also apparently extra-nucleolar (5, 6). Although it has been suggested that the polymerases have different cellular functions, for more conclusive investigations specific inhibitors would be very useful.While it is generally accepted that cycloheximide interrupts protein synthesis in eukaryotes, there have also been reports that it has some effect on the synthesis of RNA (7-10). The antibiotic rifampicin and rifamycins in general have been shown to specifically inhibit procaryotic RNA polymerases (11-13).The purpose of this study was to examine the RNA synthesis machinery in the primitive water mold Blastocladiella emersonii. Since the results indicated the presence of multiple RNA polymerases, another objective was to observe the effects of cycloheximide, alpha-amanitin, and rifampicin on in vitro RNA synthesis to determine whether specific inhibitors may exist for the three polymerases.MATERIALS AND METHODS Growth, harvesting, and disruption of cells Single-generation cultures of B. emersonii (Strain 49-1) were grown in aerated carboys at 230C according to Horgen and Griffin (14). Vegetative, ordinary colorless thalli were harvested by filtration and were washed with glass distilled water. The thalli were suspended in homogenizing fluid (15) and were disrupted with a French pressure cell. The cultures and the enzyme preparations were monitored for microbial contamination by plating on peptone-yeast extract-glucose agar (PYG) with negative results. ChemicalsMedium components were purchased from Difco Laboratories, Detroit, Mich. The unlabeled nucleoside triphosphates and dithiothreitol were purchased from Sigma Chemical Co., St. Louis. The [3H]ATP was obtained from New England Nuclear Corp., Boston. Salmon sperm DNA and enzyme grade ammonium sulfate were obtained from Mann Research, N.Y. Alpha-amanitin was purchased from Henley and Co., N.Y., rifampicin and cycloheximide (actidione) from Calbiochem, Los Angeles. Other chemicals used were reagent grade.Isolation of crude nuclear pellet Cell wall debris was removed by filtration through Miracloth and the homogenate was centrifuged for 20 min at 6000 X g.The resulting crude nuclear pellet was used for enzyme isolation. Further nuclear purification by centrifugation through 4 molal sucrose (15) did not increase the purity of the enzyme preparations.Purification of RNA polymerase RNA polymerase was partially purified by ammonium sulfate fractionation followed by DEAE-cellulose column chromatography after Mertelsmann and Matthaei (16)...

Effect of the exotoxin of Bacillus thuringiensis on normal and ecdysone-stimulated ribonucleic acid polymerase activity in intact nuclei from the fat body of Sarcophaga bullata larvae

“…1-3 and in Beebee (1972) show that the properties ofthe RNA polymerase are similar to those observed in nuclei isolated from a wide range of other species (Widnell & Tata, 1964;Roeder & Rutter, 1969;Horgen & Griffin, 1971), including a related insect (Sekeris et al, 1965;Congote et al, 1969;Shaaya & Sekeris, 1971). The pattern of inhibition of the polymerase activities by a-amanitin, recorded in Table 1 Shaaya & Sekeris (1971) fornuclei from theepidermis of Calliphora erythrocephala, and is qualitatively comparable with the pattern observed in nuclei from other species.…”

Cycloheximide Is Not a Specific Inhibitor of Protein Synthesis in Vivo

“…The rate of killing appears to be at least partially independent from these other effects since it is increased by the increase in CH concentration. The results of Horgen and Griffin suggest that cycloheximide can directly inhibit RNA synthesis in vitro (13).…”

“…The others are mutants derived from arg 2 (24). One of these, ts 13, is a mutant conditionally defective in the initiation of protein synthesis. The lesion of the other mutant, ts 1, is cific radioactivities of 1.68 Ci/mmole or 7 mCi/mmole, respectively.…”

Characteristics of DNA‐dependent RNA polymerase activity from isolated yeast nuclei

“…None of the enzymes is affected by the antibiotics rifampicin and cycloheximide at concentrations up to 5/ag/ml and 200 /ag/ml, respectively. The effect of cycloheximide on the polymerase activities was investigated, because it has been reported [17] that this antibiotic specifically inhibits the RNA polymerase I (A) from Blastocladiella emersonii, and is without effect on RNA polymerases II (B) and III (C). In the case of the yeast polymerases, however, cycloheximide does not have any effect on the activity.…”