Mechanism of inhibition of Escherichia coli RNA polymerase by captan.

Captan (N-trichloromethylthiocyclohex-4-ene-1,2-dicarboximide) was shown to inhibit RNA synthesis in vitro catalysed by Escherichia coli RNA polymerase. Incorporation of [gamma-32P]ATP and [gamma-32P]GTP was inhibited by captan to the same extent as overall RNA synthesis. The ratio of [3H]UTP incorporation to that of [gamma-32P]ATP or of [gamma-32P]GTP in control and captan-treated samples indicated that initiation was inhibited, but the length of RNA chains being synthesized was not altered by captan treatment. Limited-substrate assays in which re-initiation of RNA chains did not occur also showed that captan had no effect on the elongation reaction. Studies which measured the interaction of RNA polymerase with template DNA revealed that the binding of enzyme to DNA was inhibited by captan. Glycerol-gradient sedimentation of the captan-treated RNA polymerase indicated that the inhibition of the enzyme was irreversible and did not result in dissociation of its subunits. These data are consistent with a mechanism in which RNA polymerase activity was irreversibly altered by captan, resulting in an inability of the enzyme to bind to the template. This interaction was probably at the DNA-binding site on the polymerase and did not involve reaction of captan with the DNA template.     

A polymerase activity forming 5S and pre-4S RNA in isolated HeLa cell nuclei

Isolated HeLa cell nuclei are capable of synthesizing 5S and pre-4S RNA. The labeling of these low molecular weight species has been compared with the labeling of nucleolar RNA and nuclear heterogeneous RNA. The 5S and pre-4S RNA molecules made in vitro were identified by their mobility on SDS acrylamide gels and by the sensitivity of pre-4S RNA to enzymes which cleave it in vitro to 4S RNA. Their mobilities and cleavage properties are similar to the RNA made in vivo. Unlike the nuclear heterogeneous RNA, the synthesis of the two small molecular weight RNAs is resistant to α-amanitin.A large proportion of 4S RNA labeled in vitro appears to be formed de novo. The ratio of the terminal uridine to the internal uridine 3′-monophosphate remains constant with time, even though there is linear incorporation into the pre-4S RNA in the isolated nuclei.Production of the nucleolar RNA and pre-4S RNA has been compared in the presence of various ions. The pre-4S RNA synthesis has a sharper maximum for (NH₄)SO₄ and MgCl₂ than does the synthesis of nucleolar RNA. The in vitro synthesis of pre-4S is more sensitive to ellipticine and pCMB than the production of nucleolar RNA. These differences between the production of pre-4S RNA and nucleolar RNA are discussed with respect to in vitro reinitiation and the possibility that different polymerases are involved in their synthesis.     

Biochemical effects of bleomycin A2 on Novikoff hepatoma ascites cells.

Purified nucleolar DNA was markedly degraded at a concentration of 13 mug/ml by bleomycin A2; bleomycin concentrations 20-30 times greater were required to degrade nucleoplasmic DNA. Whole nuclear DNA was degraded to only a small extent at 13 mug/ml but was markedly degraded at higher bleomycin concentrations. Treatment of the various types of DNA with high concentrations of bleomycin A2 produced low molecular weight (approximately 6S) fragments that were no longer sensitive to degradation by bleomycin A2. Hybridization studies demonstrated a loss of ribosomal DNA sequences from nucleolar DNA treated with bleomycin A2 in vitro. Studies on RNA synthesis in Novikoff hepatoma ascites cells in vitro showed there was a decreased uptake of 32Pi into high molecular weight nuclear RNA in the presence of bleomycin A2. These results indicate that nucleolar function is inhibited by a direct effect of bleomycin A2 on nucleolar DNA.     

THE REGULATION OF RNA SYNTHESIS AND PROCESSING IN THE NUCLEOLUS DURING INHIBITION OF PROTEIN SYNTHESIS

The effect of protein synthesis inhibition by cycloheximide on nucleolar RNA synthesis and processing has been studied in HeLa cells. Synthesis of 45S RNA precursor falls rapidly after administration of the drug. However, the nucleolar content of 45S RNA remains relatively constant for at least 1 hr because the time required for cleavage of the precursor molecule into its products is lengthened after treatment with cycloheximide. The efficiency of transformation of 45S RNA to 32S RNA remains constant with approximately one molecule of the 32S RNA produced for each cleavage of a molecule of 45S RNA. However, shortly after the cessation of protein synthesis the formation of 18S RNA becomes abortive. The amount of 32S RNA present in the nucleolus remains relatively constant. After long periods of protein synthesis inhibition the 28S RNA continues to be synthesized and exported to the cytoplasm but at a greatly reduced rate. When the protein synthesis inhibitor is removed, a prompt, although partial, recovery in the synthesis rate of 45S RNA occurs. The various aspects of RNA synthesis regulation and processing are discussed.     

Multiple nucleoli and enhanced nucleolar activity in the nurse cells of the insect ovary

Origin and function of the nucleolar apparatus in nurse cell nuclei of Calliphora erythrocephala have been investigated by cytological and autoradiographic methods in some inbred lines of laboratory blowflies with well paired polytene chromosomes in the nurse cell nuclei. Besides the nucleolus at chromosome VI large numbers of multiple free nucleoli develop in the highly polyploidized nurse cells during oocyte growth. The nucleoli incorporate H³-uridine in a considerable amount producing a homogeneous and RNase-sensitive label even after short time incubation. Their capacity of RNA synthesis is independent of their spatial relationships to other nuclear components. DNA particles in the nucleoli could be identified by the Feulgen reaction and by fluorescence staining with N,N'-diethylpseudoisocya-ninchloride, which also demonstrates the existence of own templates for autonomous RNA synthesis. There are indications that the nucleolus' own DNA is produced by gene amplification beyond the level of endomitotic polyploidization in the nurse cell nuclei. A quantitative estimation of grain density in the autoradiograms shows a rigorous shift of rRNA synthesis: at least 72% of all newly synthesized macromolecular RNA in nurse cell nuclei as contrasted to 13 % of nucleolar RNA synthesis in bristle forming cells with a similar degree of polyploidy. It seems that the nurse cell nuclei of Calliphora in addition to polyploidization increase their template capacity for synthesizing rRNA in a similar way as has repeatedly been demonstrated for Amphibia. Cytological and physiological peculiarities of the nurse cells have been discussed from the viewpoint of their functional similarity to the oocyte nucleus.     

Comparison of the Virion Polymerase of Reovirus with the Enzyme Purified from Reovirus-Infected Cells

Reovirus has in its protein coat an enzyme which catalyzes the net synthesis of the three size classes of virus-specific, single-stranded ribonucleic acid (RNA). For synthesis of 24, 19, and 14S single-stranded RNA, Mn(++) was the preferred divalent cation, and ammonium sulfate at an optimal concentration of 4.2% of saturation was an absolute requirement. During synthesis, the parental double-stranded RNA was conserved in the viral core and the newly synthesized completed RNA chains were released as free RNA. The viral cores synthesizing RNA had properties consistent with the presence of nascent RNA on their outer surface. The enzyme-template complex from the infected cells described in an earlier paper was comprised of viral cores already active in the in vivo synthesis of single-stranded RNA. This pool of viral cores was newly made during infection, and exponential increase in the number of particles in this pool, as detected by the increase in enzymatic activity, occurred 2 hr earlier than that in mature virus.     

5-Methyltryptamine decreases net accumulation of 32P into the polyphosphoinositides from [gamma-32P]ATP in a cell-free system from blowfly salivary glands. Activation of breakdown of the newly synthesized [32P]polyphosphoinositides

Incubation of blowfly salivary gland homogenates with 30 microM [gamma-32P]ATP resulted in a rapid, Mg2+-dependent, synthesis of [32P]polyphosphoinositides and [32P]phosphatidic acid. 5-Methyltryptamine, in the presence of 10 microM guanosine 5'-(3-O-thio)trisphosphate, reduced the net accumulation of 32P label into phosphatidylinositol-4,5-P2 and phosphatidylinositol-4-P by 35 and 20%, respectively. 5-Methyltryptamine did not affect synthesis of [32P]phosphatidic acid. Phosphorylation of polyphosphoinositides was not affected by 5-methyltryptamine. In membranes labeled in vitro with [gamma-32P]ATP, 5-methyltryptamine stimulated a rapid breakdown of the [32P]polyphosphoinositides. These results indicate that in blowfly salivary gland homogenates, hormone stimulates breakdown of the newly synthesized polyphosphoinositides. In the presence of hormone, the rate of polyphosphoinositide synthesis does not compensate for the rate of polyphosphoinositide degradation.     

Nuclear structures in the ovarioles of the butterfly Laspeyresia pomonella. Sex chromatin in trophocytes

The nuclear structures in the ovarioles have been studied in Laspeyresia pomonella by means of light and electron microscopy, autoradiography (RNA and DNA synthesis) and molecular hybridization in situ. The karyosphere was shown to form in oocyte nuclei at the beginning of oocyte growth. Numerous protein granules appeared in close contact with the karyosphere chromosomes; the true nucleolus was absent and the whole nucleus was inactive in RNA synthesis. A special attention was paid to studying nuclear structures in trophocytes. Numerous complex nucleoli actively synthesizing RNA formed in highly endopolyploid nuclei of trophocytes. Besides, each trophocyte had a spheroid vacuolized body of DNA which developed from one of meiotic bivalents soon after trophocyte differentiation and increased in diameter up to 10-15 mu. The DNA body in trophocytes and follicle cells was in close contact with the nucleolar material. Ribosomal DNA was present in these bodies as was shown by molecular hybridization in situ. A suggestion is put forward to the effect that the DNA bodies take part in the formation of complex nucleolar apparatus of trophocytes. On the basis of both the author's and literary data, a conclusion is drawn that DNA spheres in trophocytes and follicle cells are sex chromatin bodies formed, however, by both the X- and Y-chromosomes, rather than by one Y-chromosome.     

Nuclear structures in the telotrophic ovarioles of nocturnal ground beetles (Tenebrionidae, Polyphaga). II. The oocyte nucleus of Blaps lethifera and Gnaptor spinimanus. Light optical data]

Changes in the nuclear structures and their participation in RNA synthesis in the growing oocytes were followed in two species of beetles Blaps lethifera and Gnaptor spinimanus. In the oocytes of both the species, the chromosomes join into the karyosphere following the short-term lampbrush stage. A large capsule appears around the karayosphere which consists of the fibrous substance, granules and karyosphere nucleoli. The latter form in the karyosphere and contain RNP but they are not true nucleoli since they do not include 3H-uridine. RNA synthesis on the chromosomes, active at the lampbrush stage, falls markedly following their joining into the karyosphere. The oocyte nuclei of these beetles are, thus, characterized by the absence of RNA synthesizing nucleolar system and, as compared with the trophocytes, by the low level of RNA synthesis on the chromosomes.