Accumulation of Xanthosine by Escherichia coli in the Presence of Angustmycin C

Effect of angustmycin C, an adenosine analogue having an unusual sugar linked to adenine, on Escherichia coli was studied. It inhibited biosynthesis of RNA and DNA preferentially and xanthosine was excreted from the inhibited cells into the medium. During the course of its growth with addition of the antibiotic, the specific activity of inosine-5′-phosphate dehydrogenase of E. coli cells increased six times. These effects could be explained by the inhibitory effect of angustmycin C on xanthosine-5′-phosphate aminase which resulted in decreased level of IMP-dehydrogenase. Accumulation of xanthosine induced by the antibiotic reached the concentration of 940 μg/ml under an appropriate condition.     

The effect of a new antibiotic, cryptosporiopsin, on RNA synthesis in L-cells.

The effect of cryptosporiopsin on RNA synthesis in L-cells was studied as part of an investigation on the mechanism of action and potential toxicity of the antibiotic in mammalian cells. RNA synthesis in vitro was tested in intact isolated L-cell nuclei, in conjunction with selective inhibitors of nucleolar and nucleoplasmic RNA synthetic activities; It was found that only the nucleoplasmic activity (polymerase II), was inhibited by cryptosporiopsin and that the drug showed no effect on the activity of the nucleolar enzyme (polymerase I). RNA synthesis in vivo was tested using double labelling with I114-C]guanine and [3-H]-uridine in an attempt at discriminating between G+C nucleolar trna and high A+U nucleoplasmic RNA synthesis. Results revealed that the uptake of these precursors into both types of RNA was inhibited by cryptosporiopsin in intact cells. Measurements of the nucleotide pools in these cells indicated that the antibiotic affects uptak and phosphorylation of nucleosides and nucleotides, especially the production of ATP; These results suggest that the uptake inhibition observed in vivo could be due, at least in part, to energy and/or precursor shortage.     

Studies on Substances Which Increase RNA Content of Yeast Cells

Active substances which increased RNA content and RNA productivity in yeast culture without affecting the growth rate of yeast were investigated.

The remarkable effect of zinc ion on RNA accumulation was found in flask cultures of Candida utilis.

The active substance of culture of Saccharomyces cerevisiae was isolated from the culture filtrate of Streptomyces sp. S–22 and it was identified as anisomycin, an antiprotozoal and antifungal antibiotic. The effect of anisomycin on the enhancement of yeast RNA formation was shown only with the Saccharomyces genus, which was more sensitive to the antibiotic than other genus. This phenomenon was exhibited only in the case of anisomycin and cycloheximide, whose modes of action were similar among various antibiotics. The ratio of four nucleotides in RNA fraction was almost equal to that of ribosomal RNA.     

Studies on binding of toromycin, an antitumor antibiotic, to DNA

Toromycin, an antitumor, bactericidal and antiviral compound, was found to bind to DNA in such a way as to interfere with the dissociation of double helix at an elevated temperature. The antibiotic did not introduce strand scission into DNA. Single-strand-specific nuclease S1-susceptibility of negatively supercoiled DNA was not influenced by its binding. The antibiotic was shown to bind to both of the alternating purine-pyrimidine copolymers, poly(dG-dC):poly(dG-dC) and poly(dA-dT):poly(dA-dT). The unique C-glycoside molecule of toromycin interacted with single-stranded DNA, but was found to have no affinity for RNA.     

Existence of Hyphal Extension Factor and Hyphal Extension Inhibitor in the Hyphae of Rhizoctonia solani

Brefeldin A (BFA) is an antibiotic having diverse biological effects such as antifungal, antiviral and antitumor activities. The effect of BFA on biosynthesis of cellular components was examined to elucidate the mode of action of BFA using C. albicans IAM 4888.

When C. albicans was grown in the presence of BFA, cells became rounded and enlarged several times larger than the untreated control cells. Cell walls of the treated cells became irregular and a number of Sudan III-stainable lipid droplets was formed in the cytoplasm. Accompanying these morphological changes, a marked alteration occurred in the cellular lipid composition; neutral lipids increased whereas phospholipid decreased. [¹⁴C]Acetate incorporation into the lipid fraction proceeded in accordance with the growth in the presence of BFA. On the other hand, [³²P]orthophosphate incorporation into phospholipid was severely inhibited. Incorporation of radiolabeled precursors into DNA, RNA and protein was not affected on a cell weight basis.     

Einbau von Uridin und Orotsäure in plastidäre ribosomale RNA von Chlorella nach Antibiotica-Behandlung

Zusammenfassung Chlorella pyrenoidosa inkorporiert unter normalen Anzuchtbedingungen kurzfristig angebotenes Uridin fast ausschließlich in plastidäre ribosomale RNA. Es lassen sich rasch markierte Ribosomen und deren Untereinheiten von 70 S, 50 S und 30 S nachweisen. Diese Markierung wird durch Rifampin in geringen Konzentrationen bereits nach wenigen Minuten unterbunden. Auf das Zellwachstum hat Rifampin bei heterotropher Anzucht dagegen auch in höheren Konzentrationen keinen Einfluß. Chloramphenicol hemmt den kurzfristigen Uridin-Einbau in ribosomale Partikeln von 70 S, 50 S und 30 S, nur geringfügig dagegen denjenigen in ribosomale RNA. Auch die Wirkung des Chloramphenicols tritt rasch ein. Cycloheximid beeinflußt den Kurzzeit-Einbau von Uridin in ribosomale Partikeln und in RNA nicht, wenn die Inkubationszeit 60 min nicht überschreitet.Die Markierung der Nucleinsäuren von Chlorella mit 6-(14C)-Orotsäure zeigt vergleichbare Empfindlichkeiten gegen die drei Antibiotica wie der Einbau von 6-(14C)-Uridin und 5-(3H)-Uridin.

---

Incorporation of uridine and of orotate into chloroplast ribosome RNA of Chlorella after treatment with antibiotics

Summary Normal grown cells of Chlorella pyrenoidosa incorporate uridine exclusively into chloroplast ribosomal RNA after short time labeling. With sucrose gradient separation, labeled ribosomal particles of 70 S, 50 S and 30 S can be shown. This labeling is prevented by rifampin in low concentrations after a few minutes. At the same concentration of the antibiotic and also with 10-fold higher concentration, no effect on heterotrophic cell growth is observed. This indicates clearly that mitochondria cannot be influenced by rifampin. Chloramphenicol also inhibits the formation of uridine labeled ribosomal particles of 70 S, 50 S and 30 S. In the presence of this antibiotic, some labeled ribosomal RNA is formed. Also the effect of chloramphenicol can be shown after short incubation periods. Cycloheximide treatment of the cells within 30 and 60 min and up to the 10-fold concentration of protein synthesis inhibition (Morris, 1967) results in no effect on labeling of ribosomal RNA and of ribosomal particles in Chlorella with uridine. Only after prolonged treatment of the cells with cycloheximide is some effect on uridine incorporation observed.The comparison of the incorporation patterns of 6-(14C)-orotate, (6-14C)-uridine and 5-(3H)-uridine into nucleic acids in the presence of rifampin, chloramphenicol and cycloheximide shows some similarities. After 60 min incubation with the precursors, the incorporation is reduced by all three antibiotics. In rifampin treated cells, orotate and both uridines are preferentially incorporated into DNA. With chloramphenicol, the relative incorporation of orotate and of uridine into the 5 S and the 16 S RNA is higher as compared with the 23 S RNA. Cycloheximide results in an increase in the relative incorporation of orotate as well of uridine into DNA. The similarities of the effects of the three antibotics indicate that the preferential incorporation of uridine into chloroplast ribosomes of Chlorella is not due to a compartmentation of the uridine-UMP-pathway.

---

Abkürzungen BisMSB bis(O-Methylstyryl)-Benzol - PPO 2,5-Diphenyloxazol - MAK-Säule Säule aus methyliertem Albumin mit Kieselgur     

Silence of streptomycin 6-phosphotransferase gene derived by incubation at a high temperature inStreptomyces griseus

Summary The bald mutants from streptomycin (SM)-producingStreptomyces griseus 2247 obtained by incubation at high temperature (36° C), designated as HT strains, lost resistance to their own antibiotic and scarcely produced the antibiotic. Although SM susceptibility in the mutant was due to loss of SM 6-phosphotransferase activity produced in the cell, the gene coding for the enzyme cloned from an HT strain was surely expressed inS. lividans 1326 as a host. Northern blot analysis showed that the corresponding RNA is not detected in the mutant, indicating that though the gene encoding SM 6-phosphotransferase, at least, the structural gene is not deleted in the cell, the expression is silent.     

Nucleic Acids Synthesis of Nuclear Polyhedrosis Virus in Cultured Embryonic Cells of Silkworm

Embryos of the silkworm, Bombyx mori L., were dispersed by trypsin and the dissociated cells were cultured for infection with nuclear polyhedrosis virus (NPV) of the silkworm. The monolayer and suspension cultures were infected with NPV. RNA and DNA syntheses in the normal and NPV-infected cells were measured by incorporation of ³²P into RNA and DNA fractions. RNA and DNA syntheses in the cells after infection significantly increased over those in control cells (mock infection). The effects of actinomycin D, chloramphenicol and mitomycin C on RNA and DNA syntheses in infected cells were examined. The syntheses were inhibited by the antibiotics. It was suggested that the cellular DNA synthesis was inhibited by the viral infection, because the mitomycin C-resistant DNA synthesis was found in the normal cells but not in the infected cells treated with mitomycin C. The rate of DNA synthesis induced by NPV was immediately dropped to that of control cells by addition of chloramphenicol, while the RNA synthesis induced by NPV was not affected for 6 hr after the addition of chloramphenicol. If the antibiotic did not affect the size of precursor pools, this event suggested that the RNA polymerase concerned with viral RNA synthesis was more stable than the DNA polymerase participating in the viral DNA synthesis. The viral DNA as templates for RNA and DNA syntheses was decomposed by mitomycin C.     

Production of extracellular proteins,cellulases and antifungal metabolites by Chaetomium globosum Kunze ex. Fr.

Chaetomium globosum has been well-known potential antagonist of several seed and soilborne fungus. Eight isolates of C. globosum were obtained from different sources and were identified by morphological characters. C. globosum isolates examined for the presence of extra cellular proteins, cellulases and antifungal metabolites in culture filtrate by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), thin-layer chromatography and high-performance liquid chromatography. Variation in the mycelial protein of C. globosum isolates was noted in the SDS-PAGE analysis. Different C. globosum isolates that showed more number of bands in protein profile was further screened for the production of cellulases in culture filtrate. Cellulase activity of C. globosum isolates revealed that maximum activity was observed in the isolate Cg-6 after 11?days of incubation, while Cg-2 had least activity. C. globosum isolates were tested for antibiotic production, among which three isolates viz. Cg-6, Cg-7 and Cg-5 were found to produce the antibiotic Chaetoglobosin A in the culture filtrate. The antibiotic Chaetoglobosin A appeared blue colour under UV spectrum with a wavelength of 250?nm.     

Inhibition of cell division inMyxococcus xanthus by mecillinam

Increase in cell numbers is inhibited by adding mecillinam to vegetative cultures ofMyxococcus xanthus. However, both cell length and volume continue to increase in the presence of the antibiotic. Different size classes of cells increase in proportion to their initial size. Incorporation ofmeso-diamino[¹⁴C]pimelic acid into peptidoglycan and [³H]uridine into RNA is not immediately affected by mecillinam. It is suggested that mecillinam inhibits the formation of new sites of wall synthesis, and these are necessary for cell division to occur.     

An embryogenic suspension cell culture system for Agrobacterium-mediated transformation of citrus

A method for the genetic transformation of several citrus cultivars is described, including cultivars observed to be recalcitrant to conventional epicotyl-mediated transformation. Embryogenic cell suspension cultures, established from unfertilized ovules were used as target tissues for Agrobacterium-mediated transformation. Several modifications were made to the culture environment to investigate factors required for efficient transfer of the T-DNA and the subsequent regeneration of transgenic citrus plants. It was determined that co-cultivation of citrus cells and Agrobacterium in EME medium supplemented with maltose (EME-M) and 100 μM acetosyringone for 5 days at 25°C was optimum for transformation of each of the citrus cultivars. Efficient selection was obtained and escapes were prevented when the antibiotic hygromycin B was used as a selection antibiotic following transformation with an Agrobacterium strain containing hptII in the T-DNA region. Transgenic embryo regeneration and development was enhanced in medium that contained a liquid overlay consisting of a 1:2 mixture of 0.6 M BH3 and 0.15 M EME-M media. PCR and Southern blot analyses confirmed the presence of the T-DNA and the stable integration into the genome of regenerated plants, while RT-PCR demonstrated variable amounts of RNA being transcribed in different transgenic lines. This protocol can create an avenue for insertion of useful traits into any polyembryonic citrus cultivar that can be established as embryogenic cell suspension cultures, including popular specialty mandarins and seedless cultivars.     

A novel strategy for obtaining kanamycin resistance in Arabidopsis thaliana by silencing an endogenous gene encoding a putative chloroplast transporter

The use of bacterial antibiotic resistance markers in transgenic plants raises concerns about horizontal gene transfer to soil bacteria. We report here that kanamycin resistance in Arabidopsis thaliana can be achieved by silencing an endogenous gene encoding a putative chloroplast transporter, which presumably imports kanamycin into chloroplasts to interfere with ribosomal RNA. Homologs of the transporter exist in other plant species, suggesting this strategy may be generally useful for selecting transformed plant cells.     

Announcement

Phosphate concentration was found to control the biosynthesis of the antibiotic candicidin by resting cells of Streptomyces griseus. Phosphate concentrations above 1 mM decreased the rate of incorporation of [¹⁴C]propionate and [¹⁴C]p-aminobenzoic acid into candicidin in relation to the concentration of phosphate. The inhibitory effect of phosphate on incorporation of labeled precursors into candicidin was not caused by inhibition of cellular uptake of precursors. Protein synthesis, sensitive to chloramphenicol, was not affected by phosphate levels that inhibit antibiotic synthesis. Similarly, phosphate concentrations inhibitory to antibiotic synthesis did not affect rifampinsensitive RNA synthesis.     

Ribonuclease from Alcaligenes eutrophus: Activation by heat treatment,partial purification and some properties

Summary Heat treatment (10 min at 65° C) of cells of Alcaligenes eutrophus, which resulted in the release of RNA degradadation products into the medium, was found to activate cellular ribonucleases. Two ribonucleases degrading yeast RNA were found, one localized in the periplasmic space and the other in the soluble fraction of the ribosomes. Compared to non-heated cells, in the heat-treated cells the former enzyme, the cell debris ribonuclease, was present at an eightfold increased specific activity, and the latter, the cytoplasmic ribonuclease, was present at a fourfold increased specific activity. This increase was due to the inactivation of a thermolabile inhibitor and to denaturation of part of the soluble protein during heat treatment. With respect to their properties the enzymes were similar; they had endonuclease activity and hydrolysed only RNA. They were heat-stable, resistent to trypsin, highly sensitive to a ribonuclease inhibitor isolated from the same bacterium and were partially inhibited by ATP and GTP. These properties provided a partial explanation for the mechanism of the release of RNA dagradation products from A. eutrophus cells after heat treatment.Abbreviations DNA Deoxyribonucleic acid - RNA Ribonucleic acid - tRNA Transfer RNA - DNase Deoxyribonuclease - RNase Ribonuclease - d-RNase debris RNase - c-RNase cytoplasmic RNase     

In vitro studies of cerebral cortical RNA and nucleotide metabolism in hypothyroidism.

—Thyroid hormone deficiency induced during the neonatal period in the rat, resulted in an enhanced incorporation of [2-¹⁴C]uridine and [8-¹⁴C]adenosine in vitro into cerebral cortical RNA at 25 days of age. An examination of the acid-soluble pool constituents separated by polyethyleneiminecellulose TLC, revealed that all phosphorylated derivatives were more highly labelled compared to controls. These differences were not apparent at a lower incubation temperature (4°C). When the average specific activity of precursor pool ATP labelled from adenosine was utilized for the calculation of the rate of RNA synthesis, no change was observed in hypothyroidism. The results are compatible with a maturational-dependent increase in nucleoside transport and rate of phosphorylation in hypothyroidism which is reflected in the stimulated incorporation into cerebral RNA. The apparent normal rate of RNA synthesis coupled with a diminished cellular RNA concentration in thyroid hormone deficiency, suggests an increased RNA turnover. Experiments with actinomycin D revealed no apparent difference in the rate of decay of rapidly-labelled (nuclear) RNA. The possibility is discussed that the processing of nuclear RNA, the formation of stable ribosomal complexes and events at the translational level are subject to modification in developing hypothyroid rats.     

The production, isolation, and characterization of a grisein-like sideromycin complex

Streplomyces griscus var. X-2455 produces an antibiotic complex which is active in vitro against a number of gram-positive and gram-negative bacteria and in mice against systemic infections caused by K, pneumoniae and D, pneumoniac. In view of the favorable chemotherapeutic index and the broad in vitro spectrum of crude concentrates, isolation of the pure antibiotic complex and the individual constituents was undertaken. The antibiotics referred to as Ho 5-2667, Ro 7-7730, and Ho 7-7731 can be differentiated by tle, ultraviolet light absorption spectra, and in vitro antibacterial activities. They all contain iron and may be classified as sideromycins. From antibiotic concentrates an antibacterially inactive substance was isolated and identified as N-acetyltyramine.     

Auxin-stimulated RNA Synthesis in Oat Coleoptile Cells

Using oat coleoptile segments the following results were obtained. Ten mg/l auxin (indole-3-acetic acid) increased the incorporation of uracil-2-¹⁴C and orthophosphate-³²P into RNA fraction during a relatively short incubation period. Stimulation of ³²P incorporation due to auxin was found only in the region heavier than ribosomal RNA, probably in the messenger RNA region. The stimulation of uracil-2-¹⁴C incorporation into RNA caused by auxin was not influenced by the presence of 0.3 M mannitol which prevents osmotically the water absorption of cells. It is concluded that auxin primarily stimulates the biosynthesis of RNA, possibly messenger, in oat coleoptile cells.     

Induction of Antibiotic Resistance in Paramecium tetraurelia by the Bacterial Gene APH-3'-II

We have generated a transformation marker for Paramecium using a Paramecium expression vector (pPXV) and the open reading frame (ORF) of the bacterial antibiotic resistance gene aminoglycoside 3'-phosphotransferase-II (APH-3'-II or neo^r) from the transposon Tn5. The expression vector contained a small multiple cloning site between the 5' and 3' non-coding regions of the calmodulin gene, and Tetrahymena telomere sequences for the stability of the plasmid in Paramecium. After the neo^r ORF was inserted, the plasmid was referred to as pPXV-NEO. Delivery of approximately 10–20 picoliters of linearized PXV-NEO at > 2000 copies/pl into the macronucleus effected 100% transformation. Southern and Northern blot hybridization showed the presence of neo^r-specific DNA and RNA, respectively, in all of the transformed clones but not in the untransformed clones. The degree of resistance to G-418, and the concentrations of neo^r-specific DNA and neo^r-specific RNA in the clones were proportional to the concentration of the vector injected. We have demonstrated that when the linearized plasmid was injected into the macronucleus, the prokaryotic sequence conferred an antibiotic resistance to Paramecium despite codon-usage differences.     

Reduced hnRNPA3 increases C9orf72 repeat RNA levels and dipeptide‐repeat protein deposition

Intronic hexanucleotide (G₄C₂) repeat expansions in C9orf72 are genetically associated with frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The repeat RNA accumulates within RNA foci but is also translated into disease characterizing dipeptide repeat proteins (DPR). Repeat‐dependent toxicity may affect nuclear import. hnRNPA3 is a heterogeneous nuclear ribonucleoprotein, which specifically binds to the G₄C₂ repeat RNA. We now report that a reduction of nuclear hnRNPA3 leads to an increase of the repeat RNA as well as DPR production and deposition in primary neurons and a novel tissue culture model that reproduces features of the C9orf72 pathology. In fibroblasts derived from patients carrying extended C9orf72 repeats, nuclear RNA foci accumulated upon reduction of hnRNPA3. Neurons in the hippocampus of C9orf72 patients are frequently devoid of hnRNPA3. Reduced nuclear hnRNPA3 in the hippocampus of patients with extended C9orf72 repeats correlates with increased DPR deposition. Thus, reduced hnRNPA3 expression in C9orf72 cases leads to increased levels of the repeat RNA as well as enhanced production and deposition of DPR proteins and RNA foci.