Synthetic Template Poly Research Articles

Efficient translation of synthetic and natural mRNAs in an mRNA-dependent cell-free system from the dimorphic fungus Candida albicans

An mRNA-dependent cell-free translation system has been developed from the human pathogenic fungus Candida albicans using either S30 or S100 lysates prepared from glass-bead-disrupted whole cells. Translation of the synthetic template poly(U) in this system is highly efficient at temperatures up to 37 degrees C and is ATP-dependent. Studies using a range of elongation-specific inhibitors suggest that the mechanism of translational elongation in C. albicans is similar to that of another yeast, Saccharomyces cerevisiae. A micrococcal-nuclease-treated C. albicans S100 lysate was able to translate exogenously-supplied homologous mRNAs, and a range of heterologous natural mRNAs, using an initiation mechanism that is inhibited by the antibiotic edeine and the 5' cap analogue 7-methylguanosine 5'-monophosphate (m7GMP). As with cell-free lysates prepared from S. cerevisiae, the C. albicans lysate is unable to initiate translation upon natural mRNAs at temperatures above 20 degrees C.

Acyclic guanosine analogs as inhibitors of human cytomegalovirus.

The acyclic guanosine analogs R- and S-enantiomers of 9-(3,4-dihydroxybutyl)guanine [(R)- and (S)-DHBG], 9-(4-hydroxybutyl)guanine (HBG), and 9-(2-hydroxyethoxymethyl)guanine (ACV) were examined for their effects on human cytomegalovirus (CMV) replication and on CMV DNA synthesis in cell culture as well as for their ability as triphosphates to interact with CMV DNA polymerase. Production of early CMV antigens was not affected. All analogs inhibited CMV DNA synthesis and late viral antigen synthesis. Primary CMV isolates were less susceptible to all tested analogs than was the laboratory strain CMV Ad.169. The triphosphate of ACV was the most potent inhibitor of CMV DNA polymerase, with an observed Ki of 0.0076 microM. The corresponding Ki values of the triphosphates of (R)-DHBG, (S)-DHBG, and HBG were 3.5, 13.0 and 0.23 microM, respectively. All triphosphates of the analogs given above inhibited CMV DNA polymerase in a competitive manner with respect to dGTP. The triphosphates of the analogs also inhibited reactions when the synthetic template poly(dC)oligo(dG)12-18 was used, whereas no inhibition was observed with poly(dA)oligo(dT)12-18. None of the triphosphate analogs supported DNA synthesis in the absence of dGTP, showing that no analog was an alternative substrate to dGTP.

The effects of paromomycin on the fidelity of translation in a yeast cell-free system

The effects of the aminoglycoside antibiotic paromomycin on the fidelity of translation of the synthetic template poly(U), and two natural mRNAs (rabbit globin mRNA and Brome Mosaic virus RNA), were examined in an mRNA-dependent cell-free system from the yeast Saccharomyces cerevisiae. At antibiotic concentrations that did not inhibit translation (100 μM) optimal mistranslation of all three templates was observed, with the effects declining at higher antibiotic concentrations. Synthesis of the opal termination read-through protein of rabbit β-globin mRNA was induced by paromomycin, but only in lysates prepared from a [psi +] strain of yeast. The antibiotic did not induce detectable levels of either ochre or amber read-through, but did induce general misreading of Brome Mosaic virus RNA to the same degree in both [psi +] and [psi −] lysates. This misreading was enhanced by addition of the polyamine spermidine.

Effects of retinoic acid on protein synthesis in cultured melanoma cells.

Retinoic acid reduces the growth rate of mouse S91 melanoma cells in culture and increases the proportion of cells in the G1 phase of the cell cycle. Because of the integral role protein synthesis has been shown to play in growth control we studied the effect of retinoic acid on the protein synthesis machinery with a cell-free system developed from the melanoma cells. This system was capable of translating endogenous mRNA, exogenous globin mRNA, and the synthetic template poly(U). Of the above activities of the protein synthesis system only the translation of endogenous mRNA was reduced significantly in the cell-free system prepared from retinoic acid-treated cells. Analyses of the amount and function of RNA revealed that treatment with retinoic acid leads to reductions in total RNA content, in the proportion of ribosomes in polysomes, in the amount of poly(A)RNA, and in the amount of polysome-associated mRNA. All these effects of retinoic acid contribute to the decrease in protein synthesis activity of treated cells. Two-dimensional electrophoresis analysis of L-[35S]methionine-labeled proteins produced by untreated and treated cells revealed only a few quantitative differences. We suggest that retinoic acid-induced suppression of protein synthesis activity may be the cause for growth inhibition.

Isolation of a soluble and template-dependent foot-and-mouth disease virus RNA polymerase

A large-scale purification method for the soluble RNA-dependent RNA polymerase from foot-and-mouth disease virus (FMDV)-infected BHK 21 cells is described. A cytoplasmic extract made 2.75 hr postinfection (fraction 1b) was chromatographed on DEAE-cellulose (fraction 2), poly(U)-Sepharose (fraction 3a, b) and sized on Ultrogel AcA 44 (fraction 4). Fraction 3a, b contained the viral protein P56 (the VIA antigen) plus small amounts of its precursor P72. The enzyme is capable of transcribing the synthetic template poly(A) primed with (Up) 5 U by a poly(U) polymerase activity. Furthermore, fraction 3a, b transcribed FMDV RNA with an RNA polymerase activity which did not require oligoribouridylate priming. These chromatographic procedures separated P72 from P56. P72 alone appeared to be transcriptionally inactive in vitro. Gel filtration of fraction 3a resulted in a high yield of pure P56. This fractionation step led to a considerable loss of transcribing activity.

Early Effects of Thyrotropin on Ribonucleic Acid Transcription in the Thyroid*

Previous studies on the effects of TSH on the incorporation of labeled precursors into RNA have demonstrated an increase in the percentage of label in the phosphorylated nucleotide precursors of RNA. To avoid this nonspecific effect of TSH, we chose to measure RNA transcription in a system which uses exogenous phosphorylated precursors. Isolated nuclei were prepared from the thyroids of dogs which had been injected 90 min earlier with TSH. TSH caused a mean increase of 168% above control in total transcriptional activity, 193% in polymerase II-mediated (alpha-amanitin-sensitive) activity, and 155% in RNA polymerase I- and III-mediated (alpha-amanitin-resistant) activity. To determine whether part of this early effect of TSH was due to increased activity of RNA polymerase, incubations were also carried out in the presence of actinomycin D, which blocks transcription of endogenous template but does not inhibit transcription of added synthetic template poly deoxyadenylic-deoxythymidylic acid (dA.dT). Under these conditions, about half of the TSH-induced increase in the transcription of endogenous template could still be detected. Thus part of the early effects of TSH on RNA synthesis appears to be mediated through increased polymerase activity as well as enhanced template activity.

Reverse transcriptase activity per virion for avian myeloblastosis virus and Rauscher murine leukemia virus.

We have measured reverse transcriptase enzyme activity per virus particle for samples of avian myeloblastosis virus (BAI strain) and murine leukemia virus (RAUSCHER) USing the synthetic template poly(rC)-oligo(dG). Absolute virus concentrations were determined directly by laser beat frequency spectroscopy. Enzyme activity per virion was determined from the slope of the activity plotted as a function of virus concentration. With this reverse transcriptase assay, the minimum activity (expressed as picomoles of dGTP incorporated/virion per hour) is estimated at (28.1 +/- 4.2) X 10(-7) for avian myeloblastosis virus and (1.1 +/- 0.2) X 10(-7) for murine leukemia virus. The sensitivity of this assay, which is determined by the level of incorporated radioactivity measurable above background, is 2.5 X 10(-4) virions for avian myeloblastosis virus (with dGTP specific activity of 8.9 Ci/mmol) and 88 X 10(-4) virions for murine leukemia virus (with dGTP specific activity of 6.52 CI/mmol). These results show that although reverse transcriptase assays can obviously be used to measure relative virus concentrations of equally purified samples of the same virus, they can be very misleading when used to compare the concentrations of different virus species.

Mechanism of N-Hydroxy-2-acetylaminofluorene Inhibition of Rat Hepatic Ribonucleic Acid Synthesis

Abstract This study attempts to identify the type and mechanism of RNA synthesis inhibited by N-hydroxy-2-acetylaminofluorene (N-OH-AAF). Two hours after the drug injection, the hepatic nuclear RNA synthesis was significantly inhibited when measured in intact nuclei in vitro. After the nuclei were sonicated and fractionated into nucleolar and nucleoplasmic fractions, it was found that the inhibitory effect was very slight in the nucleoplasmic fraction and was very extensive in the nucleolar fraction, where the inhibition by N-OH-AAF reached 90% 1 hour after the injection. Furthermore, when the endogenous template function of the nucleoli was abolished by actinomycin D and the RNA polymerase activity was determined in the presence of the exogenous synthetic template poly[d(A-T)], the polymerase activities of the nucleoli from the control and the carcinogen-treated animals became identical. These studies indicate that the inhibition of RNA synthesis, after acute N-OH-AAF treatment in vivo, is predominantly ribosomal RNA of nucleolar origin and is due to the impairment of the nucleolar DNA template function rather than to RNA polymerase per se.