A New Method Based on the Use of Diethyl Pyrocarbonate as a Nuclease Inhibitor for the Extraction of Undegraded Nucleic Acid from Plant Tissues

Solymosy, Ferenc; Fedorcsák, I.; Gulyás, Anna; Farkas, G. L.; Ehrenberg, L.

doi:10.1111/j.1432-1033.1968.tb00401.x

Cited by 241 publications

(60 citation statements)

References 20 publications

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“…RNase TI oligonucleotides were located by autoradiography, excised, crushed and eluted overnight at 37 °C in a solution of 0.5 M-ammonium acetate, 0.1 mM-EDTA, 50 gg tRNA, 3~ (v/v) diethylpyrocarbonate (Solymosy et al, 1968). The eluted oligonucleotide was precipitated with 2.5 vol.…”

Section: Methodsmentioning

confidence: 99%

Genetic and Epidemiological Studies of Dengue Type 2 Viruses by Hybridization Using Synthetic Deoxyoligonucleotides as Probes

Kerschner¹,

Vorndam²,

Monath³

et al. 1986

Journal of General Virology

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SUMMARYA rapid nucleic acid hybridization procedure was developed for examining the genotypic variation of dengue type 2 viruses (DEN 2) having distinct RNase T1 fingerprints and isolated from different geographical areas. Synthetic DNA hybridization probes were constructed complementary in nucleotide sequence to coaimon and unique RNase TI oligonucleotides of topotype viruses from Puerto Rico/South Pacific, Jamaica, the Seychelles, Thailand/Burma and Africa. Hybridization probes with both type-and topotype-specific reactivities were observed, as were probes specific for two or more of the DEN 2 topotypes. These results confirm geographical movement of topotype virus strains and suggest possible origins. Detection of DEN 2 RNA by hybridization is a rapid and reproducible method that can be modified and applied as a viable alternative to the laborious T1 oligonucleotide fingerprinting.

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Section: Methodsmentioning

confidence: 99%

Genetic and Epidemiological Studies of Dengue Type 2 Viruses by Hybridization Using Synthetic Deoxyoligonucleotides as Probes

Kerschner¹,

Vorndam²,

Monath³

et al. 1986

Journal of General Virology

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“…The sucrose solutions were treated with diethylpyrocarbonate to inactivate ribonucleases following the procedure described by Solymosy et al (26) except that double-strength sucrose only was treated, and was then diluted with double strength buffer which had been prepared in diethylpyrocarbonate-treated water. To avoid changes in pH buffer solutions were not treated.…”

Section: Methodsmentioning

confidence: 99%

Studies on Phytoalexins

Biggs

1972

Plant Physiol.

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Actinomycin D stimulated phaseollin production in endocarp tissues of the French bean (Phaseolus vulgaris L.), maximum production being obtained with 25 to 30 micrograms per milliliter of antibiotic. Under these conditions, net incorporation of 3H-uridine into total cell ribonucleic acid was inhibited by more than 80% over a 6-hour induction period. If allowance was made for a 2-hour lag in the action of actinomycin D, inhibition of incorporation was greater than 95%. Contrary to other reports, no evidence was obtained of an increased formation of any specific ribonucleic acid fraction. Actinomycin D applied in the cold (4 C) was not found to be effective in stimulating phaseollin production. When applied in this way, actinomycin D did not affect induction of phaseollin by a fungal peptide, Monilicolin A, although ribonucleic acid synthesis was inhibited by more than 95%. It is suggested that the induced formation of phytoalexins may not be dependent on increased ribonucleic acid synthesis as has previously been claimed.Other experiments indicated that the apparent effects of actinomycin D on ribonucleic acid synthesis could be influenced by the choice of precursor used to label ribonucleic acid and by the order of addition of precursor and antibiotic to the plant tissue. These effects were only observed in the period immediately following application of actinomycin D. It is suggested that such effects could critically influence the results obtained in short term experiments and may explain some differences in reported action of actinomycin D from different laboratories.

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“…(B) Dodecyl Sulfate Phenol Method, was done according to Itoh and Hirai [21], with the modification described earlier [15].…”

Section: Extraction Of Nucleic Acids For Assay Of Template Activitymentioning

confidence: 99%

“…Nucleic acid was estimated according to the procedure published earlier [15]. The determination of DNA nucleotides present in the trichloroacetic acid extract was done by the indole reaction [23].…”

Section: Quantitative Determination Of Nucleic Acidmentioning

confidence: 99%

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On the Extraction of Ribonucleic Acid with Template Activity from Barley Embryos Using Diethyl Pyrocarbonate as Nuclease Inhibitor

1969

Self Cite

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Using diethyl pyrocarbonate as enzyme inhibitor, RNA was extracted from embryos of germinating barley kernels. The template activity of this RNA was tested by its capacity to stimulate [14C]phenylalanine incorporation into trichloroacetic acid insoluble protein synthesized in an Escherichia coli cell free system. The template activity was compared with that of RNA isolated from barley embryos by other methods of extraction. The RNA extracted by the ðyl pyrocarbonate method, omitting sodium dodecyl sulfate, was found to have better template activity than the others, and was equivalent or superior to tobacco mosaic virus RNA isolated by the phenol method. Sucrose density gradient sedimentation of the RNA yielded fractions with still higher specific template activity. Reextraction with phenol of the RNA extracted by the diethyl pyrocarbonate method reduced the template activity of the RNA. Native and denatured DNA were also found to decrease the template activity of RNA. RNA extracted from kernels germinated in the presence of 5-fluorodeoxyuridine had a reduced template activity indicating that this compound may inhibit mRNA synthesis, possibly indirectly by blocking strand separation in DNA synthesis. [7]. The results obtained are however confusing and evade critical comparison mainly because (a) the cell free protein synthesising systems and the experimental conditions employed by various authors are different; (b) the procedures of isolation of mRNA are different although in most of the cases the extraction of mRNA was done by various versions of the "hot phenol" method [S], a procedure known to lead to aggregation and partial degradation of RNA [9] ; in addition ribonuclease activity may survive treatment by phenol [lo, 111 which implies an isolation of RNA and especially mRNA degraded to M e r e n t extents; and (c) in most of the experiments no proper standards (such as poly U, or viral RNA) were used; hence they do not permit an evaluation of the quantity of mRNA isolated.Unusual Abbreviations. FUdR, 5-fluorodeoxyuridine; mRNA, messenger RNA; poly U, polyuridylic acid; TMV, tobacco mosaic virus; tRNA, transfer RNA.The greatest obstacle in the isolation of mRNA is its degradation by the ribonuclease [12] during extraction. (According to our observations even bentonite, which is certainly not a specific adsorbent for ribonuclease, is ineffective in completely inhibiting this effect.) A method for the isolation of undegraded nucleic acids, utilizing diethyl pyrocarbonate as a protein denaturing general enzyme inhibitor [13,14] has been described earlier [I51 and it has also been shown that diethyl pyrocarbonate inhibits deoxyribonuclease and ribonuclease without affecting the substrate properties of the nucleic acids [16] and bacterial DNA treated with diethyl pyrocarbonate retains its biological activity [17]. Under the conditions used for the extraction of tissue RNA, diethyl pyrocarbonate leaves transfer RNA (tRNA) functionally intact [18]. I n the present paper we describe attempts to use diethyl. pyroc...

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A New Method Based on the Use of Diethyl Pyrocarbonate as a Nuclease Inhibitor for the Extraction of Undegraded Nucleic Acid from Plant Tissues

Cited by 241 publications

References 20 publications

Genetic and Epidemiological Studies of Dengue Type 2 Viruses by Hybridization Using Synthetic Deoxyoligonucleotides as Probes

Genetic and Epidemiological Studies of Dengue Type 2 Viruses by Hybridization Using Synthetic Deoxyoligonucleotides as Probes

Studies on Phytoalexins

On the Extraction of Ribonucleic Acid with Template Activity from Barley Embryos Using Diethyl Pyrocarbonate as Nuclease Inhibitor

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