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

Abstract

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 &ethyl 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...

Synthesis by ribonuclease a of codons containing modified nucleosides in the ‘wobble’ position

“…The crucial step in using the reverse of the normal reaction of RNase A is to stop the hydrolytic action of the enzyme and thus prevent degradation of the products formed. Initially we used diethylpyrocarbonate to inactive the RNase [8], but abandoned its use later on when we discovered that it reacts at higher concentrations with a number of nu- cleosides, especially adenosine. Therefore, we stopped enzyme action by lowering the pH of the reaction mixture to pH 2.5.…”

Effect of Diethylpyrocarbonate on the Antiviral and Interferon-Inducing Activities of Viral and Nonviral Agents

“…When an influenza virus (A2/Japan 305) was similarly treated, its hemagglutination titer was reduced from 4096 U/ VOL. 9,1976 678 YAMAMURA AND COCHRAN ml to less than 4 U/ml (Table 2). Infectivity in tissue culture cells (CEF) was similarly reduced.…”

“…Diethylpyrocarbonate (DEPC) is a potent inhibitor of enzymes, and its inhibitory effect on nucleases in particular (7) has been used for preparing ribonucleic acid (RNA) with intact biological activities (5,9,26). Treatment of intact virus particles with DEPC results in a loss of infectivity due to denaturation of virion proteins (8,21,23), but viral nucleic acids extracted from DEPC-inactivated virions are still infective (8,21,23).…”

The Effects of Diethyl Pyrocarbonate on the Stability and Activity of Plant Polyribosomes

“…Weeks and Marcus (18) showed that much of the degradation could be prevented by isolation with diethyl pyrocarbonate, a compound previously used by Fedorcsak et al (2,3,16,17) as a nuclease inhibitor during the isolation of nucleic acids. The results of Weeks and Marcus (18) showed that there was a small number of monoribosomes and a much larger population of subunits in DEP'-treated preparations and that DEP inhibited the ability of the ribosomes to support in vitro amino acid incorporation.…”