Matthys Staehelin

Isolation of mammalian transfer RNA

Abstract A simple and rapid method is described for the large scale preparation of mammalian tRNA which involves phenol extraction, ion-exchange chromatography and isopropanol precipitation. The tRNA is shown to contain its full complement of terminal adenosine.

An improved method for the separation and quantitation of the modified nucleosides of transfer RNA.

A method is described which allows a very efficient determination of the modified nucleosides of tRNA. The technique involves enzymatic degradation of the tRNA to nucleosides at pH 7.6 and their separation by two-dimensional thin-layer chromatography on cellulose-coated aluminum foils. Based on the analysis of two mammalian tRNAs it is shown that the technique is suitable for the determination of chemically unstable nucleosides as well as the ribose-methylated compounds. At least 36 of the 45 known modified nucleosides can be separated and quantitatively determined by the method described. This procedure is especially suitable for the estimation of the nucleoside composition of unlabeled tRNAs as well as for studying the post-transcriptional modifications of tRNA.

The rifamycins--relation of chemical structure and action on RNA polymerase.

Abstract The interaction of RNA polymerase with various rifamycin derivatives has been studied. It could be shown that the macrocyclic ring is the part of the molecule responsible for the complex formation and inhibition of RNA polymerase. Very slight modifications, such as oxidation in the case of rifamycin Y or hydrogenation of double bonds, alter the sterical shape of the ring in such a way that it no longer shows optimal binding to the acceptor site of the enzyme. Changes in other parts of the molecule have little effect on the direct action on RNA polymerase but may affect the permeation into intact bacterial cells.

The preparation of rat-liver soluble ribonucleic acid

Abstract A method for the preparation of rat-liver sRNA is described, where contamination with other RNAs and DNA is minimized. Sephadex G-200 chromatography was used to free the RNA of most high- and low-molecular-weight contaminants. Ratliver sRNA purified by this method has approximately twice the serine acceptor activity as yeast sRNA, using a rat-liver enzyme preparation. The method is suitable for the preparation of gram quantities of sRNA.

Base sequences in S-RNA

Nucleotide sequences have been studied in pancreatic RNase digests of soluble RNA. Various sequences containing “odd” nucleotides have been identified. The finding of sequences with more than one “odd” nucleotide, such as 2-dimethyl guanyl-5-ribosyl uracil phosphate and 1-methyl guanyl-2-methyl guanyl cytidylic acid, is evidence for a clustering of the unusual bases in certain parts of the molecule. The frequency with which these two sequences occur, i.e. about 0·3 residue per average chain, indicates their presence in more than one individual amino acid-acceptor S-RNA. Three sequences containing unusual bases have been found in S-RNA enriched in serine acceptor activity: guanyl-5-methyl cytidine, a tetra- and pentanucleotide which both carry 5-ribosyl uracil phosphate as the pyrimidine nucleotide and which contain, in addition to guanosine and adenosine, inosine and dimethyl guanosine, respectively.

Column Chromatography of Oligonucleotides and Polynucleotides

Publisher Summary The chapter discusses the column chromatography of oligonucleotides and polynucleotides. The separation of oligonucleotide and polynucleotide material on certain other adsorbents that have been introduced more recently, such as diethylaminoethyl cellulose (DEAE-cellulose) and methylated serum albumin, is described in this chapter. The former offers wide possibilities in the fractionation of oligonucleotides and the latter deserves special attention, because of its ability to separate nucleic acids of high molecular weight by column chromatography. The chromatographic separation of oligonucleotides, according to chain length, is discussed in this chapter. Many components can be isolated from an RNase digest, by chromatography on DEAE-cellulose, at a slightly alkaline pH. The chromatographic separation of the oligonucleotides on sephadex is also discussed in this chapter. Sephadex is a cross-linked dextran that, in principle, allows the separation of substances according to the molecular size. Chromatographic separation of nucleic acids, such as chromatographic separation on substituted cellulose anion exchangers and chromatographic separation of nucleic acids on methylated albumin columns, are also discusses in this chapter.

Minor dinucleotides of rat-liver transfer RNA

Abstract From a pancreatic ribonuclease digest of rat-liver tRNA, several minor dinucleotides have been isolated and characterized. The quantitative distribution indicates that some of them, e.g. m 2 2 G-ψ and m 2 2 G-C ★ , are constituents of more than one tRNA species whereas others, such as Cm-C and ψm-U, occur in less than 1% of all tRNA molecules. Several minor nucleotides have been shown to occur in more than one dinucleotide, whereas others known to occur in considerable amounts in tRNA could not be detected in dinucleotides. An unknown cationic nucleoside is described which was found in two dinucleotides.

Comparative study of different preparations of yeast soluble ribonucleic acid

Abstract Soluble RNA was isolated from bakers yeast by means of phenol, NaCl, and sodium dodecyl sulfate extractions. Phenol and dodecyl sulfate extraction yielded highly active soluble RNA preparations whereas extraction with NaCl resulted in a partially degraded nucleic acid. The extraction with sodium dodecyl sulfate resulted in a heterogeneous preparation which contained two components of 4.01 S and 1.04 S, respectively. Its leucine acceptor activity was considerably greater than that of phenol-soluble RNA. Upon purification on DEAE-cellulose the heavy component could be isolated; it had a lower specific activity than the crude material and corresponded in all respects to the phenol component.

Effects of benzoctamine (30803-Ba, TACITINR), a new psychoactive drug, on catecholamine metabolism

Abstract Benzoctamine(TACITIN R ), 1-methylamino-methyldibenzo [b,e]bicyclo[2.2.2] octadiene, is a new psychoactive agent which possesses tranquillizing properties. Its effects on catecholamine metabolism have been studied. Benzoctamine produced no marked change in the catecholamine concentration of various rat organs after either single or repeated treatment and did not inhibit monoamine oxidase or catechol- O -methyltransferase activities in liver and brain. It enhanced markedly the incorporation of [ 3 H]tyrosine into [ 3 H]catecholamines in brain and adrenals. It accelerated also the disappearance rate of intracisternally administered [ 3 H]noradrenaline. [ 3 H]noradrena-line uptake in the rat heart and brain was not inhibited. It is concluded that benzoctamine most probably increases the turnover rate of catecholamines.

Guanethidine uptake and noradrenaline depletion in noradrenaline storage particles of the rat heart

Abstract Rats received an intravenous injection of [3H]noradrenaline or of [3H]guanethidine. The noradrenaline storage particles of the heart were isolated by sucrose density gradient centrifugation. Their analysis confirmed that both agents are taken up into subcellular granules by a reserpine-sensitive process and showed that they have a similar pattern of subcellular distribution into two types of gradients. The amounts of [3H]guanethidine found in the granules 30 min and 5 hr after its intravenous injection were compared with the loss of noradrenaline from these granules after intravenous injection of the same dose of unlabelled guanethidine. After 30 min an appreciable amount of guanethidine was found, whereas no significant loss of noradrenaline could be detected. Between 30 min and 5 hr, the granules lost an amount of noradrenaline which was greater than the supplementary amount of guanethidine taken up. Since the rate of neuronal uptake of guanethidine is not correlated with the rate of noradrenaline loss, it is concluded that the noradrenaline depletion caused by guanethidine in the rat heart is not due to a simple displacement mechanism. Chromatographic experiments carried out in two solvent systems failed to demonstrate the presence of metabolites in the granule fraction, whereas at least one metabolite was detected in the coarser particles. This suggests that the guanethidine metabolites play at best a minor role in the mechanism of action of guanethidine.