UBC Theses and Dissertations
Studies on yeast soluble-ribonucleic acid Millward, Stewart
Part I of this thesis describes the digestion of bakers' yeast s-RNA at 0° and at room temperature in the presence of various concentrations of magnesium ions and at several different s-RNA : enzyme ratios (w/w). Digestion of s-RNA with pancreatic ribonuclease (ratio, 2000:1, (w/w) ) in the presence of 0. 2 M magnesium and at 0°, followed by chromatographic analysis, indicated that about 75% of the ultraviolet absorbing material was of medium to high molecular weight. Analysis of the nucleotide composition of the fraction containing the intermediate size oligonucleotides showed that fraction to be enriched in the odd bases ψ, T, 1-methylG, N, N-dimethylG and several other unidentified nucleotides. This result was incompatible with the 'hairpin' models for s-RNA, proposed by McCully and Cantoni (1962). The presence of spermine in the digestion mixture clearly masked the catalytic action of pancreatic ribonuclease, albeit to a lesser extent than magnesium ions, contrary to a report by Thomas and Hubst (1963) in their study of the affect of spermine on the catalytic activity of E. coli ribonuclease toward E. coli ribosomal RNA. The studies described in Part II of this thesis are concerned with the fractionation of mixed s-RNA from yeast. Part II-A describes a new chromatographic material consisting of a bifunctional mercuri-dioxane derivative attached through a thiol group to the cellulose matrix. A previous report (Eldjarn and Jellum, 1963) had shown that HS-proteins could be fractionated on an organomercurial-cellulose (Material I) derived from aminoethyl-cellulose. The studies reported, here suggest that ¹⁴C-cysteinyl-s-RNA cannot be retained on Material I because the salt concentration required to overcome its ion-exchange properties preclude any interaction between the mercury of Material I and the sulfhydryl group of ¹⁴C-cysteinyl-s-RNA. The present study describes how this disadvantage was overcome by preparing an organomercurial-cellulose devoid of ion-exchange properties (Material II). Preliminary studies showed that Material II can retain the radioactivity associated with ¹⁴C-cysteinyl-s-RNA. These studies suggested that Material II might fractionate nucleic acids according to their base composition. Part II-B describes some of the attempts to fractionate yeast s-RNA by column partition chromatography. In general, resolution of acceptor activities of s-RNA, as well as the recovery of biological activity, was poor. Part II-C describes some of the author's contributions to the studies carried out in this laboratory on the fractionation of yeast s-RNA on another new chromatographic material, benzoylated-DEAE-cellulose (Material III). Certain variables (such as, magnesium ion concentration and pH), which can be manipulated during rechromatography of s-RNA on Material III, are discussed in light of the chromatographic behaviour of glycine and other acceptor RNA's. The forces responsible for this fractionation are also discussed. Part II-D describes a chemical procedure for the isolation of glycine s-RNA which, when combined with the chromatographic procedures described in Part II-C, afforded glycine-specific s-RNA, in high purity. Observations on the effect of magnesium ion during enzymatic synthesis of glycyl-s-RNA are discussed. The advantages and possible consequences of using purified aminoacyl-s-RNA synthetase enzymes for the preparation of the aminoacyl-s-RNA's are also discussed. The need to prepare large quantities of aminoacyl-s-RNA synthetases derives from their indispensable role in the development of chemical methods for purifying amino acid-specific s-RNA's (see Part II-D). Grinding of yeast cells with glass beads, which is the usual method employed for disrupting yeast cells, was found to be inadequate. Lysis of yeast cells with toluene at 37° was found to be a partial solution to this problem. Although previous studies showed that most of the synthetase enzymes were destroyed by this treatment (von Tigerstrom and Tener, 1967), investigations described in Part III of this thesis, show that most of the synthetase activities can be preserved by controlling the pH of the toluene-yeast mixture. However, there were still several synthetase activities missing in the cell-free extracts prepared by the warm toluene method. Preparation of large quantities of yeast cell-free extract containing all of the aminoacyl-s-RNA synthetases was finally achieved by treatment of the yeast cells with toluene containing excess dry ice followed by an incubation period at 3-5°. Partial purification of the aminoacyl-s-RNA synthetases was achieved by chromatographing the cell-free preparation on hydroxyl-apatite. The presence of 40% glycerol in the eluting buffers was found to be essential for the preservation of most of the synthetase activities. When the cell-free preparations were chromatographed on hydroxy-apatite, most of the protein, but only a few of the synthetases were eluted by a linear gradient of phosphate buffer to 0.20 M. Most of the synthetases were eluted only when the column was washed with ammonium sulfate. The significance of this observation is discussed.
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