Document Type

Dissertation - Open Access

Award Date

1972

Degree Name

Doctor of Philosophy (PhD)

Department

Agronomy

First Advisor

Donald G. Kenefick

Abstract

Several lines of evidence have shown that genetic variability exists within the barley species (Hordeum vulgare L.) with respect to the kinetics and specificity of yeast ribonucleic acid degradation. Initial efforts demonstrated that ribonuclease (RNase), in the crude soluble protein fraction from the cultivar Dicktoo, was inhibited by KC1 and phosphate. RNase activity was enhanced if heated to 60 C for short periods. In contrast, RNase in the crude soluble protein of the cultivar Tennessee Winter showed no altered response by either KC1 or phosphate. Maximal RNase activity was at pH 5.0 for extracts of both cultivars (0.16 M KC1), but after desalting the protein fraction on Sephadex G-25, RNase activity was maximized at pH 7.0 (without KC1). Various chromatographic substances were used in attempts to purify RNase of the crude soluble protein fraction. The column chromatographic sequence ultimately selected for the crude protein fractionation was as follows: Sephadex G-25, Sephadex DEAE A-50 (with KC1 in the elutant) and Bio-Gel P-100 (which separated the cyclizing RNase peaks I and II). RNase peak I was further purified by the column sequence of Bio-Gel P-100 (eluted with 0.2 M KC1), and Sephadex DEAE A-50 (eluted with a 0 to 0.4 M KC1 linear gradient). The enzyme from the last step was desalted and rechromatographed on a Bio-Gel J>-60 column. Characterization of the rechromatographed RNase peak I and the RNase peak II of the first Bio-Gel P-100 step showed that each reacted differently to alterations of KC1 and pH in an assay with yeast RNA. The final purification steps showed that total RNase activity extracted from the Tennessee Winter tissue was higher than for Dicktoo tissue. RNase peak I (pHmax. = 7. 0) exhibited more activity than RNase peak II for the Tennessee Winter preparations, but the reverse order of activity was shown for the two peaks from Dicktoo preparations, i.e. - higher activity for RNase peak II (pHmax. = 5.0). A comparison of degradation rates of synthetic polynucleotides, Poly A, Poly C, Poly G, Poly I and Poly U by RNase peak I from the two cultivars, showed no activity for Poly U. The protein from Tennessee Winter barley was distinguished from that of Dicktoo preparations by its increased activity with Poly A and Poly G, while activity on Poly I and Poly C was slightly greater for the Dicktoo derived enzyme.

Library of Congress Subject Headings

Ribosomes
Barley

Format

application/pdf

Number of Pages

89

Publisher

South Dakota State University

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