Isolation and Partial Characterization of Transfer RNAs from Astragalus bisulcatus

Abstract

A procedure has been developed for the isolation of transfer RNA from the selenium accumulator plant Astragalus bisukatus. This material appears free of interfering phenolic compounds, has a high guanosine to cytidine ratio, shows a major and modified nucleoside composition characteristic of plant transfer RNAs, and exhibits chromatographic and electrophoretic properties similar to transfer RNAs from other weUl studied bacterial and plant systems. RNAs isolated from A. bisukatus seedlings incubated in the presence of 75Se indicate some incorporation of radioactivity into the transfer RNAs, but at extremely low levels. The transfer RNAs were active in accepting amino acids, although their over-all levels of activity appeared low when compared with those from a homologous Eschenrhis coHl aminoacylation reaction system. Selenium accumulator plants, such as Astragalus bisulcatus, grown on seleniferous soils, absorb massive amounts of inorganic selenium, much of which is converted to water-soluble, low mol wt organic compounds (selenocystathionine, Se-methylselenocysteine, dimethyl selenide, selenohomocystine). Trace quantities of selenium appear to be required for proper growth and development of the plants (19,22). Unlike nonaccumulator plants, where considerable amounts of selenium can be incorporated into protein in the forms of selenomethionine and selenocysteine (13), only limited amounts of selenium-containing amino acids appear to be incorporated into proteins of accumulator plants. The control mechanisms that exist in the accumulator species to limit the extent of substitution of seleno-for sulfur-containing amino acids are unknown (22). In order to examine the aminoacylation reaction as a possible control point, it is necessary to have relatively pure and biologically active tRNA. We have developed a procedure for the isolation of tRNAs from A. bisulcatus. These were shown to be active in accepting normal amino acids and were characterized by chemical, chromatographic, electrophoretic, and spectral methods. Several different types of nucleoside analyses were carried out to determine major and modified base compositions, and to examine the tRNAs for the possible presence of selenium-containing nucleosides-which have been isolated from Escherichia coli tRNAs (6,18 Phenol (J. T. Baker) and m-cresol (Eastman) were distilled under reduced pressure and stored at 4 C in the dark. All solutions were prepared with degassed, deionized H20 and all other chemicals and reagents were either analytical or reagent grade.Plant Material. Mature seed pods were harvested from A. bisulcatus plants growing wild near Laramie and Medicine Bow, Wyoming, in areas of high soil selenium. After air-drying, they were processed through a Wiley Mill to break the pods, and Clipper Reaper Grain Seed and Bean Cleaner to separate seeds from chaff. The seeds were stored in plastic jars at -20 C. Samples of dry seeds were homogenized in water and aliquots analyzed for selenium content (12), which ranged from 25 to 146 jig selenium pe...

Identification and synthesis of a naturally occurring selenonucleoside in bacterial tRNAs: 5-[(methylamino)methyl]-2-selenouridine

“…Is 4-Selenouridine a tRNA Component? Although earlier reports indicated that 4-selenouridine is a constituent of E. coli tRNAs (Hoffman & McConnell, 1974;Prasado Rao & Cherayil, 1974;Young & Kaiser, 1979), the ion-exchange and thin-layer chromatographic procedures available at the time lacked the resolving power necessary to distinguish between selenonucleosides and the numerous degradation products generated by the prolonged alkaline digestion procedures that were employed. In the present studies using rapid enzymic digestion procedures and the much more powerful analytical methods now available, we have not detected 4-selenouridine in the tRNA populations of E. coli, C. sticklandii, or M. vannielii.…”

Selenium in Plants: Uptake, Functions, and Environmental Toxicity

“…Transfer-RNAs (tRNAs) that contain Se appear to be ubiquitous among all groups of organisms (Mayland et aI., 1989). In plants, Young and Kaiser (1979) detected tRNA, which incorporated some Se, in the Seaccumulator Astragalus bisulcatus. In a variety of higher plants and Chlamydomonas, Wen et al, (1988) demonstrated 75Se-tRNA upon supplying the plants with [ 75S eJ selenite.…”

Selenium-containing tRNAGlu from Clostridium sticklandii: correlation of aminoacylation with selenium content.

“…When selenium was detected earlier in the tRNAs ofEscherichia coli (3,4) in the form of selenium-modified bases, it was thought to have resulted from the nonspecific substitution of the element for sulfur, but recent studies show that selenium in these tRNAs is a normal component (5) and is incorporated by means ofa highly specific process (6). The tRNAs of two strictly anaerobic bacteria, Clostrtidium sticklandii and Methanococcus vannielii, have a higher content of selenium and this is distributed among four to six seleno-tRNA species (7,8).…”