The Fractionation of Transfer Ribonucleic Acid from Roots of Pea Seedlings

Total aminoacylation of glycine and leucine transfer RNAs was compared between chilled and nonchilled hypocotyls of 7-day-old soybean seedlings. Total charging was greater for both specific transfer RNAs from nonchilled sources. Isoaccepting transfer RNA species for glycine and leucine were fractionated using reverse phase column chromatography. Leucyltransfer RNAs were fractionated into six distinct fractions with relatively small shifts appearing in specific fractions between chilled and nonchilied sources. Glycyl-transfer RNAs were fractionated into two distinct fractions with major shifts appearing for both fractions between chilled and nonchilled sources.It is known that RNA and protein metabolism relate closely to chilling injury and frost resistance of plants (13). Dure (8) suggests that translational control of protein synthesis is functional in higher plant development, while Anderson and Gilbert have shown tRNAs to be involved in translational control of hemoglobin synthesis (3). Further, quantitative and qualitative changes in isoaccepting tRNAs of plants have been related to changes in physiological state (2, 26) and environmental factors (6). The purpose of this investigation was to determine if aminoacylation of isoaccepting tRNAs is related to chilling stress in soybean seedlings. MATERIALS AND METHODSSoybean Tissue. Soybean (Glycine max L.) seeds were germinated and grown in vermiculite at 28 C with a day length of 15 hr and a light intensity of 800 ft-c. After 6 days, treated seedlings were placed at 5 C for 1 day while control seedlings were maintained at 28 C. Hypocotyls of nonchilled and chilled seedlings were excised for tRNA preparation.Other groups of soybean seeds were germinated and grown in the dark at 28 C. After 5 days, hypocotyls were excised for aminoacyl-tRNA synthetase preparation. Five-day-old etiolated hypocotyls were found to yield greater aminoacyl-tRNA synthetase activity than 7-day-old green hypocotyls. bean hypocotyl tRNA sources to eliminate enzyme activity as a variable during chilling.Transfer RNA Preparation. Transfer RNAs were isolated and purified from chilled and nonchilled soybean hypocotyls by a modification of the method of Vanderhoef et al. (24).Hypocotyl tissues were homogenized in a Waring Blendor in 0.1 M sodium acetate buffer, pH 4.5, 50 mm NaCl, 20 mM MgCI2, 10 mg/ml of bentonite, and 0.3% SDS' (w/v). After homogenates were filtered through double-layered cheesecloth, filtrates were centrifuged for 15 min at 30,000g. An equal volume of water-saturated phenol was added to supernatant solutions. After samples were stirred for 1 hr at 4 C and centrifuged for 15 min at 30,000g, aqueous layers were removed and volumes were determined. RNA was precipitated from aqueous solutions by addition of 2.5 volumes of 95% ethanol at -20 C. Precipitates were centrifuged at 30,000g for 15 min and resuspended in 50 ml of buffer A: 20 mm sodium acetate buffer, pH 4.5, 50 mm NaCl, and 10 mi MgCI2. Following centrifugation of resultant solutions for 5 min at 10,000g, su...

Section: Resultssupporting

confidence: 73%

Isoaccepting Transfer Ribonucleic Acids during Chilling Stress in Soybean Seedling Hypocotyls

Yang¹,

Brown²

1974

“…Three distinct tRNATY' species have been detected in pea seedling roots (26). That none of the three species was artifactual was indicated by several experiments: (a) an aqueous tRNA solution was heated to 85 C, then quick cooled prior to charging to determine the presence of internally damaged (and, hence, chromatographicaily distinct) molecules (24).…”

Section: Discussionmentioning

confidence: 99%

“…These procedures have been previously described (26 Examinaton of similar micrographs of sections taken from ase incubation time. B: "C-tRNA was added to the mitochondria throughout the final mitochondrial pellet showed no contami-mixture for various time periods, the total tRNA subsequently was nation by either proplastids or bacteria.…”

Section: Methodsmentioning

confidence: 99%

Mitochondrial Tyrosyl Transfer Ribonucleic Acid in Soybean Seedlings

Meng

Vanderhoef

1972

Self Cite

Soybean seedlings were examined for the presence of mitochondrial tRNA. Tyrosyl transfer tRNAs from whole cells, from a well characterized mitochondrial preparation, and from a snake venom phosphodiesterase-treated mitochondrial preparation, were compared by reverse phase chromatography. It was concluded that none of the three previously reported tRNATYr species were mitochondrial. Rather, a fourth tRNATYr species, eluting somewhat later, was of mitochondrial origin. Mitochondrial tRNATYr was chromatographically similar to Escherichia coli tRNATYr.Transfer RNA and tRNA synthetases have previously been purified from mitochondria. Barnett and his colleagues (2, 3) pioneered in this area when their studies of species specificity in the aminoacyl transfer reaction led to the discovery of the localization of certain tRNAs and their synthetases in Neurospora crassa mitochondria (4)(5)(6)(7)(8)(12)(13)(14). Mitochondrial tRNA and/or synthetases have also been discovered in yeast (1,10,11,18,21,29), Tetrahymena (22,23), and rat liver (9, 21).Suyama reported that "a large portion of mitochondrial sRNA is not transcribed from mitochondria DNA" in Tetrahymena; it is clear, however, that mitochondrial DNA does transcribe tRNAL"U and tRNAFM-t in yeast (I 1, 18 were discarded, and the remainder of the seedling was stored in ice-cold 1 % sucrose for not longer than 2 hr before use for extraction of tRNA, enzymes, or mitochondria.Extraction of Mitochondria. A modified method of Flowers and Hanson (16) was used. Tissue was diced with razor blades in 1.5 vol homogenization medium (0.4 M sucrose, 50 mM KH2PO4, 5 mM Na2EDTA, adjusted to pH 7.6 with crystalline tris). A PT1O-35 Polytron homogenizer (generator PT20/ST, setting 3) was used to further disrupt the cells. The homogenate was filtered through four layers of cheesecloth, then centrifuged 10 min at 1,000g. The supernatant fluid was recentrifuged 7 min at 31,000g. The resulting pellet was suspended in 0.4 M sucrose and centrifuged 10 min at 1,000g. The supernatant fluid was saved, the pellet resuspended in 0.4 M sucrose, centrifuged 10 min at 1,OOOg, and that supernatant fluid combined with the first. The combined supernatant fluids were underlaid with 0.6 M sucrose in a centrifuge tube and centrifuged 25 min at 10,000g. The supernatant fluid was discarded and the mitochondria-rich pellet was immediately quick-frozen at -80 C and stored at -20 C for future tRNA extraction.

“…These possibilities have led to much recent work on the tRNAs ot prokaryotic and eukaryotic cells (3,4,11,16,17,21,22,25,27). Changes isoaccepting tRNAs during growth and differentiation have been sought as possible control points of translation.…”

Section: Lysine-trnas From Black-eyed Peasmentioning

confidence: 99%

“…According to several recent reports, plant tissues exhibit chromatographically detectable changes in isoaccepting species of tRNA during growth and differentiation, and it is inferred that these changes may be important in the growth process (3,6,22,25 …”

mentioning

confidence: 99%

The Coding Properties of Lysine-accepting Transfer Ribonucleic Acids from Black-eyed Peas

Hague

Kofoid

1971

It has been suggested that protein synthesis may be regulated at translation by the particular complement of tRNAs and aminoacyl-tRNA synthetases present in the cell (2,18,20). According to several recent reports, plant tissues exhibit chromatographically detectable changes in isoaccepting species of tRNA during growth and differentiation, and it is inferred that these changes may be important in the growth process (3,6,22,25