Demonstration and characterization of a DNA-like RNA in excised plant tissue

When bacteria are subjected to step-down conditions, there is an enhanced production of a messenger-type of RNA for a short time after the shift. A cultural shift, which appears to be similar to step-down, is Extraction and Determination of Total Nucleic Acid. Nucleic acids were extracted as described by Key and Shannon (15) with the modification that after hydrolysis of the RNA the contaminating carbohydrates were removed with Dowex-1 resin, as suggested by Smillie and Krotkov (30). The gametophytes from individual cultures were collected by filtration and then homogenized in 5 ml of ice-cold water with a mortar and pestle. Four-milliliter aliquots of this homogenate were precipitated in duplicate with perchloric acid (0.2 N final concentration) in ice for 30 min, washed two times with 0.2 perchloric acid at 0 to 4 C. and then defatted for 30 min at 37 C with a 2:2:1 (v/v) mixture of ethanol:ether:chloroform. The pellet was then subjected to hydrolysis in 0.3 N KOH for 18 hr at 37 C, acidified to about 0.3 N with perchloric acid, and centrifuged in the cold. The supernatant, containing the RNA nucleotides, was removed, and the residue was washed with cold 0.2 N perchloric acid. The supernatant and washings were then adjusted to pH 8 with KOH, chilled, and centrifuged in the cold. The residue was then washed two times with cold water. The combined supernatants were then placed on a column of Dowex 1 X 8, chloride form, 200 to 400 mesh. The column was washed with 10 mm NaCl, and the nucleotides were eluted with a HCl-NaCl solution (20 ml of 10 N HCl + 5.6 g of NaCl in 240 ml of water). RNA was then estimated with orcinol reagent according to the technique of Lin and Schjeide (17). The DNA was extracted from the KClO, precipitate by hydrolysis at 70 C for 40 min with 0.5 N perchloric acid. After hydrolysis the material was neutralized with KOH, centrifuged to remove KClO,, and the DNA content of the supernatant was determined by the diphenylamine reaction (4).To determine the base composition of the RNA, cultures previously labeled with 32P-orthophosphate were subjected to extraction and hydrolvsis as described above. (Unlabeled RNA 914 www.plantphysiol.org on May 10, 2018 -Published by Downloaded from

Section: Methodsmentioning

confidence: 56%

“…The combined supernatants were then placed on a column of Dowex 1 X 8, chloride form, 200 to 400 mesh. The column was washed with 10 mm NaCl, and the nucleotides were eluted with a HCl-NaCl solution (20 ml [12]). The peak tubes for each nucleotide were combined, and an aliquot was removed for counting.…”

Section: Methodsmentioning

confidence: 99%

Ribonucleic Acid Synthesis Associated with a Developmental Change in the Gametophyte of Pteridium aquilinum

Sobota

1972

“…In the DNA peak isolated from shoots labeled for 16 like RNA) or messenger RNA (5,18). In yeast and mammalian cells this peak has been designated as Q1-RNA; it sediments around 45 S and has been shown to be a precursor for ribosomal RNA (9, 32, 40, 42).…”

Section: Resultsmentioning

confidence: 99%

“…12) or by treatment with SLS, and separated on sucrose gradients. This labeled RNA was polydis- perse (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) 14, without deproteinization to avoid degradation and also to minim-ize loss). The fractions "mRNA" and TB-RNA each contained 41 % of the counts, the rest being in transfer RNA and ribosomal RNA.…”

Section: Resultsmentioning

confidence: 99%

Characterization of Rapidly Labeled Ribonucleic Acid from Dwarf Peas

Johri

Varner

1970

The ribonucleic acid synthesized by excised shoots of dwarf pea (Pisum sativum L. cv. Progress No. 9) during short labeling periods has been characterized. Thirty per cent of the total 32Pi incorporated in 1 hour is found in the ribosomal fraction. This labeled RNA was polydisperse (6-18 Svedberg units) and after chromatography on a methylated albumin-kieselguhr column about 80% of the radioactivity appeared in two peaks. One of these appeared on the shoulder of heavy ribosomal RNA ("mRNA") while the other was tenaciously bound to the column (TB-RNA).In the presence of high NaCl concentration, about half of the polydisperse RNA interacted with ribosomal RNA and eluted as "mRNA" while the remainder eluted as TB-RNA.This interaction in the presence of salt seems to result in the alteration of secondary structure because the "mRNA" fraction had a high sedimentation coefficient (45-50 Svedberg units). The polydisperse RNA approaches DNA in low cytidylate and guanylate content. After short periods of labeling TB-RNA showed higher adenylate content than "mRNA." The radioactivity from the "mRNA" peak can be chased, and these counts may represent a class of shortlived messenger RNA molecules with an average half-life of 10 to 15 minutes. The other component, TB-RNA, could not be chased and accumulated radioactivity during the chase period.In bacteria and viruses the information for the sequence of amino acids in a polypeptide chain is encoded into messenger RNA. Presence of a similar fraction has been suggested in some higher plants (5,6,21,26,39). All of our knowledge about messenger RNA from higher plants is presumptive and based on analogy to microorganisms; in no case has this fraction been purified or characterized, nor has its rate of synthesis or its stability been studied.We chose a dwarf pea as the experimental material because we have been investigating the RNA synthesized by nuclei isolated from shoots of this plant (19), and the knowledge obtained from the system in vivo may be applicable to the results obtained with isolated nuclei. MATERIALS AND METHODS Uptake and Incorporation of Labeled Precursors. Dwarf pea seeds (Pisum sativum L., cv. Progress No. 9) were surfacesterilized for 7 to 10 min in 1% sodium hypochlorite, implanted in 500-ml Erlenmeyer flasks containing moist, sterile vermiculite, and allowed to germinate at 23 C in continuous light. At the end of 5 or 6 days, shoots 0.8 to 1 cm long were excised and 10 or 15 shoots were incubated in 25-ml Erlenmeyer flasks under aseptic conditions with labeled uridine or 32Pj (carrier-free H3u2PO4, neutralized) in 2 ml of water containing 100 ,ug of chloramphenicol. Flasks were transferred to a shaker, and, after incubation for various times, the shoots were washed thoroughly in ice-cold 0.05 M KH2PO4 and deionized water and were then extracted for nucleic acids.In experiments designed to study the relationship between uptake and incorporation, the labeled shoots were homogenized in ethanol, 80% (v/v,) and the nucleic acids were determined (33). The ...

“…Pods were then placed in the dark at 22 C. After POLYPEPTIDES AS PISATIN INDUCERS and treated 15 min later with water. After 30 min the two samples were combined and the RNA was extracted at 60 C by the phenol method (24) and fractionated by linear 5 to 40% sucrose density gradient centrifugation. Centrifugation was performed in a SW-50-L rotor for 18 hr at 32,000 rpm at 0 to 4 C. The ratios were corrected for counting efficiencies and relative counts administered as described previously (38).…”

Section: Methodsmentioning

confidence: 99%

Mode of Pisatin Induction

Hadwiger¹,

Jafri²,

Broembsen³

et al. 1974

Increases in phenylalanine ammonia lyase activity and pisatin synthesis were induced in excised pea pods (a) by basic polypeptides such as protamine, histone, lysozyme, cytochrome c, and ribonuclease; (b) by the polyamines spermine, spermidine, cadaverine, and putrescine, and (c) by the synthetic oligopeptides poly-L-lysine, poly-DL-ornithine, and poly-Poly-L-lysine (1 milligram per milliliter, molecular weight 7,200) was utilized as a model inducer of pisatin and phenylalanine ammonia lyase. The poly-L-lysine-induced responses could be inhibited by adding the RNA synthesis inhibitors cordycepin or a-amanitin to the pods prior to or at the time of inducer application. Cordycepin added 1.5 hours after inducer no longer completely inhibited induction. The application of poly-L-lysine was shown to characteristically change the rate of RNA synthesis within 30 minutes. Ultrastructural changes in pea nuclei were detected within 3 hours, and gross changes in nuclear morphology were apparent at 14 hours after inducer application. The physical appearance of uranyl acetate-stained chromatin isolated from poly-L-lysine 2 hours after inducer application differed from that of water-treated tissues. The template properties of chromatin extracted from pods 3 hours after inducer application were consistently superior to control chromatin when assayed with Escherichia coli RNA polymerase (without sigma factor). Chromatin from poly-L-lysine-induced tissue also bound 49% more actinomycin D-'H.The DNA-complexing properties of inducer compounds and the induced changes in the template and dye-binding properties of pea chromatin formed the basis for a proposed mode of action for phytoalexin induction.The functions of numerous polypeptide hormones (5) have been characterized in animal systems; however, interest in the potential role of polypeptides in regulating plant processes is still limited. There is reason to believe that numerous polypeptide components remain to be discovered which can influence plant cell processes (46). Phytoalexin induction in pea tissue has been employed as one measure of the effects of chemically defined compounds on the regulation of cell processes (12,(15)(16)(17)(18)(19)(20)(21)38). We now report that basic peptides can induce gene-controlled responses, and we demonstrate that this gene-activating potential exists for basic compounds (2, 41, 47) which occur in plants and other higher organisms.The synthetic compound, poly-L-lysine, was chosen as a model inducer because of its amino acid residue uniformity and chemical simplicity. This paper examines the phytoalexininducing effect of poly-L-lysine in relation to (a) alteration of the fine structure of nuclei and cell membranes, (b) template activity and dye-binding capacity of isolated chromatin, (c) the in vivo rate of RNA synthesis, and (d) the dependency of the induction process on RNA and protein synthesis.Our previous hypothesis (20, 38) that regulation of gene expression can occur as a result of changes in the conformation of specific segment...