Isolierung und Charakterisierung schnell markierter RNS von Euglena gracilis mit Hilfe analytischer und präparativer Elektrophorese in Polyacrylamid-Gelen

Zusammenfassung MAK-Säulenchromatographie der Gesamtnucleinsäuren aus autotrophen und gebleichten Zellen von Euglena gracilis, welche für 2 Std mit ³²P-Orthophosphat markiert wurden, liefert 6 Komponenten: niedermolekulare RNS (I–III), DNS (IV) und hochmolekulare RNS (V, VI). Das in der DNS-Region eluierte Material konnte mittels Gelfiltration in ³²P-DNS, in eine ³²P-RNS mit hoher spezifischer Aktivität sowie in ³²P-markierte Polyphosphate aufgetrennt werde. Außerdem fanden sich letztere in der ³²P-RNS-Fraktion, die relativ fest an die MAK-Säule gebunden bleibt. Eine weitaus bessere Auftrennung der einzelnen RNS-Komponenten gelang mit der Elektrophorese in Polyacrylamid-Gelen. So erschienen in 9.5% Gel 5 Komponenten, darunter die 3 niedermolekularen I–III, welche bei MAK-Chromatographie auftreten. Sie wurden als 4 S Transfer-RNS (I), 5 S ribosomale RNS (II) und 6 S RNS (III) identifiziert. Die hochmolekulare RNS wurde bei Auftrennung in 2,6% Gel in 6 Banden zerlegt. Die der ribosomalen RNS fanden sich als Hauptbanden in der 24 S und 20 S Region des Gels. Aufgrund ihrer Position konnten für die übrigen Komponenten Sedimentationskoeffizienten zwischen 18 S und 9 S berechnet werden. Das elektrophoretische Trennmuster der Gesamtnucleinsäuren aus gebleichten Zellen war sehr ähnlich, wenngleich quantitative Unterschiede zwischen den einzelnen Komponenten bestanden. Bei der Fraktionierung der Nucleinsäuren durch Gel-Elektrophorese im präparativen Maßstab fiel für jede markierte RNS-Komponente genügend Material an, um eine Rechromatographie an MAK und die Bestimmung der Basenzusammensetzung durchzuführen. Außer Transfer-RNS und 5 S RNS wurden 2 Komponenten in 9,5% Gel isoliert, deren Zusammensetzung ribosomaler RNS entsprach. Eine weitere niedermolekulare Komponente wurde als die schnell markierte RNS identifiziert, welche gemeinsam mit der DNS von der MAK-Säule eluiert wird. Die präparative Gel-Elektrophorese der ³²P-markierten hochmolekularen RNS in 2,6% Gel lieferte neben mehreren ribosomalen Species auch ³²P-RNS mit einer hohen spezifischen Aktivität.

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Isolation and characterization of rapidly labelled RNA from Euglena gracilis by means

Summary MAK column chromatography of total nucleic acids from autotrophic and bleached cells of Euglena gracilis cultured with ³²P_i for 2 h resulted in the separation of six labelled components: low molecular RNA (I–III), DNA (IV) and high molecular RNA (V, VI). Gel filtration of the material eluted in the DNA region revealed the presence of ³²P-RNA with a high specific activity and of ³²P-labelled polyphosphates in addition to ³²P-DNA. ³²P-polyphosphates were also found among the labelled RNA tenaciously bound to the MAK column. A far better resolution of the RNA components, however, was achieved by polyacrylamide-gel electrophoresis. On a 9.5% gel five main fractions were resolved among which appeared the components I–III isolated by MAK chromatography. They were identified as 4 S transfer RNA (I), 5 S ribosomal RNA (II) and 6 S RNA (III). The high molecular RNA gave rise to six bands when a 2.6% gel was used. From these the ribosomal RNA migrated as two bands in the 24 S and 20 S region of the gel. Based upon these values sedimentation coefficients from 18 S to 9 S were calculated for the others. The electrophoretic pattern of total nucleic acids from bleached cells was rather similar; only quantitative differences were observed. Fractionation of the nucleic acids by polyacrylamide-gel electrophoresis on a preparative scale provided enough material of each labelled RNA component to perform a rechromatography on MAK and to determine the base composition. Besides the 4 S transfer RNA and the 5 S RNA two RNA components with a ribosomal type base composition were isolated on a 9.5% gel. Another one was identified as the rapidly labelled RNA which is eluted with the DNA from the MAK column. Preparative gel electrophoresis of the labelled high molecular RNA (2.6% gel) revealed the presence of several ribosomal species in addition to ³²P-RNA components with a high specific activity.

     

Effect of different conditions of light on the nucleic acid fractions in cotyledons ofChenopodium rubrutn L.

The nucleic acid fractions in cotyledons of young Chenopodium rubrum plants exposed to continuous light, continuous darkness and short (8 h) day, respectively and labelled with³²P 24 h prior to harvesting were studied by means of chromatography on MAK columns. Some parameters of cotyledon growth (dry weight, cotyledon area, occurrence of mitoses) were also investigated. The changes in the nucleic acid fractions agreed with the dynamics of cotyledon growth. In continuous light the content of all fractions increased. The radioactivity of DNA and s-RNA did not undergo any great changes and only r-RNA increased. The specific activity of r-RNA increased slightly, that of soluble RNA and DNA was reduced. In continuous darkness the content of all the fractions did not undergo any great changes. The radioactivity as well as the specific activity of all fractions decreased. In short day the content of the nucleic acid fractions did not change conspicuously. Only the specific activity of s-RNA increased in a noticeable way while the radioactivity of r-RNA and the specific activity of DNA decreased and this of r-RNA did not change. The changes in nucleic acid metabolism were partially connected with changes in³²P uptake which depended upon light conditions but they were not merely a consequence of this fact. Obviously, there also exists a more direct relationship between nucleic acid synthesis and growth.     

Effects of Phosfon-S on Nucleic Acid Metabolism in Pisum sativum Alaska

Phosfon-S, a substance which inhibits stem elongation, alters nucleic acid metabolism in Pisum sativum Alaska. Methylated albumin kieselguhr (MAK) columns were used to fractionate ³²P-labeled nucleic acids. Phosfon-S treatment of the plants resulted in a decrease in soluble RNA and an increase in ribosomal RNA. Specific activities of the various nucleic acid fractions were lower as a result of treatment. The nucleic acids from treated tissues were more resistant to RNase degradation, and endogenous RNase activity was lower in treated tissues. When RNase treated nucleic acids were fractionated on MAK columns, the DNA-RNA fractions from treated plants had a higher specific activity than that of the control, which was not true before nuclease treatment. Spectrophotometric examination of this fraction revealed a difference in absorption spectra, possibly indicating a Phosfon-S nucleic acid complex. It is suggested that these alterations in nucleic acid metabolism could in turn alter a wide variety of metabolic processes, resulting in retarded growth.     

Poly- und Metaphosphate in höheren Pflanzen (Lemnaceae)

Summary After pulse-labelling with [³²P]orthophosphate of higher plants (Lemna perpusilla (Torrey) and Lemna gibba (L.)) which were previously grown in a P-free medium for 24 h, the [³²P]-radioactivity is partially incorporated into condensed inorganic phosphates. The isolation of the latter was achieved by joint extraction with the nucleic acids and fractionation on methylated serum albumin and Kieselgur (MAK). Two-dimensional thin layer chromatography on a mixture of cellulose and microcrystalline cellulose revealed that ³²P-radioactivity was present in linear oligophosphates with 2 to 7 residues as well as in cyclic tri-, tetra-, penta- and hexametaphosphates. Among the low-molecular condensed inorganic phosphates the trimetaphosphate contained significantly more radioactivity than the other compounds. It is supposeded that the condensed phosphates are involved in the regulation of the ATP-ADP-P_i-system.     

EFFECT OF GIBBERELLIC ACID ON ELONGATION AND NUCLEIC ACID SYNTHESIS IN ETIOLATED ALASKA PEA EPICOTYL

The following results were obtained using etiolated Alaska pea epicotyls. Gibberellic acid (GA) had the remarkable effect on the elongation in part I (elongating region) of epicotyls, whereas it had little effect on that in part II (mature region) of epicotyls. In cortex of part I and II of epicotyls, the cell number in longitudinal direction was hardly affected by GA. On the increase in width of epicotyls, GA was hardly effective in any parts of epicotyls. In both part I and II GA enhanced the incorporation of ³²P into all nucleic acid fractions prepared by methylated albumin kieselguhr (MAK) columns, i.e. sRNA, DNA and rRNA + mRNA. In part I the net increase in DNA and RNA content during the incubation period was slightly promoted by GA, whereas in part II the net decrease in both nucleic acids content was slightly promoted by GA. The relationship between GA-induced growth and nucleic acid synthesis is discussed.     

Properties of Single- and Double-stranded Ribonucleic Acid from Barley Plants Infected with Bromegrass Mosaic Virus

Incorporation of ³²P into nucleic acids in barley plants infected with bromegrass mosaic virus (BMV) was analyzed by chromatography on methylated albumin kieselguhr (MAK) columns. Treatment with actinomycin D reduced the synthesis of ribosomal ribonucleic acid (RNA) to low levels and allowed the detection of the three components of BMV-RNA in vivo. The kinetic study on ³²P incorporation into these BMV-RNA components suggested that a single cleavage occurred in some of the intact RNA shortly after completion of its synthesis, giving rise to the small and medium components. Chromatographic analyses also revealed a double-stranded, ribonuclease-resistant RNA which has been purified by differential extraction, sucrose-density gradient centrifugation, and MAK column chromatography. This RNA sediments at approximately 14S, is alkali-labile, and has a sharp thermal transition with a T_m of 96 C in 0.1 × standard saline citrate buffer, as determined by susceptibility to ribonuclease. The RNA is absent in uninfected barley plants.     

Isolierung und Charakterisierung schnell markierter,hochmolekularer RNS aus frei suspendierten Calluszellen der Petersilie (Petroselinum sativum)

Summary By culturing of callus tissue originating from root explants of Petroselinum sativum in a synthetic liquid medium under aeration, freely suspended single cells and small clusters consisting of mostly five cells were obtained. The rapidly dividing cells did not exhibit any morphogenesis. Their nucleic acid metabolism was investigated by pulse experiments with ³²P-orthophosphate. Rapidly labelled RNA was prominently found associated with high molecular RNA. During the fractionation of the total nucleic acids on MAK columns it was eluted after the ribosomal RNA components. Its base ratio, however, differed from the latter in that the AMP content was higher than the GMP content. Sucrose gradient centrifugation and polyacrylamide gel electrophoresis resulted in the separation of the ribosomal RNA from the rapidly labelled RNA, thus proving the higher molecular weight of the latter. Based upon the migration in the gel a sedimentation coefficient of approximately 32S was calculated. The possible function of the heavy rapidly labelled RNA component as precursor of ribosomal RNA is discussed.     

The labelling of nucleic acids by radioactive precursors in the blue-green algae Anacystis nidulans and Synechocystis aquatilis Sanv.

Summary The labelling of nucleic acids of growing cells of the blue-green algae Anacystis nidulans and Synechocystis aquatilis by radioactive precursors has been studies. A. nidulans cells most actively incorporate radioactivity from [2-¹⁴C]uracil into both RNA and DNA, while S. aquatilis cells incorporate most effectively [2-¹⁴C]uracil and [2-¹⁴C]thymine.Deoxyadenosine does not affect incorporation of label from [2-¹⁴C]thymidine into DNA, but weakly inhibits [2-¹⁴C]thymine incorporation into both nucleic acids and significantly suppresses the incorporation of [2-¹⁴C]uracil.The radioactivity from [2-¹⁴C]uracil and [2-¹⁴C]thymine is found in RNA uracil and cytosine and DNA thymine and cytosine. The radioactivity of [2-¹⁴C]thymidine is incorporated into DNA thymine and cytosine. These results and data of comparative studies of nucleic acid labelling by [2-¹⁴C]thymine and [5-methyl-¹⁴C]thymine suggest that the incorporation of thymine and thymidine into nucleic acids of A. nidulans and S. aquatilis is accompanied by demethylation of these precursors. In this respect blue-green algae resemble fungi and certain green algae.     

Changes in Nucleic Acid Fractions of Seed Components of Red Pine (Pinus resinosa Ait.) during Germination

Changes in nucleic acid fractions of Pinus resinosa during seed germination were studied. At various stages of seed germination, embryos and megagametophytes were surgically separated and nucleic acids were extracted separately by a phenol-method. Total nucleic acids were fractionated on single-layer methylated albumin kieselguhr (MAK) columns. Total nucleic acids in embryos increased significantly 2 days after seeds were moistened, whereas, in megagametophytes, total nucleic acids stayed almost at a constant level until they degenerated at the time of shedding. In embryos, ribosomal RNAs (rRNA) increased 2 days after seeds were sown, whereas soluble RNA (sRNA) increased at 3 days. By comparison, nucleic acid fractions of megagametophytes did not show any quantitative changes during germination, except that rRNA fractions decreased shortly before shedding of seed coats. In dormant embryos the proportion of DNA was high and the proportions of sRNA and rRNA were low, whereas in megagametophytes at dormancy the proportions were completely reversed. As seed germination progressed, proportions of nucleic acid fractions in embryos changed significantly. In megagametophytes, although proportions of individual fractions remained almost constant throughout the experimental period, incorporation of ³²P into sRNA and rRNA of megagametophytes indicated turnover of these fractions.     

Germination of Phaseolus vulgaris III. The role of nucleic acid and protein synthesis in the initiation of axis elongation

Excised embryonic axes of Phaseolus vulgaris L. (var. WhiteMarrowfat) begin cell elongation after approximately 4 hr ofincubation at 26°C. The incorporation of ³²P into nucleicacids and phenylalanine-l-¹⁴C into protein markedly increasesduring the 4th hr of incubation, prior to initiation of cellelongation. CH, which inhibits incorporation of phenylalanine-l-¹⁴C intoprotein by 93% during the 2nd hr after its addition, completelyprevents the initiation of axis elongation if added up to 2hr after the beginning of imbibition. Actinomycin D reducesthe fresh weight increase of the axes, and inhibits both ³²Pincorporation into nucleic acids and phenylalanine-l-¹⁴C incorporationinto protein. 5-FU inhibits ³²P incorporation into nucleic acidsbut not phenylalanine-l-¹⁴C incorporation into protein or thefresh weight increase of the axes. MAK column chromatography indicates that actinomycin D inhibitsthe synthesis of all types of nucleic acids to about the sameextent, while 5-FU almost completely inhibits the accumulationof ³²P in ribosomal RNA with lesser but significant inhibitoryeffects on accumulation of ³²P in tRNA. The results suggest an absolute requirement for protein synthesisprior to initiation of cell elongation and at least a partialrequirement for synthesis of nucleic acid species other thanribosomal RNA, tRNA and DNA. The kinetic data suggest that theaxes develop a greatly increased capacity for nucleic acid andprotein synthesis prior to initiation of axis elongation. ¹This research was supported by NSF grant GB 4145 and a grantfrom the U. S. Forest Service. (Received December 16, 1968; )     

Nucleic acid metabolism in cold-treated wheat embryos

The incorporation of ₃₂P into nucleic acid fractions separatedon a MAK column was compared for normally germinated and cold-treatedwheat embryos. ₃₂P accumulation in DNA fraction was decreasedby cold treatment, although that in the RNA fractions was slightlypromoted. The synthesis of the fraction, probably mRNA, elutedafter the peak of heavy rRNA was enhanced in cold-treated embryosand suppressed when the embryos were cold-treated in the presenceof 8-azaguanine, an inhibitor of vernalization. (Received May 2, 1975; )     

A detailed evaluation of the possible contribution of bacteria to radioactive precursor incorporation into nucleic acids of plant tissues

An investigation of the possible contribution of bacteria to the labeling patterns of soybean seedling nucleic acid was made. The results using sucrose gradient, MAK column, and acrylamide gel electrophoretic fractionation together with base composition analyses of nucleic acid preparations show that contaminating bacteria do not contribute to the incorporation of ³²P-orthophosphate into the RNA of excised hypocotyl or soybean root tip. Sterile, non-sterile, and CM-treated soybean hypocotyl synthesize D-RNA to the same extent. The contaminating bacteria do not synthesize an AMP-rich RNA. The G-C rich ³²P-DNA component of the soybean tissues used in these studied results, at least primarily, from the incorporation by contaminating bacteria. CM can be used successfully to eliminate the contribution of bacteria to the labeling of nucleic acids by etiolated plant tissues. Bacterial counts, although valuable, are not sufficient to determine if contaminating bacteria will significantly contribute to nucleic acid labeling in plants.     

Effect of trifluralin on growth, morphology, and nucleic Acid synthesis

Roots and shoots of corn seedlings (Zea mays L. var. Dixie 18) germinated in trifluralin (α,α,α-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine) solutions are characterized by radial enlargement of the cortical cells and by multinucleate cells in the meristematic regions. Trifluralin inhibits elongation of Avena coleoptile sections at concentrations of 0.1 μm to 10 μm. Synthesis of DNA, RNA, and protein is suppressed in the root tips while no significant effect is noticeable in the shoots of corn germinated in trifluralin. A ³²P time-course study of 48, 72, and 96 hours utilizing phenol extraction and MAK column separation of corn root and shoot nucleic acids showed suppression of ³²P incorporation in the treated roots; however, the 72 and 96 hour treated shoots incorporated a much greater amount than the control with most of the increased incorporation found in the sRNA and DNA fractions. The increased activity in the DNA may be due to a high G-C type DNA. No selective suppression or enhancement of any particular RNA species was noticed in the treated plants.     

Incorporation of label from radioactive uridine into the stylar nucleic acids of Lilium longiflorum thunb. as affected by heat, 6-methylpurine and actinomycin D

Summary 5-³H-uridine injected into the stylar canal of detached lily stigma-styles was taken up initially into the rapidly-labeled-RNA of the nucleic acid profile of a methylated albumin kieselguhr (MAK) column but with increasing time was found in all portions of the RNA profile, but not in the DNA. Heat treatment of the style before injection of 5-³H-uridine greatly reduced the rate of incorporation of label into and the ultimate amount of label found in the RNA species of the lily style. Translocation of 5-³H-uridine through the ovary into heattreated pistils and the injection of 5-³H-uridine into styles which had been incubated for 1 or 2 days after heat treatment resulted in stylar nucleic acids more highly labeled than nucleic acids in control styles, with an incorporation pattern different than control styles. Heat treatment of lily pistils resulted in detectable changes in the proportion of stylar RNA species as separated on MAK columns and measured as absorbance units. Actinomycin D and 6-methylpurine treated styles incorporated label from a stylar injection of radioactive uridine in patterns different than each other, different than heat-treated styles and different than non-treated styles. 6-methylpurine and heat treatment of styles only slightly reduced the rate at which 5-³H-uridine was removed from the stylar canal into the stylar tissue.Paper number 8917 of the Scientific Journal Series, Minn. Agr. Exp. Sta., St. Paul, MN 55108.     

Using FAM labeled DNA oligos to do RNA electrophoretic mobility shift assay

The electrophoretic mobility shift assay is a useful tool to identify proteins and nucleic acids interactions. Traditionally, the nucleic acids fragments are end-labeled with ³²P. We present here the use of fluorescent methodologies for studies of RNA in place of radioactivity. The method is highly sensitive and quantitative with the use of an infrared fluorescence imaging system. Fluorescently labeled primers can be used to monitor protein–RNA interactions by fluorescent mobility shift assays. The simplicity and validity of this approach may have more advantages than that of previous methods that traditionally used hazardous radioisotopes. This method was successfully tested in the study of the interactions between MS2 Coat Protein and its RNA target.     

Chromium-51 distribution in tissues and extracts of Leptospermum scoparium

Summary Leptospermum scoparium plants cultured in solutions containing Na₂ ⁵¹CrO₄ accumulated most of the absorbed radioactivity in the roots. About one third of the root radioactivity was soluble in 80% ethanol in the form of three ⁵¹Cr-complexes, the predominant one being identified as trioxalatochromate (III) ion. These complexes were also present in stem and leaf extracts. ⁵¹Cr distribution was examined in various chemical fractions; protein and nucleic acids were especially low in radioactivity.     

Localization of ribosomal cistrons in metaphase chromosomes of Vicia faba (L.)

Tritiated ribosomal RNA (rRNA) was prepared from the roots of Vicia faba after incubation in ³H-uridine. Separation of the nucleic acids by MAK chromatography yielded fractions of specific activity of 4–5 × 10⁵ dpm/μg. 4 + 5S, 18S and 25S RNA fractions were used for cytological hybridization on squash preparations of Vicia faba root tip meristems. Autoradiographs of the 18S and 25S RNA preparations exhibited a clear labelling in the secondary constriction of the satellite (SAT) chromosomes after exposition times of 28 weeks.     

Selektive Hemmung der Synthese der AMP-reichen RNS durch α-Amanitin in Zellen höherer Pflanzen

Summary The effect of -amanitin on the synthesis of AMP-rich RNA has been investigated. After incubation of freely suspended callus cells of parsley with the toxin and pulse labelling (30 min) with ³²P-orthophosphate, the high AMP content of the RNA component eluted from MAK columns behind the 25 S-RNA disappears. The base ratio of this RNA becomes ribosomal (CMP 20.1, AMP 26.5, GMP 28.4, UMP 25.0). Polyacrylamide gel electrophoresis of the high molecular RNA shows that radioactivity is incorporated only into the 32 S-RNA. At higher -amanitin concentrations the total nucleic acid synthesis is reduced. In this case only the high molecular RNA (32 S-RNA) is produced.     

Preparation of DNA and four different RNA species from rat brain. A new RNA fraction and a new characteristic of the various RNA's

A new modification is described for the fractionation of nucleic acids from brain. The cold phenol method followed by a hot extraction was combined with ‘salt-precipitation’ and chromatography on Sephadex and Agarose gels. The method enables the isolation of five highly purified fractions in large amounts: DNA; high molecular weight salt-soluble RNA, a hitherto undescribed fraction; rRNA; residual RNA (mRNA) and sRNA. All fractions showed, after 9 h in vivo incubation with ³²P, different specific radioactivities and/or characteristic base compositions. The single mononucleotides of each RNA fraction differed markedly in their ³²P-labelling rate. This was found to be a new characteristic, for these differences were in no way similar in the various RNAs, but specific and constant for each fraction. An assay of base composition only by ³²P-labelling was therefore not possible.