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.

     

Characterization of rapidly labelled RNA associated with DNA in Chlorella

Summary After pulse-labelling with ³²P-orthophosphate and fractionation of the nucleic acids from synchronously cultured cells of the green alga Chlorella pyrenoidosa on methylated serum albumin and kieselgur (MAK) the DNA contained a species of ³²P-RNA. About 3% of the total ³²P radioactivity incorporated in the cells' RNA were confined to this DNA-associated component. Its base ratio differed significantly from that of soluble and ribosomal RNA but varied only slightly during the life-cycle of the cells. About 4% of the DNA-associated ³²P-RNA resisted ribonuclease digestion suggesting a stable binding of RNA to DNA in the form of a complex. Gel filtration and sucrose gradient centrifugation of the nucleic acids isolated in the DNA region during previous MAK column chromatography resulted in a separation of most of the ³²P-RNA from the DNA. The remaining ribonuclease-resistent but alkali labile ³²P radioactivity bound to the latter was in the order of 4%. No evidence has been obtained so far that it represents rapidly synthesized RNA associated with DNA in stable and functional complex.     

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.     

Metabolism of nucleic acids in wheat roots in dependence on nutritive conditions

Influence on individual types (fractions) of nucleic acids (NA) was studied in roots of wheat plants grown in various cultivation media, namely in distilled water, in sodium humate and in a nutrient solution. NA’s were prepared by means of the phenol technique. Using separation on a methylalbumine column (MAK) five fractions were obtained, namely: fraction I.— low molecular weight substances, fraction II.—soluble RNA, fraction III.—DNA-RNA, and the ribosomal RNA in two fractions, IV.—(l r-RNA) and V.—(h r-RNA). Of the NA fractions investigated, the r-RNA fraction was noticeably influenced by the kind of nutrition, its amount varying in a certain proportion to the growth intensity affected by the cultivation medium. The other NA fractions were not apparently affected. The metabolical turnover of the individual NA types (as observed from the specific³²P activity) was considerably influenced by the kind of nutrition as well. The specific³²P activity in all NA fractions of wheat roots cultivated in a nutrient solution was approximately double that in roots of wheat plants cultivated in distilled water and Na-humate. Changes in the specific³²P activity of r-RNA were again considerably evident. With regard to root growth their relative values appeared in an inverse proportion to the changes in the r-RNA content. The specific³²P activity decreased with increasing growth intensity. Besides changes in the r-RNA fraction, changes in fraction I. were apparent. An especially striking decrease in the specific³²P activity was found in roots of plants grown in H₂O, namely by about one order in comparison with its specific activity in fraction I. from roots of plants grown in Na-humate and in a nutrient solution.     

Changes in Nucleic Acid Metabolism of Excised Barley Leaves During Senescence and the Effect of Kinetin on these Changes

The changes in the amount, rale of synthesis and the nucleotide composition of different RNA fractions in excised barley leaves floated on water or kinetin (10 mg/l) in the dark were examined. In excised leaves floated on water all nucleic acid components declined and these declines were retarded by kinetin. Barley leaves floated on water showed a stimulation of ³²P incorporation into various RNA fractions within 48 hours followed by a decline after 96–144 hours. The leaves floated on kinetin, however, showed an even higher incorporation of ³²P into UNA by 48 hours which remained at a comparatively higher level throughout the experiment. In spite of the above changes in RNA synthesis significant differences in the ³²P sucrose gradient profiles or in the ³²P nucleotide composition of UNA from water and kinetin floated leaves were not noted. The results of this study show that important changes in nucleic acid metabolism occur during the early stages of leaf senescence and that alterations in nucleic acid metabolism during senescence and during kinetin treatment may involve quantitative and only subtle qualitative changes.     

The identification of a heat-shock protein complex in chloroplasts of barley leaves

In vivo radiolabeling of chloroplast proteins in barley (Hordeum vulgare L. cv Corvette) leaves and their separation by one-dimensional electrophoresis revealed at least seven heat-shock proteins between 24 and 94 kD, of which most have not been previously identified in this C₃ species. Fractionation into stromal and thylakoid membrane components showed that all chloroplast heat-shock proteins were synthesized on cytoplasmic ribosomes, translocated into the chloroplast, and located in the stroma. Examination of stromal preparations by native (nondissociating) polyacrylamide gel electrophoresis revealed the presence of a high-molecular mass heat-shock protein complex in barley. This complex was estimated to be 250 to 265 kD in size. Dissociation by denaturing polyacrylamide gel electrophoresis revealed a single protein component, a 32-kD heat-shock protein. The synthesis of this protein and the formation of the heat-shock protein complex were dependent on functional cytoplasmic ribosomes. Immunological studies showed that the heat-shock protein complex did not contain any proteins homologous to the α-subunit of ribulose bisphosphate carboxylase oxygenase subunit-binding protein. Other features about the complex included the absence of nucleic acid (RNA or DNA) and its nondissociation in the presence of Mg²⁺/ATP. These results suggest that the heat-shock protein complex in barley chloroplasts is a homogeneous octamer of 32-kD subunits.     

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.     

Effects of decenylsuccinic Acid on p uptake and translocation by barley and winter wheat

After 3 days of exposure to 10⁻³ and 10⁻⁴ M decenylsuccinic acid, winter wheat plants wilted and died. Decenylsuccinate at 10⁻³ M inhibited ³²P uptake by barley roots and wheat roots and resulted in significant (P ≤ 0.05) leakage of previously absorbed ³²P and total phosphorus (barley roots). Decenylsuccinate effects on ³²P uptake and retention were attributed to increased permeability resulting from injury. Decenylsuccinate at 10⁻⁴ M did not inhibit root uptake of ³²P but decreased movement into the shoot. This could be interpreted as an indication of reduced transpiration or inhibition of ³²P loading into the transpiration stream. Decenylsuccinate did not increase cold hardiness in winter wheat in a nonhardening environment.     

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; )     

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.     

RNA synthesis in germinating red bean seeds

Nucleic acids from ³²P-labelled germinating red bean seeds wereinvestigated by means of MAK column chromatography. 1) In cotyledons,synthesis of D-RNA occurred in the early stages of germination,3 to 24 hr after the onset of imbibition. ³²P was also incorporatedinto rRNA continuously at rather a moderate rate. DNA-RNA hybridizationexperiments revealed that the proportion of heterogeneous RNA(D-RNA) to rRNA decreased gradually. Nucleotide analysis suggestedthat tRNA was synthesized de novo, and that its CCA-end exchangewas remarkable at early stages of germination. 2) In embryos,however, the incorporation of ³²P into rRNA was very much greaterthan into D-RNA, and the exchange reaction at CCA-end of tRNAwas not detected. The role of D-RNA, found in cotyledons inthe initial stages of germination, was discussed. ¹Present address: Research Institute for Biochemical Regulation,Faculty of Agriculture, Nagoya University, Chikusa-ku, Nagoya,Japan. (Received May 10, 1972; )     

Changes in the transcription pattern of flax cotyledons after inoculation with flax rust

The rate of ³²P incorporation into RNA fractions of flax cotyledons (Linum usitatissimum L. var. Bison) was found to increase two- to three-fold by 48h after inoculation with flax rust [Melampsora lini (Pers.) Lev., race no. 3]. This was accompanied by a change in the nucleotide composition of the newly transcribed sodium chloride-soluble RNA fraction. A comparison of the nucleotide composition of the RNA synthesized in the host–parasite complex at different stages of development indicated the preferential synthesis of one or more molecular species of RNA with a high A+U/G+C ratio at a relatively early stage of infection. Treatment of healthy plants with indol-3-ylacetic acid also resulted in a substantial stimulation in the rate of ³²P incorporation into RNA but this was not accompanied by a detectable change in the nucleotide ratios of the newly synthesized RNA. These results suggest that the synthesis of one of more additional RNA species or the augmented synthesis of certain species of RNA may be a specific phenomenon elicited by host–pathogen interaction.     

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.     

Plasmids controlling exclusion of the K2 killer double-stranded RNA plasmid of yeast

Saccharomyces strains of two types (K₁⁺R₁⁺ and K₂⁺R₂⁺) kill each other and K^?R^?-sensitive strains by secreting protein toxins. K₁ killer strains carry a 1.25 × 10⁶ dalton double-stranded RNA plasmid, [KIL-k₁], while K₂ killers have a 1.0 × 10⁶ dalton double-stranded RNA plasmid, [KIL-k₁]. Mating [KIL-k₁] haploids with [KIL-k₂] haploids yields only [KIL-k₁] diploids, that is, [KIL-k₁] excludes [KIL-k₂]. [EXL], a new non-Mendellan genetic element from a nonkiller strain, excludes [KIL-k₂] but does not exclude [KIL-k₂]. A second new non-Mendelian genetic element, called [NEX], when present prevents [EXL] from excluding [KIL-k₂]. [NEX] does not prevent [KIL-k₁] or [KIL-s₁] (a suppressive mutant of [KIL-k₁]) from excluding [KIL-k₂]. A chromosomal gene, called MKT1, is needed for maintenance of [KIL-k₂] if [NEX] is present. In the absence of [NEX], [KIL-k₂] does not need MKT1. [KIL-k₁] does not need MKT1 even if [NEX] is present. [EXL] replication depends on at least the products of MAK1, MAK3, MAK10and PET18. [NEX]replication depends on MAK3 but is independent of MAK4, MAKE, MAK27 and MKT1.     

On the nature of RNA synthesized in pollen tubes ofNicotiana alata

The RNA formed in pollen tubes during 4 hours of growthin vitro was resolved by chromatography on methylated albumine on kieselguhr (MAK) into three principal fractions. Acoording to the labelling from uracil-¹⁴C about 11% was eluted with tRNA and 5 S RNA (low molecular weight RNA), 76% just after rRNA (D-RNA) and nearly 14% was recovered from the column by SDS at 35 °C (TB-RNA). In the presence of actinomycin D at concentration of 30 μg ml^-1 the synthesis of the three classes of RNA was inhibited by 71%, 97% and 70% respectively. On sucrose density gradient the radioactive low molecular weight RNA sedimented at 4 S-5 S which suggests that one or both of these RNA species are synthesized in pollen tubes. The D-RNA eluted from the MAK column is polydisperse in size exhibiting a wide range of sedimentation values up to about 35 S with a large peak at 9 S-10 S and two smaller peaks at 14 S-15 S and at about 23 S. The rapid labelling and the polydisperse rather low molecular weight character suggest that the D-RNA is a heterogeneous population of mRNA. The sedimentation profile of TB-RNA was similar to that of D-RNA. The RNA synthesized in the presence of³²PBO3-₄ or uracil-¹⁴C exhibited no radioactivity peaks corresponding to sedimentation peaks of rRNA.     

Biochemical evidence of DNA transport from the silk gland to the fat body of the silkworm, Bombyx mori

β-DNA, a component of DNA found in the pupal fat body of the silkworm, Bombyx mori, has the same GC content but a smaller molecular weight than typical silkworm DNA (α-DNA). Its origin and time of synthesis were studied by MAK column chromatography of phenol extracts after labelling with radioactive precursors.The DNA components of the fat body changed greatly during the early pupal stage, the β-DNA showing a striking increase relative to α-DNA. Thymidine-6-³H and phosphoric acid-³²P injected into the animals 1 day before analysis caused labelling of α-DNA, but not of β-DNA of the fat body, indicating that β-DNA was not synthesized during the stage of its appearance in the fat body.On the other hand, injection of thymidine-6-³H into 2-day-old fifth instar larvae, when DNA of the silk gland was being actively synthesized, gave high incorporation of the isotope into β-DNA of the pupal fat body. The sudden appearance of highly labelled β-DNA in the fat body during the early pupal stage as well as the occurrence of β-DNA in both the silk gland and fat body suggested that DNA might move from the silk gland to the fat body.It is possible that the fat body stores DNA as a nutrient from the degenerating silk gland.     

Incorporation of cytokinin N-benzyladenine into tobacco callus transfer ribonucleic Acid and ribosomal ribonucleic Acid preparations

The incorporation of the cytokinin N⁶-benzyladenine into tobacco (Nicotiana tabacum) callus tRNA and rRNA preparations isolated from tissue grown on medium containing either N⁶-benzyladenine-8-¹⁴C or N⁶-benzyladenine-8-¹⁴C: benzene-³H(G) has been examined. N⁶-benzyladenine was incorporated into both the tRNA and rRNA preparations as the intact base. Over 90% of the radioactive N⁶-benzyladenosine recovered from the RNA preparations was associated with the rRNA. Purification of the crude rRNA by either MAK chromatography or Sephadex G-200 gel filtration had no effect on the N⁶-benzyladenosine content of the RNA preparation. The distribution of N⁶-benzyladenosine moieties in tobacco callus tRNA fractionated by BD-cellulose chromatography did not correspond to the distribution of ribosylzeatin activity. N⁶-benzyladenosine was released from the rRNA preparation by treatment with venom phosphodiesterase and phosphatase, ribonuclease T₂ and phosphatase, or ribonuclease T₂ and a 3′-nucleotidase. N⁶-benzyladenosine was not released from the RNA preparation by treatment with either ribonuclease T₂ or phosphatase alone or by successive treatment with ribonuclease T₂ and a 5′-nucleotidase. Brief treatment of the rRNA preparation with ribonuclease T₁ and pancreatic ribonuclease converted the N⁶-benzyladenosine moieties into an ethyl alcohol soluble form. On the basis of these and earlier results, the N⁶-benzyladenosine recovered from the tobacco callus RNA preparations appears to be present as a constituent of RNA and not as a nonpolynucleotide contaminant.     

Abscission: the role of RNA synthesis

Ethylene stimulated the incorporation of ³²P into RNA in the abscission zone of bean explants (Phaseolus vulgaris L. var. Red Kidney). The enhancement was observed in all fractions separated by methylated albumin kieselguhr column chromatography, although the magnitude of the increase was not the same for each fraction. Differential extraction of the nucleic acids indicated that the ethylene stimulation was confined to the fraction extracted with sodium lauryl sulfate, with the increase mainly in Fraction III (Ribosomal RNA) and Fraction IV (Messenger RNA). Actinomycin D, which blocks ethylene-stimulated abscission, inhibited ³²P incorporation into all column fractions. 5-Fluorouracil, which blocked 50% of the ethylene-enhanced ³²P incorporation, did not inhibit ethylene-enhanced abscission. The results indicate that ethylene may regulate abscission through control of specific RNA's.