Incorporation of mevalonic Acid into ribosylzeatin in tobacco callus ribonucleic Acid preparations

The incorporation of ¹⁴C-2-mevalonic acid into transfer RNA and ribosomal RNA (high molecular weight RNA) in rapidly growing, cytokinin-dependent tobacco (Nicotiana tabacum var. Wisconsin No. 38) callus cultures has been investigated. Approximately 40% of the label incorporated into transfer RNA was present in a ribonucleoside with chromatographic properties identical to those of cis-ribosylzeatin. The remainder of the label in the transfer RNA appears to be nonspecific incorporation resulting from degradation and metabolism of ¹⁴C-2-mevalonic acid by the tobacco callus tissue. Although the total radioactivity incorporated into ribosomal RNA was roughly the same as in transfer RNA, the specific radioactivity of the transfer RNA was about four times higher than that of the ribosomal RNA, and the ribosomal RNA labeling could be distinguished from the cytokinin labeling observed in transfer RNA. The distributions of the ¹⁴C-2-mevalonic acid label and cytokinin activity in tobacco callus transfer RNA fractionated by benzoylated diethylaminoethylcellulose chromatography indicate that at least two cytokinin-containing transfer RNA species are present in this tissue.     

Comparison of an Avian Osteopetrosis Virus with an Avian Lymphomatosis Virus by RNA-DNA Hybridization

Myeloblastosis-associated virus (MAV)-2(0), a virus which was derived from avian myeloblastosis virus and induced a high incidence of osteopetrosis, was compared with avian lymphomatosis virus 5938, a recent field isolate which induced a high incidence of lymphomatosis. The following information was obtained. (i) MAV-2(0) induced osteopetrosis, nephroblastoma, and a very low incidence of hepatocellular carcinoma. No difference was seen in the oncogenic spectrum of end point and plaque-purified MAV-2(0). (ii) ¹²⁵I-labeled RNA sequences from MAV-2(0) formed hybrids with DNA extracted from osteopetrotic bone at a rate suggesting five proviral copies per haploid cell genome. The extent of hybridization of MAV-2(0) RNA with DNA from osteopetrotic tissue was more extensive (87%) than was observed in reactions with DNA from uninfected chicken embryos (52%). (iii) Competition of unlabeled viral RNA in hybridization reactions between the radioactive RNA from the two viruses and their respective proviral sequences present in tumor tissues showed that 15 to 20% of the viral sequences detected in these reactions were unshared. In contrast, no differences were detected in competition analyses of RNA sequences from the two viruses detected in DNA of normal chicken cells. (iv) MAV-2(0) 35S RNA was indistinguishable in size from avian lymphomatosis virus 5938 35S RNA by polyacrylamide gel electrophoresis.     

Erratum

RNA polynucleotide kinase has been shown to transfer [γ³²P] from ATP to 5-OH termini of endogenous nuclear RNA. The products of this reaction have been isolated in RNA larger than 125 after in vitro incubation of mouse L cell nuclei. About 20%–30% of these 5′-OH kinase products are polyadenylated. A sizeable fraction of the [γ³²P] label from ATP is also found in internal phosphodiester bonds after 30-minute nuclear incubation in vitro. The possibility of substantial [³²P] recycling via the α position of nucleoside triphosphate was ruled out because: (1) 2mM nucleoside triphosphates in the incubation medium, (2) limited nearestneighbor distribution 3′ and 5′ to the phosphodiester bond compared with that from [α³²P] UTP, (3) different nearest-neighbor distribution for RNA molecules > 12S and 12-3S, (4) relative insensitivity of the [γ³²P] incorporation to α-amanitin as compared with total RNA synthesis, (5) internal [³²P] appearance in RNA > 12S in less than five minutes of incubation, and (6) < 0.03% to 0.6% of the total [³²P] in the α position of nucleoside triphosphates after 30 minutes of incubation. The [γ³²P] incorporation was dependent on high ATP concentration and was insensitive to competition by inorganic phosphate. These results are consistent with the levels of 5′ RNA polynucleotide kinase activity in L cell nuclei and suggest the presence of an RNA ligase that can utilize the termini generated by the 5′-OH RNA kinase in a ligation reaction.     

Characterization of the ribonucleic acid synthesized by isolated rat-liver nuclei

1. Isolated rat-liver nuclei incorporated [¹⁴C]UMP into RNA when incubated in the presence of Mg²⁺ and all four ribonucleoside triphosphates. The addition of bentonite to the system diminished the breakdown of the newly synthesized RNA. 2. AMP and CMP were incorporated in the absence of the other added triphosphates, and in the presence of deoxyribonuclease. 3. RNA synthesized in the presence of Mg²⁺ contained a high proportion of CMP and GMP, and sedimented in the regions of ribosomal RNA and of heavier molecules. About 1% of this RNA hybridized with homologous DNA, and hybrid formation was more effectively inhibited by nuclear RNA than by ribosomal RNA. 4. RNA synthesized in the presence of Mn²⁺ plus ammonium sulphate had a composition intermediate between that of ribosomal RNA and of DNA, and about 4% of this RNA formed hybrids with DNA. 5. Less than 2% of the newly synthesized RNA was capable of forming ribonuclease- and deoxyribonuclease-resistant complexes. 6. It was concluded that the newly synthesized RNA arose as a result of an asymmetric process and included both ribosomal and DNA-like species.     

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.

     

Extraction of High Quality of RNA and Construction of a Suppression Subtractive Hybridization (SSH) Library from Chestnut Rose (Rosa roxburghii Tratt)

Chestnut rose (Rosa roxburghii Tratt) is a rare fruit crop of promising economical importance in fruit and ornamental exploitation in China. Isolation of high quality RNA from chestnut rose is difficult due to its high levels of polyphenols, polysaccharides and other compounds, but a modified CTAB extraction procedure without phenol gave satisfactory results. High concentrations of PVP (2%, w/v), CTAB (2%, w/v) and β-mercaptoethanol (4%, v/v) were used in the extraction buffer to improve RNA quality. The average yield was about 200 μg RNA g⁻¹ fresh leaves. The isolated RNA was of sufficient quality for construction of suppression subtraction hybridization (SSH) library, which allowed the isolation of several pathogen-induced defense genes. Qiang Xu and Xiaopeng Wen - Contribute to this work equally Revisions requested 3 November 2005; Revisions received 18 January 2006     

Sequences coding for proteins expressed in liver and for globin in poly(A)+ and poly(A)− RNA fractions from nuclei and cytoplasm of chicken immature red blood cells

By hybridization with [³H]labeled globin cDNA the contents of globin coding sequences in total nuclear RNA, poly(A)⁺nuclear RNA, poly(A)^--nuclear RNA and polysomal RNA of chicken immature red blood cells was determined to be 0.86%, 20%, 0.42% and 1% respectively. As the poly(A)⁺-fraction comprises only about 2% of total nuclear RNA, globin coding sequences are distributed with 49% in the poly(A)⁺-fraction and with 51% in the poly(A)^--fraction.Part of the mRNA sequences which are found in liver are also transcribed in immature red blood cells. These sequences are enriched in poly(A)⁺-nuclear RNA as the globin coding sequences but their total amount in the poly(A)⁺-fraction is much smaller than in the poly(A)^--fraction.When nuclear RNA from immature red blood cells was translated in an ascites tumor cell-free system, 20% of the newly synthesized proteins were globin chains. The percentage of globin chains in the newly synthesized proteins increased to over 70% when poly(A)⁺-nuclear RNA was translated. Only about 7.5% of globin chains were found in proteins coded by poly(A)^--nuclear RNA.     

SV40 RNA: Filter hybridization for rapid isolation and characterization of rare RNAs

Modifications of the filter hybridization method for the measurement and isolation of simian virus 40 (SV40) RNA from transformed cells are described. The modified method used small (0.02 cm²) nitrocellulose filters with > 30 μg/cm² SV40 DNA applied following formaldehyde denaturation. The small volume and high DNA densities allowed hybridization to be completed in 2 h and washing after hybridization to be completed in 6 h. The washing reduced background to 1 × 10^?5 of input radioactivity without using nucleases. The efficiency of hybridization after washing was 40% or greater. These procedures have allowed the quantification of proportions of SV40 RNA in labeled RNA from transformed lines and the characterization of SV40 RNAs by electrophoresis and cell-free translation. A 3.7-kb SV40 RNA from SV80 cells was discovered in this work.     

Ruminal degradability of 15N labelled ribonucleic acid in grass

The ruminal degradation of RNA in rye grass (Lolium perenne) was studied using the bag method. A non-lactating cow (BW 550?kg) fitted with a rumen cannula was used and fed twice daily at maintenance level with a chopped grass hay-based ration containing 30% ground barley. Rye grass, labelled during growth by fertilization with ¹⁵N₂-urea (9.5 atom% ¹⁵N, 20?g N/m²), was cut at seven stages of growth and maturity and freeze-dried. RNA-N represented 6 to 17% of total N. Labelled grass samples (milled to 5.0?mm screen, 5.0?±?0.1?g DM) were incubated in polyester bags (100?×?200?mm, pore size 50?μm) in the rumen for periods of 1, 3, 6, 9, 12, 24, and 48?h. Data of N and RNA disappearances from the bags were fitted to an exponential equation to estimate parameters of degradation. The effective degradability of RNA in the rumen averaged 90?±?4%, for N it was 11% units lower (P?R ²?=?0.92). Degradability of RNA (R ²?=?0.96) and N (R ²?=?0.93) decreased with increasing fibre content of grass. Increasing the fibre content by 1% diminished the degradability of RNA and N by 1.1% units and 2.4% units, respectively (P????1, a model calculation indicates that about 9 to 19% of duodenal RNA are of dietary origin in animals fed grass. This should be taken into account for the calculation of microbial N on the basis of RNA as marker.     

Ribohomopolymer formation as a source of error in the radioactive precursor incorporation studies of nuclear DNA-dependent RNA synthesis

Isolated rat liver nuclei contain ribohomopolymer polymerases with relative activities in the following order: Poly (A) (100%) > Poly (C) (62%) > Poly (U) (34%) > Poly (G) (13%). Because these enzymes share the same substrates with the nuclear DNA-dependent RNA polymerases in nuclei, labelled precursor is therefore concurrently incorporated into both RNA and ribohomopolymer. Thus, experiments designed to study DNA-dependent RNA synthesis are subjected to error. It is estimated when [¹⁴C]ATP is used as the labelled precursor, the error is as high as 35%; [¹⁴C]CTP, 20%; [¹⁴C]UTP or [¹⁴C]GTP, 10%.     

The cytoplasmic distribution and characterization of poly(A)+RNA in oocytes and embryos of Drosophilia.

Using the presence of poly(A) tracts as a marker for mRNA, we have examined the distribution of this class of RNA between polysomes and free RNP particles. This has been done in mature oocytes and in embryos aged for various times from fertilization through to hatching of a larva. The proportion of ribosomes that are in polysomes to those that are not has been calculated. In mature oocytes, 58% of the poly(A)⁺ RNA and 72% of the ribosomes are not in polysomes. By 1 hr, this drops to 51% of the poly(A)⁺ RNA and 48% of the ribosomes. By 7 hr, a plateau is reached: 30% of each are not in polysomes. The poly(A)⁺ RNA in the cytoplasm of oocytes and 1-hr embryos is found in particles with an average size of 50S and a range of 30–70S. The poly(A)⁺ RNA ranges in size from 7 to 40S, with an average size of 22S. The polyA from this RNA is 50–200 nucleotides long with an average of 115 nucleotides. These data have allowed us to calculate that 1–2% of the total RNA is poly(A)⁺ RNA.     

Nuclear RNA in sea urchin embryos : I. Some characteristics of ribonucleoprotein complexes

Using cesium chloride gradient analysis, we have examined the kinetics of labeling and some physical properties of nuclear ribonucleoprotein complexes in sea urchins. Our results strongly indicate that these complexes are not artifacts of procedure but are definite members of chromatin that are readily distinguishable from cytoplasmic RNP complexes. The nuclear complexes can be separated into two major classes: (1) A class in which the protein to RNA ratio is at least 4:1; (2) a second class in which the protein to RNA ratio is much less. Using [³H]uridine, long-term labeling and short pulses of [³²P], we have studied the labeling kinetics of RNA in the two classes. The nuclear RNA which is associated to a high degree with protein turns over very rapidly; over 70% of the nuclear RNA labeled in a short pulse appears in this fraction, whereas the nuclear RNA synthesized in long-term pulses is present to a greater extent in complexes containing a much smaller proportion of protein. The difference in the rate of turnover of RNA in these two classes may be significant in the regulation of RNA processing in the nucleus.     

Poly(A)+ RNA synthesis in germinating pollen ofNicotiana tabacum L

Poly(A)⁺RNA is synthesized during the first hours of pollen germination and is rapidly incorporated into polysomal structures. After a 2-h pulse with uracil-¹⁴C, 42% of the transcribed fraction of polysomal RNA is polyadenylated. Following 4 h of germination the amount of the newly-made poly(A)⁺RNA decreases steadily at the rate of about 14% per h, whereas that of rapidly-labelled poly(A)⁻RNA continues to grow. Beginning 1 h of cultivation the ratio of poly(A)⁻/poly(A)⁺RNA increases exponentially. Similarly as in non-polyadenylated mRNA the main portion of the synthesized polysomal poly(A)⁺RNA sediments at a rate of 4 to 14 S and its mean size decreases slightly with the time of labelling. RNA isolated from nuclei and cell wall containing pollen tube fraction differed from the polysomal one in higher apeoific radioactivity and the polyadenylated RNA exhibited higher size distribution. The comparison of the results with earlier observations suggests the involvement of poly(A)in mRNA translation in pollen tubes.     

Biosynthesis and stability of globin mRNA in cultured erythroleukemic friend cells

Biosynthesis and stability of the mRNA population in DMSO-induced Friend erythroleukemic cells were studied after labeling the RNA with ³H-uridine and then chasing it with nonlabeled uridine. Globin RNA metabolism was studied by hybridization to excess complementary DNA covalently coupled to oligo(dT)-cellulose. After a labeling period of 120 min, 2–4% of the poly(A)-containing labeled RNA was in globin RNA; it decayed with a half-life of 16–17 hr. The rest of the poly(A)-containing RNA was composed of two kinetic populations: 85–90% decayed with a half-life of about 3 hr, while 10% decayed with a half-life of about 37 hr. The portion of globin RNA in labeled poly(A)-containing RNA behaved in an unexpected fashion during the chase period. During the initial chase period, the percentage of globin RNA increased rapidly, reaching a maximum of about 15% at 20 hr, but if subsequently declined gradually.Based on these findings, a model was built that describes the changes in the proportion of globin mRNA in poly(A)-containing RNA during continuous synthesis and after chase of the labeled RNA. It appears that if the parameters described remain constant during the maturation of erythroblasts, then this model would not account for the almost exclusive presence of globin RNA in the reticulocyte. By far the most effective way to achieve this high level of globin RNA is the destabilization of the mRNA population which is more stable than globin RNA, and not the stabilization of globin RNA itself.     

Polyribosomes and messenger RNA from RAT liver mitochondria

Summary Rat liver mitochondrial polyribosomes were isolated free from cytoplasmic ribonucleoprotein contaminations in a number of criteria (sedimentation and buoyant density patterns, ribosomal RNA composition). Heterogeneous poly A containing RNA from mitochondrial polysomes was purified by two-stage cellulose chromatography. This RNA was in vitro labelled with¹²⁵I up to specific activity ~10⁶–10⁷ cts.min^–1.µg ^–1 and used for hybridization experiments with separate complementary strands of mitochondrial DNA and nuclear DNA fragments. The proportions of mitochondrial poly A containing RNA that is complementary to heavy and light strands of mtDNA were respectively 31.5% and 8.3%. Besides, a significant RNA fraction was complementary to unique sequences of nuclear DNA (2–3 copies per haploid genome). The hybrids that were formed possessed a high T_m indicative of a perfect base pairing. A dual intracellular origin of mitochondrial messenger RNA is discussed.