Gel electrophoretic analysis of poly(riboadenylic acid)

It is shown that molecular weights and molecular-weight distributions of poly(rA), and by implication other single-stranded polynucleotides, and synthetic and natural polyelectrolytes in general, can be determined by electrophoresis in polyacrylamide gels. It is shown that fractions of very narrow molecular-weight distribution can be obtained by preparative electrophoresis of polydisperse samples. Molecular-weight calibrations based on sedimentation coefficients of such fractions are given, and in aqueous systems do not coincide with calibrations for partially base-paired RNA species. Poly(rU) fractions fall on the same calibration as poly(rA). Relations between mobilities, relative to standard markers, and molecular weight for poly(rA) over a wide range of molecular weights are given, which allow rapid molecular-weight determination on poly(rA) samples, such as the segments found in many types of messenger RNA.     

Translation of messenger RNA fractions extracted from free and membrane bound rat forebrain ribosomes in a rabbit reticulocyte cell-free system

Messenger RNA fractions were obtained from free and membrane bound rat forebrain ribosomes by alkaline phenol extractions. These RNA fractions stimulated protein synthesis in a cell-free rabbit reticulocyte system partially dependent on the addition of exogenous mRNA. The polypeptide products of protein synthesis with RNA fractions derived from free and membrane bound brain ribosomes and reticulocyte ribosomes were compared by polyacrylamide gel electrophoresis and found to have different distributions.     

Identification of rat brain polysomes synthesizing the brain specific enolase (14.3.2 protein), S100 protein and alpha and beta tubulin subunits

Polysomes prepared from frozen rat brain powder were fractionated by centrifugation in a sucrose gradient. Individual fractions were used to program a reticulocyte lysate in a run-off reaction. The products of cell-free synthesis were assayed for the brain-specific enolase (14.3.2 protein) and S100 protein by immunoprecipitation with specific antisera and for tubulin by two-dimensional electrophoresis in polyacrylamide slab gels. The relative synthesis of these proteins by unfractionated free brain polysomes were 0.1 per cent, 0.05 per cent and 0.7 per cent respectively. After centrifugation in a sucrose gradient polysomes synthesizing S100 protein were separated from those synthesizing the other two markers. There was a threefold enrichment in the specific messenger RNA activity for each of the three proteins studied in their respective peak fractions of polysomes.     

Extraction of total RNA from leaves of Eucalyptus and other woody and herbaceous plants using sodium isoascorbate

Rapid extraction of total RNA from Eucalyptus leaves is difficult due to the high content of polyphenolics and polysaccharides. A rapid and simple method was developed by using an extraction buffer containing sodium isoascorbate at a concentration of 500 mM. This method consisted of one or two chloroform extractions, one acid guanidium-phenol-chloroform extraction, and isopropanol precipitation alone. The yields of the RNA fractions were 246-1750 micrograms/g fresh weight when leaves of Eucalyptus, five other woody plants, and four herbaceous plants were used as samples. The contamination of the RNA fractions by proteins and polysaccharides was very limited as judged spectrophotometrically. When the RNA fractions were subjected to agarose gel electrophoresis, intact rRNA bands were detected. The RNA fractions could be used for RT-PCR. These results indicate that our new method achieves a simple and rapid preparation of high-quality RNA from leaves of Eucalyptus and other plant species.     

Intercellular information transfer in the immunogenesis process. VIII. Analytical separation on agar and polyacrylamide gel of RNA fractions from the spleens of immunized and intact rats

RNA isolated from the spleens of intact rats and from rats with immunized sheep red cells was fractionated through three steps: 1 - extraction from phenol nuclei at 50-55 degrees and 65-75 degrees C, 2 - calcium-phosphate chromatography, 3 - agar electrophoresis. Eight agar fractions were obtained of the spleens of immunized rats, an increased RNA content was manifested in at least three agar fractions: the first (4 S), the third (21 S) and the eighth (26 S) ones. The first and the eighth immune RNA fractions, as it was shown earlier, induce the synthesis of antibodies in the rat transplantable lymphosarcoma cell. The first agar fraction of nuclear RNA from the spleens of immunized and intact rats were additionally separated using PAAG electrophoresis. The 4 S agar RNA fraction appears to be rather heterogeneous. It contains 4 S, 4.5 S, 5 S, 5.8 S, U1, U2 and 8 SII fractions, which are low-molecular nuclear RNAs, the 4 S subfraction prevailing. It is suggested that the 4 S PAAG subfraction is most active in the synthesis of antibodies induced by the heterogeneous agar 4 S RNA.     

Monitoring differential synthesis of RNA in individual cells by capillary electrophoresis

A capillary electrophoresis (CE)-based technique is reported here to monitor differential RNA synthesis in individual Chinese hamster ovary cells at distinct stages of the cell proliferation cycle. Cell synchronization was achieved by the shake-off method, in which mitotic (M) cells were dislodged, and cells at G(1), S, and G(2) phases were harvested 2.5, 10, and 13 h, respectively, after synchronizing the mitotic cells. Thirty-two cells (eight from each phase) were analyzed by injecting each cell into the capillary, lysing it with dilute surfactant, separating the RNA by capillary electrophoresis, and detecting the peaks with laser-induced fluorescence. The results from single cells show that the total amount of RNA increased at each successive stage (from G(1) to M), while the relative synthetic rates of different RNA fractions varied with progression through the cycle. There was a threefold increase in the synthetic rate of total RNA from S to G(2), compared with G(1) to S. In addition, differential accumulation of specific RNA fractions was observed, with the low-molecular-mass fraction exhibiting a much higher synthetic rate from G(2) to M, relative to the rates of the larger ribosomal RNA (rRNA) fractions. Comparison of the large rRNA fractions with one another reveals that at S phase more 28S rRNA was accumulated than 18S rRNA, and at G(1) and M phases, the synthetic rate of 28S rRNA was slowed compared with that of 18S. Minimal sample preparation, combined with the separation power of CE and single-cell detection sensitivity of laser-induced fluorescence, results in a simple method for assessing differential accumulation of RNA from distinct individual cells.     

Incorporation of labelled precursors into the electrophoretic fractions of rat-liver ribonucleic acid

1. Incorporation of [(32)P]orthophosphate and of [2-(14)C]orotic acid into rat-liver RNA was studied by agar-gel electrophoresis by using u.v.-densitometry and radioautography of dried agar electrophoretograms. 2. During the electrophoresis some low-molecular-weight contaminants, including inorganic phosphate present in the RNA preparations, were separated from the RNA fractions. Since nucleoside mono-, di- and tri-phosphates still interfered, the RNA preparations had to be subjected to a purification procedure [Sephadex G-25 or Dowex 1 (X8)]. 3. In RNA extracted from cytoplasm, isolated microsomes or ribosomes, whatever variations were made in the phenol procedure no special rapidly labelled RNA fraction was detected other than ;soluble' RNA and the ribosomal RNA components. 4. When the whole homogenate or cytoplasmic fraction was treated only with phenol (pH6) a considerable part of the cytoplasmic RNA was not extracted. The treatment of the cytoplasmic fraction with sodium dodecyl sulphate before the addition of phenol increased the yield of the high-molecular-weight RNA and at the same time a higher specific activity was found for the faster ribosomal RNA component. 5. The presence of four distinct rapidly labelled RNA fractions was established in the RNA not extracted by phenol, and they moved slower than the ribosomal RNA. They were extracted only with the use of phenol-sodium dodecyl sulphate at an elevated temperature.     

Structure and function of rat liver polysome populations. II. Characterization of polyadenylate-containing mRNA associated with subpopulations of membrane-bound particles

Poly(A)+RNA fractions prepared from free and loosely and tightly membrane-bound polysome populations (poly(A)+RNAfree, poly(A)+RNAloose, and poly(A)+RNAtight) were used to drive cDNA in homologous and heterologous hybridization reactions. A large fraction by mass of sequences was shared among the three poly(A)+RNA populations, but shared sequences exhibited distinct frequency distributions within the different populations. 13-15 in vitro translation products of poly(A)+RNAfree and poly(A)+RNAloose detected by gel electrophoresis were shared. Most of these were produced in different relative quantities by the two RNA populations. Five or six higher mol wt polypeptides were produced by poly(A)+RNAloose that were not detected as products of either poly(A)+free or poly(A)+RNAtight. We suggest that loosely bound polysomes may not be artifactually derived as reflected in their quantitatively distinct poly(A)+RNA population. Two tightly membrane-bound RNP fractions were prepared from rat liver on the basis of their release from or retention on purified rough microsomes or a crude membrane fraction after in vitro disaggregation of polysomes with high-salt and puromycin. Homologous and heterologous hybridizations involving their poly(A)+RNA fractions revealed that a large portion by mass of sequences was shared but that these sequences exhibited distinct frequency distributions in the two fractions. The RNA fractions produced exhibited distinct frequency distributions in the two fractions. The RNA fractions produced an identical set of in vitro translation products but individual polypeptides were produced in different relative quantities. This indicates that the two RNP fractions do not arise by any random artifactual process and suggests that they may represent functionally distinct populations.     

A new method for the isolation of undegraded nuclear and cytoplasmic RNA from liver of Xenopus larvae

In order to obtain undegraded RNA from subcellular components a technique for tissue fractionation at ?20°C was developed by using 50% glycerol in the homogenization medium. RNA extracted from nuclear and cytoplasmic fractions, isolated by this method, revealed no signs of degradation after electrophoresis on exponential gels.     

Cell free synthesis of some storage protein subunits by polyribosomes and RNA isolated from developing seeds of pea (Pisum sativum L.)

Polyribosomes which have template activity in the wheat germ system have been isolated from developing pea seeds. Some of the translation products have identical mobilities to the vicilin and legumin subunits by SDS-PAGE. Certain products were specifically immunoprecipitated with antisera prepared against purified vicilin and legumin fractions. Various RNA fractions including poly A-rich RNA have also been isolated from polyribosomes and shown to direct the synthesis of polyripeptides whose properties are similar to the storage protein subunits. The results are discussed in relationship to other investigations with seed storage protein biosynthesis in vitro.Abbreviations DTT dithiothreitol - SDS-PAGE SDS-polyacrylamide gel electrophoresis - TCA tricarboxylic acid     

Two-dimensional gel electrophoretic separation of the proteins present in chromatin of Escherichia coli

The polypeptides present in 35S-labelled chromatin prepared from Escherichia coli cells, and polypeptides present in the DNA and RNA complexes obtained by micrococcal nuclease digestion of the chromatin, were analysed by two-dimensional non-equilibrium polyacrylamide gel electrophoresis. Three hundred and thirty-five 35S-labelled polypeptides were detected in the chromatin whereas the DNA- and RNA-containing fractions of the micrococcal nuclease digest contained 126 and 183 polypeptides respectively. The major basic low-molecular-weight polypeptides were found in the DNA-containing fractions.     

比格犬雌激素β受体RNA干扰实验细胞及PCR检测引物的筛选

目的 筛选用于比格犬ERβ基因RNA干扰实验的细胞和检测所用引物.方法 根据比格犬ERβ氨基末端区域和DNA结合区设计三对引物,用阳性质粒筛选最佳引物应用于RNA干扰效果检测,并利用293T、Hela和vero细胞的cDNA验证该引物的特异性.对这三种细胞内人ERβ基因的存在情况也进行了检测.结果 经过三次重复性实验之后,结果显示引物C36684扩增效率高,且没有非特异条带,该引物对293T、Hela和vero细胞cDNA扩增时同样没有非特异性条带,但293T细胞中存在人的ERβ基因.结论 引物C36684用于检测RNA干扰效率,而Hela细胞则作为RNA干扰实验的细胞工具.     

Concentration of proteins using carboxymethyldextran

Carboxymethyldextran was used to displace several proteins from DEAE-cellulose in highly concentrated form, and the concentrated protein fractions were examined directly by gel electrophoresis. The concentration of DNA-dependent RNA polymerase II demonstrates the application of the method to a labile enzyme. The concentration of goat γ-globulin by sharp elution from CM-cellulose with carboxymethyldextran is also described.     

Preparation, purification and analyses of thirteen alkali-stable dinucleotides from ribonucleic acid

Of the 16 alkali-stable dinucleotides known to be obtained by hydrolysis of commercial yeast RNA with alkali, 13 were prepared in quantities of the order of 10mg or more. The samples, with only one exception, contain at least 90% of dinucleotide, and spectroscopic constants and nucleotide-sequence determinations, although not conclusive, indicate a high degree of purity of these products. The small dinucleotide fraction in 150g of RNA hydrolysed with alkali (1-2% of the total nucleotides) was separated from the mononucleotides by stepwise ion-exchange chromatography on DEAE-cellulose columns and resolved into seven fractions containing from one to four different dinucleotides by electrophoresis on paper at pH3.0. These fractions were resolved into their constituent dinucleotides by chromatography in ammonium sulphate. Contamination of the products by impurities from the paper was minimized by washing it before using it for chromatography or electrophoresis and, by using a thick grade of paper (Whatman no. 17), it was possible to handle and purify relatively large quantities of nucleotides.     

Hymenolepis diminuta: Action of worm RNase against RNA

Hymenolepis diminuta possesses a tegumental ribonuclease (RNase) which hydrolyzes rat liver and degraded yeast RNA. Polyacrylamide gel electrophoresis and gel chromatography of rat liver RNA after incubation with intact worms demonstrated significant hydrolysis of the high molecular weight RNA fractions (28 S and 18 S), with the appearance of fractions of intermediate molecular weight (i.e., between 18 S and 4 S), as well as ethanol-soluble fractions. Hydrolysis of degraded yeast RNA (with a molecular weight of approximately 25,000) yielded a single ethanol-precipitable hydrolysis product, as well as ethanol-soluble hydrolysis products.     

Distinct oligonucleotide patterns of distinct influenza virus RNA's

Three molecular-weight fractions of influenza virus RNA (SS₁ SS₂ and SS₃) were prepared as described previously (Content &; Duesberg, 1971) and subjected to digestion with RNase T₁. Two-dimensional analyses of the digests by electrophoresis and homochromatography led to distinctive oligonucleotide patterns for each fraction of viral RNA. Almost all physically distinguishable large oligo-nucleotides of a given RNA species had a distinct base composition. It was concluded that each of the three fractions of viral RNA investigated contained different nucleotide sequences and presumably different genetic information.     

Synthesis of RNA in isolated polytene nuclei from salivary gland cells of Chironomus thummi

The incorporation of [³H]UTP into RNA by isolated polytene salivary gland nuclei of Chironomus thummi was investigated under different incubation conditions; the labeled RNA fractions were characterized by electrophoresis. The results suggested that at two characteristic ionic conditions most of the RNA synthesized was the product of RNA polymerase I or RNA polymerase II as distinguished by their differential sensitivities to α-amanitin. Electrophoretical analysis of the RNA synthesized under conditions favouring polymerase I showed that this RNA population consisted mainly of four distinct molecular weight fractions within a range between 2.8 × 10⁴ and 2.5 × 10⁶. Under conditions favouring polymerase II two fractions were detected: one with a broad molecular weight distribution around 0.4 × 10⁶ containing considerable amounts of poly(A)-bearing RNA molecules, and a second with a peak at a molecular weight of 2.8 × 10⁴.     

Separation and characterization of low-molecular-weight nuclear RNA species that inhibit cell-free protein synthesis

Various RNA fractions were isolated from nuclei of 12-day lactating rat mammary glands and examined for their ability to inhibit cell-free protein synthesis. Although total nuclear RNA was generally inactive, material contained in the poly(A)+ nuclear RNA fraction and the low-molecular-weight RNA derived from total nuclear RNA by sucrose gradient centrifugation, inhibited the translation of several mRNAs but not poly(U) or poly(A). Separation of the small nuclear RNAs by preparative polyacrylamide-urea gel electrophoresis allowed the identification of at least three active inhibitor RNA species. These differed both with respect to their ability to inhibit protein synthesis, and in their mechanism of action. While two of the RNA species inhibited elongation the other inhibited initiation of protein synthesis.