The hybridization capacity of ribonucleic acid produced during hormone action.
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ABSTRACT:
SUBMITTER: Wyatt GR
PROVIDER: S-EPMC1186782 | biostudies-other | 1968 Sep
REPOSITORIES: biostudies-other
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Project description:Rat liver nuclei were fractionated into chromatin and nucleolar fractions. Chromatin DNA, which does not form hybrids with rRNA, was, nevertheless, able to hybridize with 32P-labelled total nucleolar RNA. The optimal temperature for this hybridization was 55 degrees C when the reaction was carried out in 2 X SSC (0.3 MnaCl + 0.3 M-sodium citrate). The hybrids formed were specific, as judged by analysis of thermal elution profiles. The low Tm (73 degreesC) observed could be explained by the low amount of DNA in the filters. The lenth of the hybridized sequences was extimated as 54 mucleotide pairs. Contamination to nucleolar RNA by nucleoplasmic RNA was ruled out by showing the former was able to form more hybrids than the latter. Competition experiments showed that hybridization of nucleolar RNA, although not competed with by rRNA, suffered pronounced competition from total microsomal RNA, even though the levels of competition obtained did not equal thsoe with cold nucleolar RNA as competitor.
| S-EPMC1165264 | biostudies-other
Project description:1. The effect on RNA synthesis in rat liver of thyroidectomy and the administration of thyroid hormone, especially during its physiological latent period, was studied by determining: (a) the activity of DNA-dependent RNA polymerase in isolated nuclei; (b) the rate of synthesis of nuclear and cytoplasmic RNA in vivo; (c) polyribosomal sedimentation profiles; (d) the response of microsomes and ribonucleoprotein particles to polyuridylic acid; (e) the effect of inhibitors of RNA and protein synthesis on the biological activity of hormones. 2. The DNA-dependent RNA-polymerase activity of isolated rat-liver nuclei was lowered by thyroidectomy and stimulated by the administration of tri-iodo-l-thyronine or l-thyroxine (2-25mug./100g. body wt.) to both normal and thyroidectomized rats. In thyroidectomized rats, the activity of the Mg(2+)-activated RNA-polymerase reaction (for which the product is mainly ribosomal type of RNA) was stimulated at 10-12hr. after a single injection of tri-iodothyronine, reaching a peak value of 60-90% stimulation at 45hr. after hormone administration. The Mn(2+)/ammonium sulphate-activated RNA-polymerase reaction (for which the RNA product is more DNA-like) was not affected for 24hr. after hormone administration but stimulated by 30-40% at 45hr. The response of both RNA-polymerase reactions to the hormone in vivo paralleled the physiological response but the enzyme was not stimulated by the addition in vitro of the hormone to isolated nuclei. 3. Within 3-4hr. after tri-iodothyronine administration to thyroidectomized rats, the specific activity of rapidly labelled nuclear RNA, after a 10min. pulse of [6-(14)C]orotic acid, was 30-40% greater than the control values, the stimulation reaching 100 and 200% at 11 and 16hr. respectively after hormone administration. Longer exposures to [6-(14)C]orotic acid and [(32)P]phosphate showed that the hormone accelerated the synthesis of mitochondrial, microsomal (or ribosomal) and soluble RNA. The greater part of the labelled nuclear RNA was of the ribosomal type. The hormone-induced increases in the incorporation of radioactive precursors into RNA were not preceded, but followed, by enhanced uptake of the precursor. There was no change, per g. of liver, of DNA, nuclear RNA or soluble RNA, but there was a 40-60% increase in the amount of ribosomal RNA between 35 and 45hr. after a single injection of tri-iodothyronine to thyroidectomized rats. 4. Coinciding with the increase in ribosomal RNA after hormone administration was an increase in the average size and amount of polyribosomes. The newly formed ribonucleoprotein particles, or messenger RNA attached to them, or both, were more firmly bound to microsomal membranes after hormone treatment. 5. Polyuridylic acid caused a bigger stimulation of incorporation of [(14)C]phenyl-alanine by ribonucleoprotein particles, but not by microsomes, from thyroidectomized rats as compared with preparations from normal animals. The response of ribonucleoprotein particles to polyuridylic acid was lowered after tri-iodothyronine treatment of thyroidectomized rats. 6. Actinomycin D, 5-fluorouracil, puromycin and cycloheximide caused a 70-100% inhibition of the stimulatory effect of l-thyroxine and tri-iodo-l-thyronine on basal metabolic rate and growth rate in both normal and thyroidectomized animals. Administration of actinomycin D also abolished the stimulation of RNA polymerase by tri-iodothyronine. 7. It is concluded that regulation of nuclear and ribosomal RNA synthesis is an essential step leading to the biological action of thyroid hormones and that the formation of new ribosomes is an important aspect of the control of cytoplasmic protein synthesis by these hormones.
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Project description:1. Rapidly labelled RNA from Escherichia coli K 12 was characterized by hybridization to denatured E. coli DNA on cellulose nitrate membrane filters. The experiments were designed to show that, if sufficient denatured DNA is offered in a single challenge, practically all the rapidly labelled RNA will hybridize. With the technique employed, 75-80% hybridization efficiency could be obtained as a maximum. Even if an excess of DNA sites were offered, this value could not be improved upon in any single challenge of rapidly labelled RNA with denatured E. coli DNA. 2. It was confirmed that the hybridization technique can separate the rapidly labelled RNA into two fractions. One of these (30% of the total) was efficiently hybridized with the low DNA/RNA ratio (10:1, w/w) used in tests. The other fraction (70% of the total) was hybridized to DNA at low efficiencies with the DNA/RNA ratio 10:1, and was hybridized progressively more effectively as the amount of denatured DNA was increased. A practical maximum of 80% hybridization of all the rapidly labelled RNA was first achieved at a DNA/RNA ratio 210:1 (+/-10:1). This fraction was fully representative of the rapidly labelled RNA with regard to kind and relative amount of materials hybridized. 3. In competition experiments, where additions were made of unlabelled RNA prepared from E. coli DNA, DNA-dependent RNA polymerase (EC 2.7.7.6) and nucleoside 5'-triphosphates, the rapidly labelled RNA fraction hybridized at a low (10:1) DNA/RNA ratio was shown to be competitive with a product from genes other than those responsible for ribosomal RNA synthesis and thus was presumably messenger RNA. At higher DNA/rapidly labelled RNA ratios (200:1), competition with added unlabelled E. coli ribosomal RNA (without messenger RNA contaminants) lowered the hybridization of the rapidly labelled RNA from its 80% maximum to 23%. This proportion of rapidly labelled RNA was not competitive with E. coli ribosomal RNA even when the latter was in large excess. The ribosomal RNA would also not compete with the 23% rapidly labelled RNA bound to DNA at low DNA/RNA ratios. It was thus demonstrated that the major part of E. coli rapidly labelled RNA (70%) is ribosomal RNA, presumably a precursor to the RNA in mature ribosomes. 4. These studies have shown that, when earlier workers used low DNA/RNA ratios (about 10:1) in the assay of messenger RNA in bacterial rapidly labelled RNA, a reasonable estimate of this fraction was achieved. Criticisms that individual messenger RNA species may be synthesized from single DNA sites in E. coli at rates that lead to low efficiencies of messenger RNA binding at low DNA/RNA ratios are refuted. In accordance with earlier results, estimations of the messenger RNA content of E. coli in both rapidly labelled and randomly labelled RNA show that this fraction is 1.8-1.9% of the total RNA. This shows that, if any messenger RNA of relatively long life exists in E. coli, it does not contribute a measurable weight to that of rapidly labelled messenger RNA.
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Project description:Certain features of RNA-DNA hybridization can be accounted for in terms of second-order-reaction theory. These include the use of annealing kinetics to estimate RNA complexity and the occurrence of approximately linear double-reciprocal plots.
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Project description:A general equation is derived describing data of DNA-RNA hybridization in the presence of a competing self-annealing reaction of RNA. The well known double-reciprocal relation and the Scatchard equation are shown to be limiting cases of this general equation. Some new hybridization data at various temperatures are presented and analysed by using the new equation. The results can only be explained if we assume that the behavior of DNA towards single RNA molecules is the same as that towards the annealed form, (RNA12. The variation of the equilibrium constant of the hybridization reaction with temperature is small, indicating a small heat of reaction. The maximum amount of hybridized RNA at equilibrium appears to be independent of temperature.
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Project description:A simple and efficient method for hybridization and subsequent recovery of non-fragmented ribosomal RNA from the hybrid is described. The procedure involves annealing of immobilized denatured DNA bound on cellulose nitrate membrane filters to complementary RNA in 50% (v/v) formamide-0.33m-potassium chloride-10mm-tris-hydrochloric acid buffer, pH7.4, at 33 degrees for 3hr. Under these conditions no detectable changes in the sedimentation coefficients of the input RNA were detected. The RNA can subsequently be recovered quantitatively from the hybrid in intact form by incubating the filters in formamide or in 85% (v/v) dimethyl sulphoxide. The applicability of the method for the evaluation of the absolute size of ribosomal RNA cistrons in Escherichia coli DNA and for the determination of the size of messenger RNA molecules is discussed.
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Project description:Noncoding RNA (ncRNA) is a kind of RNA that plays an important role in many biological processes, diseases, and cancers, while cannot translate into proteins. With the development of next-generation sequence technology, thousands of novel RNAs with long open reading frames (ORFs, longest ORF length > 303 nt) and short ORFs (longest ORF length ? 303 nt) have been discovered in a short time. How to identify ncRNAs more precisely from novel unannotated RNAs is an important step for RNA functional analysis, RNA regulation, etc. However, most previous methods only utilize the information of sequence features. Meanwhile, most of them have focused on long-ORF RNA sequences, but not adapted to short-ORF RNA sequences. In this paper, we propose a new reliable method called NCResNet. NCResNet employs 57 hybrid features of four categories as inputs, including sequence, protein, RNA structure, and RNA physicochemical properties, and introduces feature enhancement and deep feature learning policies in a neural net model to adapt to this problem. The experiments on benchmark datasets of 8 species shows NCResNet has higher accuracy and higher Matthews correlation coefficient (MCC) compared with other state-of-the-art methods. Particularly, on four short-ORF RNA sequence datasets, specifically mouse, Saccharomyces cerevisiae, zebrafish, and cow, NCResNet achieves greater than 10 and 15% improvements over other state-of-the-art methods in terms of accuracy and MCC. Meanwhile, for long-ORF RNA sequence datasets, NCResNet also has better accuracy and MCC than other state-of-the-art methods on most test datasets. Codes and data are available at https://github.com/abcair/NCResNet.
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Project description:RNA (cRNA) was synthesized in vitro on a template of rat liver DNA and its hybridization with rat liver DNA was studied by using the nitrocellulose-filter method. Sonication of the DNA diminished its apparent capacity to hybridize with RNA by about 50%. This is not due to cross-linkage of DNA molecules, because it could be shown that less than 2% of the sonicated DNA was cross-linked. The effect is due instead to the small size of the sonicated DNA molecules. Below a single-stranded molecular weight of 5x10(5) the DNA showed a progressive loss of capacity to hybridize with decrease in molecular weight. Evidence is presented suggesting that the apparently diminished capacity of the DNA to hybridize is due to loss of hybridized DNA from the membrane filters. When cRNA at concentrations of up to 25mug/ml is annealed with sonicated total DNA, an apparent hybridization saturation value is found at which about 2.5% of the DNA is hybridized with RNA. Increasing the cRNA concentration tenfold brought about the hybridization of a second component of the DNA approximately equal in amount to the first. The renaturation of rat liver DNA was studied by measuring the fall in the extinction at 260nm and two different components of renaturation were observed within the reiterated fraction of DNA. By hybridizing cRNA with different fractions of rat DNA the two components of the hybridization curve are shown to correspond to the two components of the renaturation curve. The conclusion is drawn that at a cRNA concentration of 250mug/ml most of the reiterated fraction of rat liver DNA is hybridized after annealing for 16h under standard conditions (0.30m-sodium chloride-30mm-sodium citrate at 65 degrees C). Even with such a high cRNA concentration little or no hybridization of the slowly renaturing DNA fraction occurs. It is suggested that the most highly reiterated DNA component is poorly transcribed in vitro.
| S-EPMC1179592 | biostudies-other
Project description:When RNA is annealed in solution with a sufficiently large excess of DNA, the kinetics of DNA-RNA hybridization are relatively simple. Methods are described for following the course of both DNA renaturation and DNA-RNA hybridization in this system. To explore the characteristics of the reaction a series of model systems was used. Each one utilized DNA (sheared to constant size) from a bacterium or bacteriophage and homologous cRNA, i.e. RNA synthesized in vitro on a template of the same DNA. Temperature optima were determined for the hybridization of Escherichia coli nucleic acids in 2xSSC and 3xSSC-50% formamide buffers, and of Proteus mirabilis nucleic acids in 2xSSC buffer. Rate-constants for DNA-RNA hybridization were measured by two methods. These gave somewhat different results, but in all cases the rate-constant of DNA-RNA hybridization was clearly less than that of DNA renaturation. Thus hybridization is a slower reaction than DNA renaturation. Nevertheless, in some cases, with a high concentration of DNA and a long annealing time, 90-95% of the added RNA became resistant to ribonuclease. Experiments are described which show that it is possible to deduce the analytical complexity of DNA with reasonable accuracy from its hybridization with complementary RNA. Similarly, it is possible to estimate the reiteration frequency of multiple DNA sequences (such as ribosomal DNA) from the hybridization of the total DNA with RNA complementary to the multiple sequences. The effect on the system of various DNA/RNA ratios from 100 to 1 is described.
| S-EPMC1178362 | biostudies-other
Project description:1. When a constant amount of denatured DNA is annealed for a constant time with a series of different RNA concentrations, it is often observed that the reciprocal of the amount of RNA hybridized is linearly proportional to the reciprocal of the RNA concentration. This may be explained by assuming that an equilibrium is set up between free RNA and DNA on the one hand and DNA-RNA hybrid on the other. The hybridization of Escherichia coli DNA and ribosomal RNA was used to test this proposition. Rate constants were estimated from the initial rates of the forward and back reactions and compared with direct estimates of the dissociation constant. 2. The rate constants of the forward and back reactions were estimated to be 1.82mlmug(-1)h(-1) (160lmol(-1)s(-1)) and 0.023h(-1) (6.4x10(-6)s(-1)) respectively, giving a ratio k(2)/k(1)=0.013mugml(-1). After 24h annealing the dissociation constant was estimated to be 0.114mugml(-1), and by extrapolation to infinite time, 0.047mugml(-1). 3. It is concluded that (a) equilibrium greatly favours the hybrid complex, (b) equilibrium is not established in 24h, (c) the equilibria that were directly estimated are incompatible either with the measured rates of the forward and back reactions or with the simple formulation of the reaction that was adopted, and finally (d) for these reasons the equilibrium interpretation of the linear reciprocal relationship is unsatisfactory.
| S-EPMC1185349 | biostudies-other