Ribonucleic acid-deoxyribonucleic acid hybridization in aqueous solutions and in solutions containing formamide.
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ABSTRACT:
SUBMITTER: Gillespie S
PROVIDER: S-EPMC1178082 | biostudies-other | 1971 Nov
REPOSITORIES: biostudies-other
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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: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: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.
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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.
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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.
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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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