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The effect of reaction with formaldehyde on the sedimentation rates of ribonucleic acids

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Abstract

It has been reported that the RNA of several bacteriophages and that of the larger ribosomal sub-units of mammalian cells sediment faster in the presence of 0.1m-sodium chloride than is expected from their estimated molecular weights. The effect of blocking the hydrogen-bonding amino groups of these and other types of RNA was studied. The RNA of phage R17 no longer sedimented anomalously fast after treatment with formaldehyde. In contrast, the larger ribosomal RNA of HeLa cells appeared more aberrant than before, sedimenting faster than tobacco-mosaic-virus RNA (mol.wt. 2x10(6)) in the presence of formaldehyde. The rapidly labelled nuclear 45s RNA of HeLa cells still sedimented faster than the larger ribosomal RNA after reaction with formaldehyde, showing no evidence of disaggregation. It is suggested that both the large ribosomal RNA and the 45s RNA of HeLa cells may have a non-linear structure.

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... Physical and biological properties. The (85,158,224), analytical ultracentrifugation (69), and electrophoresis on polyacrylamide or polyacrylamide-agarose gels in the presence of a variety of denaturants to minimize the effects of secondary structure on mobility (10,130). More recently, Lee et al. (127) used nucleolytic digestion (with RNase T1) of 32P-labeled RNA in conjunction with two-dimensional fingerprint analysis to estimate the molecular weights of the RNAs isolated from poliovirus types 1 and 2. Given this impressive array of experimental approaches, it seems safe to assign a molecular weight of 2.6 x 106 ± 0.2 x 106 to picornavirion RNA. ...
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Heterogeneous nuclear RNA was extracted from sea urchin gastrulae following a ten-minute pulse with [3H]uridine. Hybridization reactions with excess sheared DNA were carried out using several methods. The fraction of the Hn[3H]RNA† in hybrids was determined by RNAase resistance and hydroxyapatite chromatography in a urea-phosphate medium. After incubation to a DNA C0t value of 40, 20 to 25% of the Hn[3H]RNA binds to hydroxyapatite. Approximately one third of these bound molecules are in repetitive DNA-RNA hybrids. The size of the bound RNA molecules measured after hybridization averages 1100 nucleotides. Up to 70% of the Hn[3H]RNA forms hybrids at high DNA/RNA ratios (130,000/1) when reactions are carried out to high C0t values. Studies of the effect of increased DNA/RNA ratio on extent of hybridization at high DNA C0t values show that at least two frequency classes of single copy transcript are present in the Hn[3H]RNA population. Both repetitive and single copy DNA transcripts are present within individual Hn[3H]RNA molecules. Calculations indicate that at least 25% and possibly all of the Hn[3H]RNA molecules contain repetitive sequence elements interspersed with non-repetitive sequences.
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Rapidly labelled RNA and ribosomal RNA of HeLa and E. coli cells may be composed of smaller subunits.
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(32)P-labeled ribonucleic acid (RNA) from purified Sindbis virus was examined for the presence of hidden breaks. Viral RNA was treated with acid at pH 2.9 or with formaldehyde and was analyzed on sucrose gradients or by polyacrylamide gel electrophoresis. The sedimentation pattern and mobility on polyacrylamide gels of the 42S RNA was unaffected by heating and quick cooling and was not altered by denaturing agents such as dimethyl sulfoxide and urea. No evidence that Sindbis RNA is a polyaggregate of fragments was obtained. It is concluded that the genome consists of a continuous length of single-stranded polynucleotide.
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Chapter
Considerable light is shed on the problems of reactivity and chemical modification of nucleic acids by reactions involving exocyclic substituents of the purine and pyrimidine bases, i. e., the amino groups of cytosine, adenine, or guanine, and the carbonyl groups of uracil, guanine, xanthine, and their derivatives, and also reactions of the sulphur atom of thio derivatives (minor components of RNA). As was mentioned previously (see Chapter 3), n electrons of the nitrogen atoms of amino groups and of the oxygen atom of carbonyl groups of the nucleic acid bases (and their derivatives) react strongly with the π electron system of the heterocyclic ring, and consequently the properties of the corresponding components of the nucleic acids differ greatly from the properties of simple amines, amides, or thioamides.
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Chapter
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Conditions for the propagation, plaque assay, and purification of radioactively labeled rhinovirus 1A are described. Purified virus was free of empty capsids. It sedimented as a single peak at a rate indistinguishable from that of type 1 poliovirus and ME virus on density gradients. Treatment at pH 4, which left ME virus intact, completely disrupted rhinovirus 1A to produce largely insoluble products. Buoyant density measurements in cesium chloride confirmed that rhinovirus 1A is denser (d = 1.386) than ME virus (d = 1.342).Analysis of purified rhinovirus 1A on SDS-polyacrylamide gels revealed five components; of these, four with apparent molecular weights of 33,800, 29,500, 26,000 and 7000, respectively, were present in roughly equimolar proportions. Roughly as much of the fifth component, weighing about 36,700 daltons, was observed. This distribution of polypeptides, which is similar to that of ME virus and of poliovirus, supports the notion that all three viruses represent a common structural archetype. Thus acid lability and high buoyant density which is characteristic of rhinoviruses cannot be attributed to any gross differences from other picornaviruses in size or number of polypeptide chains in the virion.It is proposed that the rhinoviral capsid is comprised of 60 identical four-chain subunits each weighing about 96,000 daltons. Assuming an RNA content of 30–32% this model predicts that the size of the virion is 8.4 × 106 daltons and the size of the RNA is 2.6 ± 0.1 × 106 daltons.
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The observation that repetitive and single copy sequences are interspersed in animal DNAs has suggested that repetitive sequences are adjacent to single copy structural gene sequences. To test this concept, single copy DNA sequences contiguous to interspersed repetitive sequences were prepared from sea urchin DNA by hydroxyapatite fractionation (repeat-contiguous DNA fraction). These single copy sequences included about one third of the total nonrepetitive sequence in the genome as determined by the amounts recovered during the hydroxyapatite fractionation and by reassociation kinetics. 3H-labeled mRNA from sea urchin gastrula was prepared by puromycin release from polysomes and used in DNA-driven hybridization reactions. The kinetics of mRNA hybridization reactions with excess whole DNA were carefully measured, and the rate of hybridization was found to be 3-5 times slower than the corresponding single copy DNA driver reassociation rate. The mRNA hybridized with excess repeat-contiguous DNA with similar kinetics relative to the driver DNA. At completion 80 percent of that mRNA hybridizable with whole DNA (approximately 65 percent) had reacted with the repeat-contiguous DNA fraction (50 percent). This result shows that 80-100 percent of the mRNA molecules present in sea urchin embryos are transcribed from single copy DNA sequences adjacent to interspersed repetitive sequences in the genome.
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A collection of amber mutants of bacteriophage T5 was analysed using an in vivo complementation test and assigned to 21 complementation groups. The incomplete phage structures produced by T5 amber mutant infection of the non-permissive host were examined. The mutants were allocated to four phenotypic types, as defined by an in vitro complementation test: those which produced functional heads, H; those which produced functional tails, T; those which produced inactive heads and functional tails, HI + T; and those which did not synthesize either heads or tails, 0. Functional heads and inactive heads were indistinguishable in shape and size in the electron microscope. Four different patterns of DNA metabolism were observed when different amber mutants were grown under non-permissive conditions. They were the wild-type pattern (D+) in which host DNA degradation was followed by synthesis of phage DNA, host DNA degradation without phage DNA synthesis (D0), neither host DNA degradation nor phage DNA synthesis (DD0) and host DNA degradation with slight DNA synthesis (DS). Upon infection of the non-permissive host with representatives of different complementation groups of mutants either normal lysis occurred or bacterial growth ceased without subsequent lysis. The phenotypic characteristics of the amber mutants were used for partial elucidation of the functions of the affected genes.
Article
Antibodies to the four major RNA bases were elicited by immunization with nucleoside-protein conjugates. Reactivity of RNA with antinucleoside antibody was investigated by the double-antibody technique which measures the primary interaction between antibody and antigen. In order to demonstrate a reaction between antinucleoside antibody and RNA, serum ribonuclease activity had to be eliminated, and at the levels of RNase found in the antisera this could be done with Na2SO4. In the presence of 0.2 M Na2SO4, antiadenosine (anti-A) reacted with all RNA preparations tested, except tRNA. No reaction with RNA could be detected with antibody to guanosine, cytidine, or uridine under identical conditions although these were at least as reactive with DNA as was anti-A. The specificity of RNA-anti-A interaction was characterized further by inhibition experiments. Only adenosine and adenine-containing nucleosides, nucleotides, or polynucleotides inhibited the binding of tritiated Escherichia coli RNA. The inhibitory activity of poly(A) was lost when it was complexed with poly(U) in a hydrogen-bonded duplex.
Article
THE role of formylmethionyl tRNAf (fMet-tRNAf) in the initiation of protein biosynthesis in Escherichia coli is now reasonably well understood1. A similar mechanism involving fMet-tRNAf seems to be common to all 70S type ribosomal systems including those from bacteria, chloroplasts and mitochondria2,3. The corresponding process on 808 type ribosomes is, however, as yet unknown, but there is no evidence to suggest that fMet-tRNAf or any similar blocked aminoacyl-tRNA is involved3. A mammalian cell-free system which synthesizes proteins accurately in response to added messenger RNA would facilitate the study of initiation on 80S ribosomes. An efficient cell-free system derived from mouse ascites tumour cells and primed by encephalomyocarditis (EMC) viral RNA has recently been developed (ref. 4, and M. B. M. and A. Korner, manuscript in preparation). We report here on the nature of the products synthesized.
Article
Poliovirus RNA was compared with tobacco mosaic virus (TMV), mammalian 28S and 18S ribosomal, and R17 bacteriophage RNAs. Acrylamide gel electrophoresis gave a linear relation between relative mobility of the standards and logarithm of molecular weight, and the mobility of poliovirus RNA corresponded to 2.56 ± 0.13 × 106 daltons. Sucrose-gradient centrifugation in three solvents minimising secondary structures sedimented poliovirus RNA at a rate consistent with 2.1 − 2.6 × 106 daltons.
Article
SUMMARY A particle weight of 8.5 ° × I@ for encephalomyocarditis virus was derived by combining results from (a), sedimentation velocity measurements on highly purified preparations of virus (o-I to 5 mg./ml.) in o.I M-KCI+o-o2 M-phosphate buffer, pH 8.o, which gave an S°0,~. of i62. 3 s, (b), differential sedimentation studies of virus in the same solvent but containing various ratios of D20 to H20 which gave a partial specific volume of o'678 ml./g., with results from (e), analysis of boundary spreading during low speed centrifugation of dilute virus solutions which led to a diffusion coefficient D20,~- of I'44 × Io -7 cm.2/sec. Sedimentation equilibrium studies on similar virus preparations in the same solvent gave a particle weight of 8-52 x I o ~ . This suggested a hydrated particle diameter for the virus of 29"8 nm. and a frictional ratio fifo of I. 13o which was consistent with that of a hydrated sphere containing 0.29 g. water/g, dry virus.
Article
Full-text available
SUMMARY The effect of heat and formaldehyde on the sedimentation properties of virus RNA and the RNA induced in baby hamster kidney cells by infection with foot- and-mouth disease virus has been studied. The sedimentation rate of the induced 37s RNA was reduced considerably by treating with 6% formaldehyde in O.Ol M-EDTA at 37 ° but it still sedimented faster than the 35s RNA extracted from purified virus which had been treated similarly. Part of the interjacent RNA sedi- mented at the same rate as the 35s virus RNA after similar treatment. The sedi- mentation rate of the i6s ribonuclease-resistant RNA was unaffected. After treating with formaldehyde at 7 o°, part of the ribonuclease-resistant RNA sedimented faster than virus RNA which had received similar treatment. These results show that the greater rate of sedimentation of the 37s RNA is caused by the larger size of the induced RNA compared with the 35s virus RNA and suggest that the more rapid sedimentation of the ribonuclease-resistant RNA after denatur- ing at 7 oo may be due to its existence as a circular molecule.
Article
The picornaviruses have a wide range of buoyant densities in caesium chloride. Whereas the density of the pH 3-stable viruses is 1.34 g./ml. (Mattern, 1962; Schaffer & Frommhagen, 1965), the acid-sensitive rhinoviruses and foot-and-mouth disease viruses have densities of 1.38–1.41 g./ml. (Dans, Forsyth & Chanock, 1966; Chapple & Harris, 1966; McGregor, Phillips & Mayor, 1966; Gerin et al. 1968) and 1.43 g./ml. (Trautman & Breese, 1962; Wild & Brown, 1967). Although the reason for this difference in density is not understood, it seems likely that the higher values obtained with the acid-labile group are due to reaction of the caesium ions with the more accessible RNA of these viruses (McGregor et al., 1966). Recently, however, McGregor & Mayor (1968) suggested, on the basis of comparative measurements of the strand lengths of the ribonucleoproteins isolated from strains of poliovirus and rhinovirus, that the higher buoyant density of the rhinovirus was due to the high molecular weight (4 × 106) of the virus RNA.
Article
This chapter discusses that the animal cell is known to contain a great variety of RNA molecules of varying chain length. In addition to the small tRNA's and the larger ribosomal RNA's, there are other species whose existence is ephemeral. Although some of these may represent short-lived messengers, there is sufficient evidence to conclude that certain of these types are intermediates in biosynthetic pathways leading to the formation of transfer and ribosomal RNA's. It reviews that the primary structure of these intermediates is modified in certain instances by cellular enzyme systems subsequent to the completion of its primary synthesis in the processes of “transcription.” Such modifications include “trimming” or “tailoring,” that is alteration to the length of the primary transcription product by scission mechanisms, or the alteration of primary nucleotide sequences as a result of base or sugar moiety modification. This chemical work carried out by the cell is prerequisite in the formation of certain major nucleic acid species and the molecular events involved are collectively described as maturation events. It concludes that discussions can be made for and against RNA maturation processes being beneficial to higher organisms, but at the moment these are only hypothetical and no convincing evidence is available to support any one in particular.
Article
Full-text available
Heat inactivation of transfer ribonucleic acids at 90° in a solvent of neutral pH and moderate concentration of monovalent salts leads to a 63% loss (1 hit per molecule) of three different biological functions in about 10 hours. Addition of Mg⁺⁺ increases the rate of inactivation 20-fold. While chain scission is shown to be the most important mechanism of inactivation in the presence of Mg⁺⁺, it accounts for only 20 to 50% of the inactivation in the absence of Mg⁺⁺. Whereas depurination, deamination, and aggregation have been ruled out as additional important means of heat inactivation in the absence of Mg⁺⁺, the destruction of 5,6-dihydrouridylic acid residues appears to be a quantitatively significant one.
Article
1. The physical characteristics of single- and double-stranded coliphage RNA with regard to their sedimentation behaviour in gradients of sucrose in high or low ionic conditions were examined. The effect of heat on their sedimentation characteristics was also determined. 2. Single-stranded coliphage RNA was found to exist in three different forms having sedimentation coefficients 28s, 20s and 12s. The latter two were interchangeable, depending on ionic strength. All three were almost equally infectious to spheroplasts. 3. Double-stranded coliphage RNA was found to be non-infectious to spheroplasts and had sedimentation coefficients 15s and 12s. Thermal denaturation gave rise to infectious single-stranded 12s RNA. 4. Four possible hypotheses on the mechanism of replication of coliphage RNA are discussed.
Article
The mildest treatment with ribonuclease that causes any disaggregation of the polysomes of Escherichia coli or HeLa cells simultaneously attacks the RNA of the constituent ribosomes. It is concluded that the susceptibility to ribonuclease of polysomes does not suggest that they are held together by a strand of messenger RNA. The RNA of the larger sub-unit of bacterial ribosomes has particularly sensitive regions resulting in a non-random degradation. The RNA of the smaller sub-unit of E. coli ribosomes is relatively resistant to ribonuclease attack. The same may be true of the respective sub-units of the intact HeLa-cell ribosome, but both sub-units become very sensitive to ribonuclease on dissociation from each other.
Article
The stability in mildly alkaline environments of 16-S and 23-S ribosomal RNA from Escherichia coli has been studied. It has been found that instability of 23-S RNA occurred at pH 9 in 0.05 M glycine buffer at 37°, when, in the presence of bentonite, it was almost completely converted to 16-S RNA in 3 h. At pH 10, the conversion occurred within 45 min whereas at pH 8.6, there was an appreciable amount of 23-S RNA left after 6 h incubation. Longer periods of incubation of ribosomal RNA in these buffers gave rise to a slower accumulation of material smaller than 16-S RNA. However, even after 24 h incubation at pH 9, the ribosomal RNA was still completely precipitable in cold trichloroacetic acid.
Article
The effect of temperature and formaldehyde concentration on the extent of reaction of ribonucleic acid (RNA) with formaldehyde was studied by measuring the absorbance at 270 mμ. The reaction is about 85% complete after 10 min at 63° in 1 M formaldehyde. Absorbance-temperature studies of adenosine monophosphate (AMP), cytidine monophosphate (CMP), and guanosine monophosphate (GMP) before and after reaction with formaldehyde show that the extent of reaction decreases reversibly as the temperature is raised. At 250 mμ, however, the average absorbance is almost independent of the extent of reaction. Absorption spectra at 15 and 85°, and absorbance-temperature profiles were measured for Escherichia coli and yeast transfer ribonucleic acid (tRNA), R17 phage RNA, and tobacco mosaic virus-ribonucleic acid (TMV-RNA) before and after reaction with formaldehyde. The formaldehyde derivatives have hypochromicities of 0.04-0.06 compared to values of 0.19-0.23 obtained for unreacted RNA. The absorbance-temperature changes are largely reversible and independent of wavelength or counterion concentration. A comparison of the modest hypochromicity observed with the large optical rotatory dispersion that has been reported for tRNA treated with formaldehyde suggests that the single-stranded asymmetric structures present in RNA result from stacking that has only a minor influence on the absorbance. A practical consequence of this observation is that RNA hypochromicity can be attributed largely to the double-stranded hydrogen-bonded regions of the molecule. After correcting for the contribution from single-stranded base stacking, the helical content of RNA can be estimated as 59-73% depending on the RNA. The double-stranded helical regions of RNA can be partly regenerated after removing excess formaldehyde either by dialysis or passage through a Sephadex G50 column followed by heating to 80° for 10 min. Prolonged exposure to elevated temperatures is required to effect complete reformation.
Article
1. The effect of removing Mg(2+) from a purified high-molecular-weight (1.07x10(6)) fraction of Escherichia coli ribosomal RNA was examined by ultracentrifugation, thermal denaturation and optical rotation. 2. At moderate I (0.1m-sodium chloride), EDTA at 2-50mm has little effect on RNA; at low I, 0.01-0.04 (with tris as counter-ion), two boundaries appear. 3. The leading boundary, S(20,w) about 20s, is identified with the original material with counter-ion Mg(2+) (;ionic atmosphere') removed, leading to an expanded form. 4. The slow boundary, 15-16s, is associated with a further loss of Mg(2+) and a further expansion, sensitive to EDTA concentration: it is proposed that this Mg(2+) is localized on the polynucleotide chain, i.e. ;site-bound'. 5. I is important and the EDTA effect at low I is reversible if Na(+) is added immediately after the EDTA: this Na(+) reversibility is lost on standing at 0 degrees . It is suggested that changes in the tertiary structure may be associated with this loss of reversibility. 6. Thermal-denaturation studies show that there is no loss of secondary structure associated with these changes: change in the optical-rotatory-dispersion spectrum in the region of the Cotton effect may be associated with this change in tertiary structure.
Article
The sedimentation rates of ribosomal RNA from Escherichia coli, Bacillus subtilis, etiolated pea stem, white potato tuber, rabbit liver, rabbit reticulocytes, rat liver and rat fat-pads were compared by mixing any two of the above and analyzing them independently (32P and absorbancy at 260 mμ) on sucrose gradients. By this criterion rabbit liver, rabbit reticulocytes, rat liver and rat fat-pad ribosomal RNA's were indistinguishable. Pea stem and potato tuber as well as E. coli and B. subtilis ribosomal RNA's were also indistinguishable; in other words, within each small group of organisms studied, there was no detectable difference in sedimentation rate of the ribosomal RNA's.
Article
1. A study was made of the sedimentation properties of purified preparations of the rapidly labelled RNA in the nucleus and the cytoplasm of the HeLa cell. The sedimentation of the rapidly labelled nuclear RNA was very sensitive to changes in ionic strength and bivalent cation concentration. Under the conditions usually used in sucrose-density-gradient centrifugation the rapidly labelled nuclear RNA showed extreme polydispersity, and much of it sedimented more rapidly than the 28s RNA. At low ionic strength and after removal of Mg(2+), however, the rapidly labelled nuclear RNA sedimented as a single peak at about 16s. The conversion of the polydisperse material into the 16s form did not involve degradation of the RNA, since the effect could be reversed by increasing the ionic strength of the solution. 2. The cytoplasm did not contain any RNA that showed polydisperse sedimentation under the usual conditions of sucrose-density-gradient centrifugation, or that had the same sensitivity as the rapidly labelled nuclear RNA to changes in ionic strength. All the radioactivity in the cytoplasmic RNA sedimented with the 28s, 16s and 4s components over a wide range of physical conditions, but these components did contain a labelled fraction with some of the features of the rapidly labelled nuclear RNA on columns of methylated albumin on kieselguhr. 3. In both nucleus and cytoplasm the RNA detected by ultraviolet absorption could also be converted into a 16s form by removal of bivalent cations at low ionic strength; this effect was again, within certain limits, reversible. The nuclear RNA as a whole was more susceptible to changes in ionic strength than the cytoplasmic RNA. 4. It thus appears that all the RNA in the cell, except the 4s RNA, can be prepared, without degradation, as a single peak sedimenting at about 16s. The relationship of these various 16s components to each other is discussed.
Article
Ribosomal ribonucleic acid preparations of exceptional stability were obtained from the ribosomes of Escherichia coli by a method of isolation employing phenol, sodium dodecyl sulfate, and a purified hectorite, Macaloid. The sedimentation and viscosity properties of the total ribosomal RNA and of the separated components were extensively investigated and molecular weight determinations were made by sedimentation viscosity, sedimentation equilibrium, and viscosity kinetics at elevated temperatures. Molecular weights of 1.07 × 106 and 0.55 × 106 g/mole were found for the 23 and 16 S components, respectively. The influence of RNA aggregation upon hydrodynamic parameters was evaluated and several methods (organic solvents, reaction with formaldehyde, low ionic strength, and heat) are suggested for the detection of aggregates within RNA preparations. Chromatography of undegraded ribosomal RNA upon DEAE-cellulose was found to be complicated by the formation at equilibrium of a nonelutable complex between the ion exchanger and the polynucleotide. Base composition analyses were performed upon the separated ribosomal RNA components, and slight but significant differences were found between the 23 and 16 S molecules. It is concluded that the ribosomal RNA of E. coli is composed of two classes of polyribonucleotide chains, each class being covalently continuous and thus not containing polynucleotide subunits.
Article
1.1. The molecular weights of the Jensen sarcoma ribosome and its large subunit, calculated from their sedimentation and diffusion coefficients, are 4.3·106 and 2.7·106 respectively. The molecular weights of the ribosomal RNA's, calculated from their sedimentation coefficients and intrinsic viscosities, are 1.64·106 and 0.67·106 respectively. Thus these ribosomes appear to dissociate into two subunits, of approximately two thirds and one third, each containing one strand of structural RNA that makes up half its mass.2. The large subunit exists in a variety of states whose sedimentation coefficients decrease from 57 S to 30 S as cations are removed. These changes have been attributed to changes in conformation.3. The small subunit has been found in two forms: 33 S when magnesium is present and 29 S in its absence.
Article
Hydroxymethylation of pseudouridines B and C, uridine, thymidine, inosinic acid, and polyuridylic acid by reaction with formaldehyde has been shown to take place by the use of spectrophotometric techniques. Reaction is almost instantaneous at room temperature and neutral or alkaline pH, resulting in addition to the N 1 and N 3 atoms of the pyrimidine ring, where available, and to the N 1 atom of the purine ring. Reactants and products are in rapid equilibrium such that after excess formaldehyde is removed by chromatography, no hydroxymethyl adduct remains. The equilibrium constant for reaction with uridine and inosinic acid was determined to be 2.5 and 1.7 1./mole, respectively. This reaction becomes significant when nucleic acids are studied in the presence of formaldehyde, since under these conditions an appreciable fraction of the uridylic or thymidylic acid will be derivatized.
Article
When rRNA (ribosomal ribonucleates) or sRNA (soluble ribonucleates) are hydrolyzed in alkali, the resulting hydrolysis products can be resolved into four separate classes: nucleosides (N), nucleoside 2′-and 3′-phosphates (Np), nucleoside 2′(3′),5′-diphosphates (pNp), and alkali-stable dinucleotides (NxpNp, where x denotes 2′-O-methylation). The N and pNp compounds derive from 5′-linked and 3′-linked termini, respectively, while Np and NxpNp compounds derive from internal positions of polynucleotide chains: (3′-linked terminus) pNpNpNpN·····pNpNpNpN (5′-linked terminus). In the present study, the distribution of radioactivity among these different classes of compounds in alkali hydrolysates of L cell rRNA, has been compared with corresponding data for rapidly labeled RNA from L cells. Three rapidly labeled RNA specimens were prepared from L cells that had been exposed to four tritiated nucleosides (adenosine (A), guanosine (G), cytidine (C), and uridine (U)) for 15, 30, and 90 min, while rRNA was prepared from cells exposed to the tritiated nucleosides for 24 hr. All RNA preparations were repeatedly precipitated from 2.5 M sodium chloride solution at 0°, in order to remove sRNA. The rapidly labeled RNA preparations were polydisperse and characterized by different amounts (20-75%) of material sedimenting faster than 28 S, whereas the 24-hr preparation displayed an essentially bimodal sedimentation profile, with peaks at 16 and 28 S. In the case of nucleosides, all preparations were remarkably similar with respect to the proportionate amount of radioactivity in a given nucleoside (N) relative to its homologous nucleotide (Np), i.e., A/Ap, G/Gp, C/Cp, and U/Up radioactivity ratios were similar for all preparations. By contrast, in the case of pNp and NxpNp compounds, the proportionate amounts of radioactivity in rapidly labeled RNA were much lower than for rRNA, after 15 min, but approached the proportions for rRNA, after 90 min. These empirical similarities and differences are based on the primary experimental measurements, no assumptions having been made with respect to the relative mean specific activities at terminal and nonterminal positions in the polynucleotide chains of the different preparations. As such the relations can be considered as characteristic analytical parameters, which distinguish rRNA from rapidly labeled RNA. By invoking assumptions, the primary data can be used to assess molar percentages of the different components in the RNA preparations. These data are discussed in terms of the degree to which they might reflect features of the primary structure of the polynucleotide chains, and also in terms of the extent to which they might reflect differences of mean specific activity between the termini of the polynucleotide chains in the different RNA specimens.
Article
Lower concentrations of formaldehyde are required to inactivate the infectivity of the nucleic acid from tobacco mosaic virus than are required to inactivate intact TMV. The amount of formaldehyde bound at half inactivation is about 400 and 20 molecules for one intact virus particle and its separated nucleic acid, respectively.The reaction of formaldehyde with nucleic acid appears to be specific for the amino groups of the bases. However there is little reaction in those cases where the amino groups are believed to be involved in strong hydrogen bonding as exemplified by DNA and the two-strand complex of polyadenylic and polyuridylic acids.The reaction proceeds in two steps, the first leading to a more labile form of binding, whereas after a more extensive reaction most of the formaldehyde becomes firmly bound. The formaldehyde binding of RNA is greatly decreased at higher salt concentration which is taken as evidence that fewer amino groups are free to react under these conditions. A comparison of TMV-RNA with yeast- and liver-RNA preparations shows that the virus nucleic acid is the most reactive towards formaldehyde. The significance of the quantitative evaluation of the formaldehyde binding on the structure of RNA is discussed.
Article
1.1. The formaldehyde reaction of Fraenkel-Conrat has been used as a measure of the degree of denaturation of calf thymus deoxyribonucleic acid subjected to heat or pH change. Under the proper conditions the formaldehyde reaction seems to be at least as sensitive and may be more sensitive than viscosity measurements for detecting a low degree of denaturation of deoxyribonucleic acid.2.2. Using the reaction with formaldehyde as a criterion for denaturation of deoxyribonucleic acid, it has been found that no very sharp transition temperature occurs for the conversion of native to denatured deoxyribonucleic acid unless the time of heating is kept short. Deoxyribonucleic acid will denature rather completely at 70°, by the above criterion, if heating is sufficiently prolonged.3.3. Using the criterion of reaction with formaldehyde, a reversible and an irreversible type of denaturation of deoxyribonucleic acid can be distinguished. Reversal of the reversible denaturation can be accomplished by adjusting the pH to 8.5.4.4. The reaction with formaldehyde of heat-denatured calf-thymus deoxyribonucleic acid, reisolated by alcohol precipitation from iM sodium chloride solution, has been compared with the reaction of ribonucleic acid with formaldehyde. The two materials behave in a remarkably similar manner.5.5. The possibility of using the formaldehyde reaction to decide whether a reversibly denatured deoxyribonucleic acid can act as a primer in Bollum's system for deoxyribonucleic acid synthesis has been discussed.
Article
By velocity sedimentation, in appropriate solvents, the presence of two discrete components can be demonstrated in preparations of the DNA of bacteriophage φX174. The major, faster-moving S_1 and the slower S_2 are present under conditions which exclude the possibility of hydrogen-bond formation. It can be shown, either by treatment with pancreatic deoxyribonuclease or by thermal inactivation, that S_2 is the first degradation product of S_1, formed by scission of S_1, without significant decrease in molecular weight. A subsequent chain scission in S_2, which occurs with equal likelihood, results in random fragmentation. These results are interpreted to mean that the S_1 component is a covalently linked ring structure and the S_2 component is the corresponding open-chain degradation product. Under certain conditions the S_2 component can be selectively degraded by E. coli phosphodiesterase. The digestion is not complete and there appears to be a single discontinuity, resistant to phosphodiesterase, present in the φX-DNA ring.
Article
Parental RNA of the bacteriophage R17 is converted to a double-stranded replicative intermediate which can be detected by its characteristic sedimentation rate and its resistance to pancreatic ribonuclease as early as six minutes after infection of Escherichia coli. Chloramphenicol inhibits the conversion and therefore production of the ribonuclease-resistant form must require protein synthesis. Prior to ribonuclease treatment the replicative intermediate displays a heterogeneous sedimentation characteristic in sucrose gradients, but it sediments as a homogeneous sharp peak if treated with RNase before centrifugation. Sedimentation analysis of lysates of infected cells prepared 20 minutes after infection demonstrates that all intact parental phage RNA found in the lysate sediments with 70 s ribosomes in both the single- and double-stranded forms.
Article
1. RNA has been prepared from baby hamster kidney cells by extraction with a phenol-EDTA mixture and further purified by passing through a column of Sephadex G-25 that had been equilibrated with water. 2. Aging of the total RNA extracts at 4 degrees or heating at 95 degrees followed by rapid cooling caused a conversion of 28s RNA into material sedimenting in sucrose gradients at approx. 18s. 3. When heated RNA was re-extracted with phenol the sedimentation profile was not returned to that of the unheated RNA. 4. The 28s and 18s RNA fractions were collected separately from sucrose gradients by precipitation with 2vol. of ethanol and passed through a Sephadex G-25 column equilibrated with water. 5. Heat treatment of purified 28s RNA at 95 degrees caused the sedimentation coefficient to increase to approx. 40s, whereas similar treatment of 18s RNA caused no significant increase. If the RNA was heated before the Sephadex G-25 treatment the sedimentation coefficient of the 28s and 18s RNA decreased to approx. 12s and 8s. 6. Heating mixtures of purified 28s and 18s RNA at 95 degrees caused some aggregation of 18s material with the 28s fraction.
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