No full-text available
To read the full-text of this research,
you can request a copy directly from the author.
The effect of reaction with formaldehyde on the sedimentation rates of ribonucleic acids
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.
... 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. ...
The genome of Sindbis virus, 49 s RNA, is a single, intact polynucleotide chain having a molecular weight of 4.3±0.3 × 106 daltons. This has been determined using a variety of methods including polyacrylamide gel electrophoresis, sedimentation after reaction with formaldehyde, determination of the molecular weights of the double-stranded forms of Sindbis-specific RNA and hybridization competition. The second major species of single-stranded RNA made after infection with Sindbis, 26 s RNA, has been found to have a molecular weight of 1.6 × 106 daltons as determined by sedimentation in dimethylsulfoxide. Hybridizationcompetition experiments carried out between these two species of RNA, using double-stranded forms of Sindbis RNA isolated from infected cells, showed that 26 s RNA contains only one-third of the base sequences in 49 s RNA and thus represents a unique fraction of the viral genome.
1The initiation of protein synthesis directed by the RNA from encephalomyocarditis virus in a cell-free system from Krebs II ascites cells has been investigated.2Ascites cell Met-tRNAMet donates all the internal methionyl residues of the viral-RNA-specified polypeptide, whereas little incorporation from Met-tRNAfMet is detected.3fMet-tRNAfMet, which is not normally present in the cytoplasm of eukaryotic cells, donates formylmethionine into polypeptide in response to the viral RNA to give a product which contains a single N-terminal sequence fMet-Ala-Thr-, and is identical to the normal cell-free polypeptide except for the N-terminal formylmethionyl residue.4Short-chain peptidyl-tRNA synthesised in vitro in response to encephalomyocarditis virus RNA in the presence of low concentrations of sparsomycin, which inhibits chain elongation but not initiation, contains the N-terminal sequence Met-Ala-Thr-.5This data supports the notion that unblocked Met-tRNAfMet is the universal initiator of protein synthesis in eukaryotes and donates a methionyl residue into the N-terminal position of all proteins. The initiating methionyl residue is in most cases subsequently removed from the nascent polypeptide, but this can be inhibited either by an N-terminal formyl group or by preventing the nascent polypeptide from growing long enough to be a substrate for the methionine aminopeptidase.6The data also shows that encephalomyocarditis virus RNA, which codes for at least nine stable proteins in vivo, contains only one initiation site which is expressed in vitro. The polypeptide coded for immediately after the initiation site is not virus-coat protein and is rapidly metabolised in vivo, suggesting that a lead-in polypeptide precedes the first meaningful protein sequence.
Poliovirus incubated at pH 11.0 and 37° C, rapidly loses 90% of its infectivity. This inactivation is accompanied by a fragmentation of the RNAin situ.
The RNA extracted from the inactivated particles sediments as a single peak with a sedimentation coefficient of 11S. The average molecular weight is estimated as 2.5×105.
SUMMARY Cytoplasmic extracts of HeLa cells made 1 to 2 h after infection with radioactive poliovirus contained 150-s virus particles and 130-s, perhaps partially disrupted, particles. The latter were resistant to RNase but sensitive to dodecylsulphate. Both particles were associated with fast sedimenting material from which they could be released by deoxycholate, but not by EDTA. In isopyenic gradients of sucrose in DSO the labelled particles formed a sharp band coincident with membrane-bound ribosomes at a density of 1-23 g cm"3. It is suggested that attachment of virus particles to endoplasmic reticulum may be an early stage in the infectious cycle, determining the site of subsequent steps. The inhibition of cellular protein synthesis that develops during infection affects membrane- bound as well as free polysomes and therefore does not determine the membrane-association of viral protein synthesis. It is not known why viral protein is synthesized on bound and not on free poly- somes, nor how the synthesis of host protein is inhibited while viral protein is being made. We have done experiments to test the following hypotheses: (a) that a property of the infecting virus particle, namely an affinity of the virion for the endoplasmic reticulum, determines the site of functioning of its RNA, and (b) that the inhibitory condition in infected cells is effective specifically against free polysomes, leaving only the membrane-bound system available for the synthesis of viral protein. The results are consistent with the first hypothesis but not with the second.
SUMMARY A count of specifically labelled tryptic peptides from purified poliovirus (strain Mahoney) showed that the amino acid sequence comprising its structural protein contains at least 2I and 25 uniquely placed residues ofhistidine and methionine, re- spectively. Amino acid analysis after performic acid oxidation showed a content of, respectively, 21 and 27 moles of these residues per lOOO amino acids recovered. Accordingly, poliovirus structural protein must comprise a unique sequence of about I ooo amino acids, representing about 4o ~ of the coding potential of the poliovirus genome. The correspondence of this percentage with the proportion of the polio- virus genetic map occupied by structural protein genes (48 ~o) suggests that the map represents a major part of the genome. A value of about lOOO amino acids, or lOO to 1 lO ooo daltons of protein, for the repeating structural unit, coupled with a total content of 6.o to 6-4 x Io G daltons of protein in the poliovirus particle, indicates that it contains 60 such structural units and that its triangulation number = I.
The sedimentation behaviour of RNA extracted from measles virions was compared with that from other paramyxovirions. RNA from three different strains of measles virus all had an S-value of 52.0±0.6S in 15–30% linear sucrose gradients whereas RNA from other paramyxoviruses, including the closely related distemper virus had an S-value of the order 50S. The sedimentation rate of measles virus RNA was not altered by formaldehyde treatment.
The electrophoretic fractionation in polyacrylamide gels showed identical migration rates of RNA from measles and other paramyxovirions.
A possible explanation might be that MV RNA has regions of exceptionally high GC-content allowing the formation of formalin-resistant loops, and thus sediments faster in sucrose than other paramyxoviruses RNAs.
The nucleotide composition and physical properties of the large and small mitochondrial ribosomal RNA's and of mitochondrial 4 s RNA from ovaries and eggs of Xenopus laevis are described.The G + C content of the mitochondrial RNA's is between 40 and 44%. The secondary structure of these RNA's, investigated in optical melting experiments, is much less stable than the structure of bacterial and cytoplasmic RNA's. Probably as a result of the structural peculiarities of the mitochondrial ribosomal RNA's, the electrophoretic mobilities of these RNA's depend greatly on the conditions of electrophoresis. Therefore, gel electrophoresis is not an applicable method for the determination of the molecular weights of the mitochondrial ribosomal RNA's.At cation concentrations from 0.07 to 2.5 m the sedimentation coefficient of the large mitochondrial ribosomal RNA is between 0.91 and 1.0 that of the cytoplasmic 18 s ribosomal RNA; for the small mitochondrial ribosomal RNA this ratio is between 0.66 and 0.77. To study the sedimentation properties of the denatured molecules the mitochondrial RNA's were compared to cytoplasmic and bacterial RNA's by sedimentation in dimethylsulfoxide, and after treatment with formaldehyde. These experiments led to the following molecular weight estimates for the mitochondrial RNA's: large ribosomal RNA, 5.3 × 105; small ribosomal RNA, 3.0 × 105; 4 s RNA (average), 28,000.
This chapter discusses the three main reasons for the chemical modification of nucleic acids that are available on modifying agents. These agents are used for inactivations of various kinds, for directed functional changes of nucleic acids in vivo and for the elucidation, through resulting modification, of structural and functional characteristics peculiar to synthetic or native polynucleotides. Some of these agents are useful for one, two, or all three of these purposes. One of the few agents that serve all three of them is formaldehyde. It is widely used as an inactivator of viruses to obtain vaccines and is reported to exert a cytostatic (carcinostatic) effect. It is also one of the most promising mutagenic agents affecting multicellular organisms. Formaldehyde is used extensively in structural and functional studies of nucleic acids as an agent not so much for causing denaturation as for preventing renaturation, and as a fixator of nucleic acids and nucleoproteins in electron microscope and sedimentation investigations. Recently, some progress in the structural study of formaldehyde interaction products with nucleotides and nucleic acids has been made. The chapter emphasizes on the evidence for the formation of various structures and the molecular mechanisms of biological and other effects of formaldehyde.
Rapidly-labelled nRNA, isolated after extensive purification from HeLa cells, has been examined on sucrose density-gradients in conditions which reduce aggregation and intermolecular interactions. The results, which confirm and extend earlier observations, show that the rapid-labelling heterogeneous RNA sediments in a series of broad peaks which lie within or only slightly greater than the rRNA peaks. If this RNA is precipitated from the gradient and subjected to gel electrophoresis, it behaves as if it were of very high molecular weight and gives a virtually identical pattern to nRNA which had not been subjected to sucrose-gradient centrifugation. This provides decisive evidence that degradation has not taken place to account for the lower sedimentation velocity and that this species of RNA is not, therefore, of extremely high molecular weight as currently thought.
Formaldehyde treatment was used to obtain size-dependent sedimentation of high molecular weight nucleolar and nucleoplasmic RNA from normal L1210 cells, and from cells treated with drugs thought to affect the size distributions of the RNA synthesized.Reaction with formaldehyde at 70 °C to fully substitute the RNA was accompanied by significant RNA chain scission. Chain scission was minimized by carrying out a limited reaction at 40 °C. Since the reaction at 40 °C under these conditions was incomplete, however, the possible effects of persistant compact conformations and hidden breaks in the RNA were considered. The sedimentation patterns after various reaction times at 70 °C or 40 °C generally fit a simple model for random chain scission at rates of 1.7 · 10−5 and 4.4 · 10−7 breaks per nucleotide per min, respectively. Nucleoplasmic RNA after prolonged reaction at 40 °C, however, behaved as if it contained hidden breaks that resist mild formaldehyde treatment but that become exposed after more extensive treatment.Nucleoplasmic RNA synthesized by cells treated with intercalating agents exhibited an increase in sedimentation. This and the finding of hidden breaks may be due to nucleoplasmic RNA processing by cleavage at double-stranded segments, and its inhibition by intercalating agents.Nucleoplasmic RNA synthesized by cells treated with anthramycin or nitrogen mustard, agents that bind irreversibly to DNA, exhibited reduced sedimentation which was demonstrable both after formaldehyde treatment at 40 and 70 °C. The drug effects on sedimentation distributions obtained after mild formaldehyde reaction at 40 °C did not fit simple models of chain termination or DNA inactivation, and were as if a portion of the newly synthesized nucleoplasmic RNA was not susceptible to the drug-induced sedimentation reduction. The effects measured after formaldehyde treatment at 70 °C were equivalent to the introduction of additional random chain breaks due to drug.
The cytoplasm from rapidly labeled normal, poliovirus-infected HeLa cells and mouse A9 cells prepared by four different extraction procedures was analyzed by isopycnic gradient centrifugation. In all cases the rapidly labeled RNA present in the polysomes (“heavy” particles with densities ranging from 1.51 to 1.54 g/cc) was quantitatively released as “light” ribonucleoprotein complexes (ϱ = 1.40, 1.45, and 1.38 g/cc for each cell type, respectively) undistinguishable from free light particles. The latter (which in HeLa cells sediment heterogeneously from 40S to 400S) contained rapidly labeled RNA together with proteins that were barely labeled after prolonged incubation with mixed amino acids of high specific activity.In addition A9 cells contained also 1.34 and 1.45 g/cc complexes while 1.35 g/cc mature poliovirus particles were detected in the cytoplasm of polio-infected HeLa cells.Good concordance with published data was found for the density values of particles present as major components in the cytoplasm but not for the density of rapidly labeled particles.
Sucrose gradient centrifugation of SDS-treated, purified Uukuniemi virus labeled with 32P, gave three reproducible peaks of single-stranded RNA, which sedimented at 27–29 S (L), 22 S (M), and 17–19 S (S). In polyacrylamide-agarose gels these RNAs separated into four peaks with apparent molecular weights of about 4.1 × 106 (L), 1.9 × 106 (M), 0.88 × 106 (S1), and 0.78 × 106 (S2), respectively. The major M and L RNAs, which represented about 75% of the total, were found in a constant molar ratio of 1:4.6.Treatment of the RNA species with 1 M formaldehyde or by heating to 100 ° did not result in a degradation of the larger to the smaller RNAs.The base compositions of the RNA species did not differ significantly from each other, but were clearly different from those of the ribosomal 28 S and 18 S RNAs extracted from host chick embryo cells.RNAs with identical sedimentation values and mobilities in gels were found from Uukuniemi virus-infected cells labeled with [3H]uridine in the presence of actinomycin D.
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.
Rapidly labelled RNA and ribosomal RNA of HeLa and E. coli cells may be composed of smaller subunits.
Publisher Summary This chapter discusses the structure and assembly of simple RNA bacteriophages. It mentions that the components of the phage assembly system possess the intrinsic ability of forming a specific quaternary structure— namely, the phage. The versatility of the phage RNA is examined. Its 3000 nucleotides provide specific attachment sites for RNA replicase, ribosomes, coat protein, and A-protein thus enabling transcription, translation, regulation, and initiation of assembly. Besides the specific interactions with these proteins, the RNA acts catalytically in the capsid formation by a more unspecific mechanism. A special feature of phage RNA is the existence of nucleotide stretches that can be arranged in base paired loops. These are the providing sites for RNA-protein interactions. One of these loops, for instance, is located at one of the ribosome attachment sites; another near the 5’-terminus is a candidate for interaction with A-protein or RNA replicase. The chapter concludes that phage assembly is a promising tool for the study of the nature of protein-protein and protein-nucleic acid interactions.
The effect of removing Mg2+ from 30- and 50-S ribosomal subunits and ribosomal RNA by the direct addition of EDTA at room temperature has been followed using the analytical ultracentrifuge. 50-S subunits unfolded by a cooperative process, without the dissociation of the protein and RNA moieties, via two discrete, partially unfolded species, to give a 16-S particle. Ribosome-like particles were reformed when the unfolded species were dialysed into magnesium acetate. The unfolding process could be represented as follows:
By sedimentation in 2.5 x 10(-4)m sodium ethylenediaminetetraacetate at 25 C, replicative intermediate could be separated from single-stranded ribonucleic acid, both viral and ribosomal. All of the label in the replicative intermediate after a brief pulse of titrated uridine was resistant to ribonuclease.
1. Ribosomal RNA is isolated from 87-S Euglena gracilis ribosomes under a variety of salt and temperature conditions in order to study the integrity of the 25-S RNA molecule. 2. Both polyacrylamide gel electrophoresis and zone velocity sedimentation in sucrose gradients are used to monitor the degradation process. 3. In line with earlier reports it was found that Na+ (0.2 M) in the isolation milieu stabilizes the 25-S RNA. 4. The isolated 25-S RNA is readily degraded at 37-45° independent of the ionic conditions used during the isolation procedure. 5. The 20-S RNA is stable under identical conditions. 6. Under mild degradative conditions a novel 23-S RNA is formed. We postulate it to be a distinct degradation product of the 25-S RNA. 7. This 23-S RNA is clearly resolved in the polyacrylamide gel but not necessarily in the sucrose gradient. 8. The 25-S RNA molecule seems to be partly cleaved at selected loci in situ; depending on the isolation conditions the 25-S RNA molecule may undergo further degradation during the deproteinization procedure.
(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.
Summary Ribonucleic acid from vesicular stomatitis virus (VSV) virions (B particles), defective particles (T), and infected cells was examined by polyacrylamide gel electrophoresis, and apparent molecular weights were estimated. B particle RNA of 4.0×106 daltons from Indiana strain L and from Cocal virus was distinguishable from RNA of 4.5×106 daltons from Indiana strain BT-78 and several strains of New Jersey VSV. Abundant T particle RNA of 1.0×106 daltons, was present in one Indiana L stock and was absent in a plaque purified stock. Cocal virus had T particle RNA of 0.7×106 daltons, and one stock of Ogden strain New Jersey VSV had T particle RNA of 1.2×106 daltons. Little or no T particle RNA was detectable in several other VSV stocks. Major RNA components with apparent molecular weights, 2.4, 0.66, and 0.31×106 daltons in cells infected with Indiana strain L were complementary to virion RNA. The size relationships of B particle RNA from Indiana L and New Jersey strains were examined by sucrose density gradient sedimentation of undenatured and of formaldehyde denatured RNA. Sedimentation of undenatured RNA led to a conflict with gel electrophoresis results, inferring New Jersey had a lower molecular weight than Indiana L, but upon denaturation the relation was consistent with electrophoresis results.
The protein composition of several members of the animal picornavirus group is remarkably similar. Thus, the enteroviruses polio type 1, Echo 12 and bovine entero VG-5-27 (Maizel & Summers, 1968; Korant, Lonberg-Holm & Halperen, 1970; Johnston & Martin, 1971), the cardioviruses Maus-Elberfeld and encephalomyocarditis (Rueckert, Dunker & Stoltzfus, 1969; A. T. H. Burness, personal communication), the rhinoviruses 1A and HGP (Medappa, McLean & Rueckert, 1971; E. J. Stott & R. Killington, personal communication) and several of the seven immunological types of foot-and-mouth disease virus (Burroughs et al. 1971; P. Talbot, unpublished observations) contain three polypeptides with molecular weights ranging from 37 to 24 × 103 (VP1, VP2, VP3) and a smaller polypeptide with a molecular weight in the range of 7 to 13.5 × 103 (VP4). In several instances the molar ratio of the four polypeptides has been determined and, with the exception of rhinovirus 1A (Medappa et al. 1971), the three larger polypeptides are present in equimolar amounts and the smaller polypeptide is present in a half molar amount relative to the others.
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.
We have compared the total single-copy sequences transcribed as nuclear RNA in blastula and pluteus stage embryos of the sea urchin Tripneustes gratilla by hybridization of excess nuclear RNA with purified radioactive single-copy DNA. The kinetics of hybridization of either blastula or pluteus nuclear RNA with single-copy DNA show a single pseudo-first-order reaction with 34% of the single-copy genome. From the rate of the reaction and the purity of the nuclear RNA, it can be estimated that the reacting RNAs are present on the average at a concentration of one molecule per 14 nuclei. A mixture of blastula and pluteus RNA also hybridizes with 34% of the single-copy genome, indicating that the total complexity of RNAs transcribed at both stages is no greater than transcribed at each stage alone. The identity of the sequences transcribed by blastula and pluteus embryos was further examined by fractionation of the labeled DNA into sequences complementary and not complementary to pluteus RNA. This was achieved by hybridization of single-copy DNA to high pluteus RNA Cot, and separation of the hybridized and nonhybridized DNA on hydroxylapatite. Using either the DNA complementary or noncomplementary with pluteus RNA, essentially identical amounts of RNA:DNA hybrids are formed at high RNA Cot with blastula or pluteus RNA. Gross changes in the total RNA sequences transcribed do not appear to be involved in the developmental changes between blastula and pluteus, even though 45% of the mRNA sequences change between these two stages (Galau et al., 1976).
The picornavirus group numbers over 200 distinct serotypes, isolated from a wide variety of human and animal sources, and it is likely that many others exist in nature (1), as Vet unrecognised. Various sub-classifications of this large range of viruses have been proposed (2, 3). The most recent, and probably the most generally satisfactory classification (4, 5) arranges the picornaviruses into five genera (a) Cardioviruses, including encephalomyocarditis (EMG) and mengoviruses; (b) Human Rhinoviruses; (c) Equine Rhinoviruses; (d) Foot-and-Mouth Disease viruses; (e) Enteroviruses, including polio, Coxsackie viruses etc. The members of these different genera display differences in virus particle stability in the pH range 3–7, and in their particle density in caesium chloride, as well as in RNA base composition (see chapters 1 and 3).
Acute bee paralysis and sacbrood viruses, which multiply in the honey bee have some properties similar to those of the mammalian picornaviruses. Both bee viruses infect their host via the alimentary canal and are neurotropic in adult bees, and differ fundamentally in their ecology and pathology. The two viruses also resemble the mammalian picornaviruses in some physico chemical properties. For example, they are isometric particles 28 nm in diameter, have sedimentation coefficients of 160 S and are resistant to ether. Lee & Furgala provided chemical evidence that sacbrood virus contains RNA. In this paper the authors describe other physico chemical properties of the two viruses and compare these with those of the mammalian picornaviruses.
SUMMARY Several of the physico-chemical properties of representative members of the Picornaviridae family have been examined. On the basis of their buoyant density in caesium chloride, stability at pH 3 to 7 and the base composition of the virus RNA, a division of this family of viruses into six subgroups is suggested.
Solid samples of 1,3-dihydroxymethyluracil, 3-hydroxy-methyl-1-methyluracil, and 1-hydroxymethyl-3-methyluracil were prepared, and their structures were confirmed by spectroscopic analysis. The thermodynamics and kinetics of the formation of N-hydroxy-methylated uracils in aqueous formaldehyde solutions also were studied. The equilibrium constants for formation of N-1-hydroxymethyl derivatives were approximately twice those for formation of N-3-hydroxy-methyl derivatives, and they were formed more rapidly throughout the pH 3-8 range. Substituents at C-5 of uracil had little effect on the thermodynamics of N-hydroxymethylation. The potential usefulness of N-hydroxymethyl compounds as prodrugs is discussed.
The electrophoretic mobilities of mitochondrial and cell-sap ribosomal RNAs from Saccharomyces carlsbergensis have been measured relative to the mobility of Escherichia coli rRNA in 2.4% polyacrylamide gels at various temperatures and ionic strengths. At 5° C and in 20 mM sodium acetate, 40 mM Tris-acetate, 2 mM EDTA (pH 7.8), mitochondrial and cell-sap rRNAs co-migrated and were only partially resolved from E. coli rRNA. When the temperature of electrophoresis was increased the mobility of the mitochondrial rRNAs (and to a lesser extent of the cell-sap rRNAs) decreased relative to the mobility of the E. coli rRNA species, reaching a minimum between 9 and 16° C. As a consequence the mitochondrial and cell-sap rRNAs were completely resolved in this buffer at temperatures above 9° C. Similar effects of temperature on relative electrophoretic mobility were observed in 90 mM Tris-borate buffer at pH 8.3 and the effect was even more pronounced at low ionic strength (10 mM NaCl, 5 mM Tris-HCl, 2 mM EDTA, pH 7.5).
At low ionic strength the decrease in the relative electrophoretic mobility of mitochondrial rRNA with an increase in temperature was accompanied by a decrease in sedimentation coefficient from 21–22 S (large component) and 14 S (small component) at 5° C to 16 and 11.5 S at 20° C (calculated in relation to assumed S-values of 23 and 16 S for the rRNA species of E. coli).
We conclude that the electrophoretic mobility of RNA in polyacrylamide gels is more dependent on secondary structure than previous work had suggested and that caution is needed to interpret the relative mobility of mitochondrial RNA on gels in terms of molecular weight. In addition, our results show that judicious variation of temperature and ionic strength may allow the separation on gels of RNA mixtures that cannot be resolved under standard conditions.
About 80% of the RNA molecules extracted from encephalomyocarditis (EMC) virus were bound by oligo(dT)-cellulose under conditions which bind poly(A) but not poly(C) nor ribosomal RNA. This shows that most EMC virus RNA molecules contain a poly(A) tract. Both bound and unbound fractions contained RNA molecules of apparently the same size when examined by sucrose gradient sedimentation, but the bound fraction contained an adenylic acid-rich segment of about 20 nucleotides long, whereas the unbound RNA did not. The bound RNA had 200 times the specific infectivity of the unbound RNA which suggests that the poly(A) tract present in EMC virus RNA is required for infectivity.
The three RNA species isolated from virions of Uukuniemi virus, a proposed member of the newly defined Bunyaviridae family, have been characterized by analysis of 32P-labeled ribonuclease T1 oligonucleotides separated on two-dimensional polyacrylamide gels. Each species contains unique oligonucleotides not present in the two others, indicating that the genome of this virus is segmented. Each segment appears to contain a unique primary sequence with little or no overlapping among the segments. The complexities of the RNA segments as calculated from the radioactivity in unique oligonucleotides of defined lengths are about 8000 (L RNA), 3500 (M) and 1900 (S) nucleotides. Since these values are similar to the molecular weights determined by other methods, each size class of RNA corresponds to a single molecular species. The presence of a 5' terminal pppAp ... structure in each RNA segment confirms indications from electron microscopy that the apparently circular RNA segments are not covalently closed. The absence of either a 5' terminal "cap" or 3' terminal poly(A) supports the concept that Uukuniemi virus is a negative strand virus.
Time course and "chase" experiments showed that, after incubation of acute myeloid leukemia blast cells with a labeled RNA precursor, a large proportion of radioactivity remained associated with RNA molecules larger than 45 S even after several hr. Double-labeling experiments with [5-3H]uridine and [methyl-14C]methionine indicated that unmethylated giant heterogeneous RNA larger than 45 S is processed much more slowly than the 45 S ribosomal precursor, so that relatively large amounts of fairly stable RNA of the former class accumulate in the cell. The measurement of labeled giant heterogeneous RNA molecules bound to polyuridylate-fiberglass filters showed that molecules carrying polyadenylate segments seemingly turn over faster than those lacking polyadenylate.
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.
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.
The sequence complexity of heterogeneous nuclear RNA is sea urchin gastrulas was measured by RNA-driven hybridization reactions with nonrepetitive sea urchin DNA. 28.5% of the sequence complexity of the genome is represented in the nuclear RNA. This amounts to 1.74 X 10(8) nucleotides of diverse sequence, more than 10 times the nucleotide complexity of the polysomal messenger RNA extracted from sea urchin embryos at the same stage. The complex set of nuclear RNA sequences driving this hybridization reaction was shown to be the same as the rapidly labeled hnRNA, using pulse-labeled nuclear RNA as driver.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
- N Granboulan
- R M Franklin
Granboulan, N. & Franklin, R. M. (1966). J. molec. Biol.
22, 173.
- A Rogers
Rogers, A. (1966). Biochem. J. 100, 102.
- R F Gesteland
- H Boedtker
Gesteland, R. F. & Boedtker, H. (1964). J. molec. Biol. 8'
496.
- A S M Spirin
Spirin, A. S. (1963). Progr. Nuceic Acid Ru. 1, 301.
Staehelin, M. (1958). Biochim. biophy8. Acta, 29, 410.
- E J Eyring
- J Ofengand
Eyring, E. J. & Ofengand, J. (1967). Biochemistry, 6,2500.
Fenwick, M. L. (1968). Biochem. J. 107,481.
- J H Strauss
- R L Sinsheimer
Strauss, J. H. & Sinsheimer, R. L. (1963). J. molec. Biol. 7,
43.
- R Haselkorn
- P Doty
Haselkorn, R. & Doty, P. (1961). J. biol. Chem. 236, 2738.
Kurland, C. G. (1960). J. molec. Biol. 2, 83.
- S J Martin
Martin, S. J. (1966). Biochem. J. 101, 721.
- A S Spirin
Spirin, A. S. (1961). Biokhimiya, 26, 511 (Engl. trans.
p. 454).
- R M Bock
- N.-S H Ling
Bock, R. M. & Ling, N.-S. (1954). Analyt. Chem. 26, 1543.
Boedtker, H. (1960). J. molec. Biol. 2, 171.
Boedtker, H. (1967). Biochemi8try, 6, 2718.
Biochemistry, 6, 3583
- T Tamaoki
- B G Lane
- E K Wagner
- L Katz
- S Penman
Tamaoki, T. & Lane, B. G. (1967). Biochemistry, 6, 3583.
Wagner, E. K., Katz, L. & Penman, S. (1967). Biochem.
biophys. Res. Commun. 28, 152.