31P NMR spectra of ethidium, quinacrine, and daunomycin complexes with poly(adenylic acid).poly(uridylic acid) RNA duplex and calf thymus DNA

31P NMR provides a convenient monitor of the phosphate ester backbone conformational changes upon binding of the intercalating drugs ethidium, quinacrine, and daunomycin to sonicated poly(A).poly(U) and calf thymus DNA. 31P chemical shifts can also be used to assess differences in the duplex unwinding angles in the presence of the drug. Thus a new 31P signal, 1.8-2.2 ppm downfield from the double-stranded helix signals, is observed in the ethidium ion-poly(A).poly(U) complex. This signal arises from phosphates which are in perturbed environments due to intercalation of the drug. This is in keeping with the hypothesis that the P-O ester torsional angle in phosphates linking the intercalated base pairs is more trans-like. Similar though smaller deshielding of the 31P signals is observed in sonicated poly(A).poly(U)-quinacrine complexes as well as in the daunomycin complexes. The effect of added ethidium ion, quinacrine, and daunomycin on the 31P spectra of sonicated calf thymus DNA is consistent with Wilson and Jones' (1982) earlier study. In these drug-DNA complexes the drug produces a gradual downfield shift in the DNA 31P signal without the appearance of a separate downfield peak. These differences are attributed to differences in the rate of chemical exchange of the drug between free and bound duplex states. The previous correlation of 31P chemical shift with drug duplex unwinding angle (Wilson & Jones, 1982) is confirmed for both the RNA and DNA duplexes.     

Phosphorus-31 nuclear magnetic resonance of double- and triple-helical nucleic acids. Phosphorus-31 chemical shifts as a probe of phosphorus-oxygen ester bond torsional angles

The temperature dependence to the 31P NMR spectra of poly[d(GC)] . poly [d(GC)],d(GC)4, phenylalanine tRNA (yeast) and mixtures of poly(A) + oligo(U) is presented. The 31P NMR spectra of mixtures of complementary RNA and of the poly d(GC) self-complementary DNA provide torsional information on the phosphate ester conformation in the double, triple, and "Z" helix. The increasing downfield shift with temperature of the single-strand nucleic acids provides a measure of the change in the phosphate ester conformation in the single helix to coil conversion. A separate upfield peak (20-60% of the total phosphates) is observed at lower temperatures in the oligo(U) . poly(A) mixtures which is assigned to the double helix/triple helix. Proton NMR and UV spectra confirm the presence of the multistrand forms. The 31P chemical shift for the double helix/triple helix is 0.2-0.5 ppm upfield from the chemical shift for the single helix which in turn is 1.0 ppm upfield from the chemical shift for the random coil conformation.     

5'-Terminal cap structures of oligo(uridylic acid)-containing messenger ribonucleic acid from HeLa cells: comparison with other ribonucleic acid subpopulations

The 5'-terminal cap structures of 32P-labeled oligo(uridylic acid)-containing messenger ribonucleic acid [oligo(U+)mRNA] isolated from HeLa cell polyadenylated [poly(A+)] mRNA were analyzed and compared to those of the poly(A+) mRNA. A method employing P1 nuclease, alkaline phosphatase, and adsorption to activated charcoal showed that the types of cap core (m7 GpppXm) in oligo(U+) mRNA were essentially identical with those in poly(A+) mRNA. Analysis of RNase T2 digestion products of oligo(U+) mRNA demonstrated the presence of both cap 1 (m7GpppXmpYp) and cap 2 (m7GpppXmpYmpZp) in this subpopulation, confirming its cytoplasmic location. The base compositions of these two types of caps were different from each other and nonrandom but did not differ significantly between oligo(U+) and poly(A+) m RNA. The only observed difference between the mRNA populations was a higher ratio of cap 1 and cap 2 in the former. Possible implications of these findings for the relationship between oligo(U+) mRNA and poly(A+) mRNA are discussed.     

The alternating conformation of analogues of poly[d(AT)].

Synthetic DNAs were prepared containing 6-methyl adenine (m6A) in place of adenine and 5-ethyl uracil (Et5U) or 5-methoxymethyl uracil (Mm5U) in place of thymine. All three modifications destabilized duplex DNAs to varying degrees. The binding of ethidium was studied to analogues of poly[d(AT)]. There was no evidence of cooperative binding and the "neighbour exclusion rule" was obeyed in all cases although the binding constant to poly[d(m6AT)] was approximately 6 fold higher than to poly[d(AT)]. 31P NMR spectra were recorded in increasing concentrations of CsF. Poly[d(AEt5U)] showed two well-resolved signals separated by 0.55 ppm in 1 M CsF compared to 0.32 ppm for poly[d(AT)] under identical conditions. In contrast, poly[d(AMm5U)] and poly[d(m6AT)] showed two signals separated by 0.28 ppm and 0.15 ppm respectively, only when the concentration of CsF was raised to 2 M. The signals for poly[d(AT)] in 2 M CsF were better resolved and were separated by 0.41 ppm. These results suggest that minor modifications to the bases may have conformational effects which could be recognized by DNA-binding proteins.     

A method for isolation of oligo(uridylic acid)-containing messenger ribonucleic acid from HeLa cells

When cytoplasmic polyadenylated ribonucleic acid [poly(A+)RNA] from HeLa cells was treated with ribonuclease H (RNase H) and oligodeoxythymidylate [oligo(dT)] to remove its 3'-poly(A) tail, an increased binding to poly(A)-agarose was observed. The bound material, which comprised 4-6% of the initial RNA, contained 65-80% of the oligo(uridylic acid) [oligo(U)] sequences generated by RNase T1 digestion. Oligo(U) isolated from the bound fraction was shown to be 83% U and to have a U/G ratio of 33. In contrast, oligo(U) from the unbound material was 77% U and had a U/G ratio of 13, suggesting that it is shorter and less U rich than the oligo(U) in the bound fraction. On sucrose gradients, oligo(U+)RNA consistently sedimented with a larger s value than oligo(U-) RNA. The oligo(U) content of oligo(U+) RNA suggests one oligo(U) tract of 33 nucleotides per RNA molecule of 2000-3000 residues.     

Sequence content of oligo(uridylic acid)-containing messenger ribonucleic acid from HeLa cells

Oligo(uridylic acid)-containing [oligo(U+)] RNA was isolated from poly(adenylic acid)-containing [poly(A+)] mRNA from HeLa cells by using either formaldehyde pretreatment or poly(A) removal, both of which resulted in increased accessibility of oligo(U)-rich sequences to a poly(A)-agarose affinity column. In this report, we compared the sequence content of oligo(U+) RNA with that of molecules lacking oligo(U) [oligo(U-) RNA] by their relative hybridization to cDNA reverse-transcribed from poly(A+) mRNA and by comparison of their in vitro translation products synthesized in a rabbit reticulocyte lysate. Formaldehyde-modified poly(A+) RNA, treated to remove the formol adjuncts, was inactive as a template for in vitro protein synthesis; consequently, only depolyadenylated RNA, which retains its translatability, could be used in the translation studies. The hybridization kinetic experiments revealed that oligo(U+) RNA contained most of the sequence information present in oligo(U-) RNA but at a reduced level (ca. 25%), the majority of the oligo(U+) RNA sequences being poorly represented in the cDNA. This result was supported by one- and two-dimensional gel analysis of their in vitro translation products which showed that oligo(U+) RNA, although less effective as a template for translation than oligo(U-) RNA, coded for proteins, the most abundant of which were encoded by rare messages not highly represented in oligo(U-) RNA or the total poly(A+) RNA. Although some minor products were synthesized by both oligo(U+) and oligo(U-) RNA, at least 33 proteins were unique to or highly enriched in the pattern of products directed by oligo(U+) RNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Location of oligo(uridylic acid) sequences within messenger ribonucleic acid molecules of HeLa cells

A significant fraction of the polyadenylated mRNAs of HeLa cells contain an oligo(uridylic acid) [oligo(U)] sequence of 15-30 nucleotides. Several different experimental approaches were used to determine if these oligo(U)'s occupied similar sites within all mRNAs. In one approach, poly(adenylic acid)-containing mRNAs [poly(A+) mRNAs] averaging 2800 nucleotides in length were reduced to an average size of 500 nucleotides by controlled alkaline hydrolysis. Over 20% of the oligo(U)-containing fragments isolated from the hydrolysate retained a poly(A) sequence, showing that oligo(U)'s were not exclusively located near 5' ends of mRNA although 20% were apparently close to 3' ends. To confirm these observations, oligo(U)-containing mRNA [oligo(U+) mRNA] was exposed to the 3'-exonucleolytic activity of polynucleotide phosphorylase to produce fragments containing the 5' regions of mRNA. Each of a set of fragments of decreasing length generated by increased times of exposure of the mRNAs to the enzyme was found to have about the same oligo(U) content, including the shortest that averaged 550 nucleotides. These data not only eliminated an exclusive location for oligo(U) in either 3' or 5' ends of mRNA but also suggested that oligo(U)'s might be close to the 5' ends of some mRNAs. To verify this last observation, periodate-oxidized poly(A+) mRNA was labeled at the 5' caps and at 3'-adenosine residues by sodium [3H]borohydride reduction before it was nicked 3-5 times with alkali to produce 5' and 3' end-labeled pieces that could be separated with oligo(thymidylic acid)-cellulose.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-resolution phosphorus nuclear magnetic resonance spectra of yeast phenylalanine transfer ribonucleic acid. Melting curves and relaxation effects.

In a continuation of our studies on structural effects on the 31P chemical shifts of nucleic acids, we present 31P NMR spectra of yeast phenylalanine tRNA in the presence and absence of Mg2+. Superconducting field (146 MHz) and 32-MHz 31P NMR spectra reveal approximately 15 nonhelical diester signals spread over approximately 7 ppm besides the downfield terminal 3'-phosphate monoester. In the presence of 10 mM Mg2+, most scattered and main cluster signals do not shift between 22--66 degrees C, thus supporting our earlier hypothesis that 31P chemical shifts are sensitive to phosphate ester torsional and bond angles. At 70 degrees C, all of the signals merge into a single random coil conformation signal. Similar effects are observed in the absence of Mg2+ except that the transition melting temperature is approximately 20 degrees C lower. Measured spin-lattice and spin-spin relaxation times reveal another lower temperature transition besides the thermal denaturation process. A number of the scattered peaks are shifted (0.2--1.7 ppm) and broadened between 22 and 66 degrees C in the presence of Mg2+ as a result of this conformational transition between two intact tertiary structures. The loss of the scattered peaks in the absence of Mg2+ occurs in the temperature range expected for melting of a tertiary structure. An attempt to simulate the 31P spectra of tRNA Phe based upon the X-ray crystallographically determined phosphate ester torsional agles supports the suggestion that the large shifts in the scattered peaks are due to bond angle distortions in the tertiary structure.     

The synthesis of polyadenylic acid-containing ribonucleic acid by isolated mitochondria from Ehrlich ascites cells.

The synthesis of poly(A)-containing RNA by isolated mitochondria from Ehrlich ascites cells was studied. Isolated mitochondria incorporate [3H]AMP or [3H]UTP into an RNA species that adsorbs on oligo (dT)-cellulose columns or Millipore filters. Hydrolysis of the poly(A)-containing RNA with pancreatic and T1 ribonucleases released a poly(A) sequence that had an electrophoretic mobility slightly faster than 4SE. In comparison, ascites-cell cytosolic poly(A)-containing RNA had a poly(A) tail that had an electrophoretic mobility of about 7SE. Sensitivity of the incorporation of [3H]AMP into poly(A)-containing RNA to ethidium bromide and to atractyloside and lack of sensitivity to immobilized ribonuclease added to the mitochondria after incubation indicated that the site of incorporation was mitochondrial. The poly(A)-containing RNA sedimented with a peak of about 18S, with much material of higher s value. After denaturation at 70 degrees C for 5 min the poly(A)-containing RNA separated into two components of 12S and 16S on a 5-20% (w/v) sucrose density gradient at 4 degrees C, or at 4 degrees and 25 degrees C in the presence of formaldehyde. Poly(A)-containing RNA synthesized in the presence of ethidium bromide sedimented at 5-10S in a 15-33% (w/v) sucrose density gradient at 24 degrees C. The poly(A) tail of this RNA was smaller than that synthesized in the absence of ethidium bromide. The size of the poly(A)-containing RNA (approx. 1300 nucleotides) is about the length necessary for that of mRNA species for the products of mitochondrial protein synthesis observed by ourselves and others.     

Ovalbumin messenger ribonucleic acid. Purification and fractionation on the basis of polyadenylate content by thermal elution from oligodeoxythymidylate-cellulose.

Ovalbumin messenger RNA was purified from hen oviduct by immunoprecipitation of polysomes and oligo(dT)-cellulose chromatography. Two steps were introduced to improve the separation of mRNA and rRNA by oligo(dT)-cellulose. First, aggregates of mRNA and rRNA were dissociated by heating at 65degrees for 10 min before chromatography. Second, elution of the mRNA was achieved by stepwise increases in temperature rather than by lowering the ionic strength. Ovalbumin mRNA activity was eluted primarily in RNA fractions eluting between 45degrees and 55degrees. Polyacrylamide gel electrophoresis indicated that the ovalbumin mRNA thus obtained was essentiallyyy free of rRNA. The poly(A) content of various thermally eluted fractions was assayed by two methods. In the first (Favre, A., Bertazzoni, V., Berns, A.J.M., and Bloemendal, H. (1974) Biochem. Biophys. Res. Commun. 56, 273-280), the increase in fluorescence intensity of bound ethidium bromide was used to follow the formation of double-stranded RNA during titration of the mRNA with poly(U). In the second (Bishop, J. O., Rosbash, M., and Evans, D. (1974) J. Mol. Biol. 85, 75-86), poly(A) content was derived from the radioactivity remaining acid-insoluble after annealing mRNA fractions with [3H]poly(U) and treating with ribonuclease A. Both methods indicated that ovalbumin mRNA fractions eluting at higher temperatures contained greater amounts of poly(A). Values ranged from 44 to 248 mol of AMP/mol of mRNA, assuming 2200 total nucleotide residues for ovalbumin mRNA (Shapiro, D. J., and Schimke, R. T. (1975) J. Biol. Chem. 250, 1759-1764). Translational specific activities in a rabbit reticulocyte lysate system were essentiall constant for all fractions. From the binding of ethidium bromide it could be estimated that approximately 50% of the nucleotide residues in ovalbumin mRNA are base-paired.     

1H NMR study of an ethidium dimer poly(dA-dT) complex: evidence of a transition between bis and monointercalation

Comparative 1H NMR and optical studies of the interaction between poly(dA-dT), ethidium bromide (Et) and ethidium dimer (Et2) in 0.7 M NaCl are reported as a function of the temperature. Denaturation of the complexes followed at both polynucleotide and drug levels leads to a biphasic melting process for poly(dA-dT) complexed with ethidium dimer (t1/2 = 75 degrees C; 93 degrees C) but a monophasic one in poly(dA-dT): ethidium bromide complex (t1/2 = 74 degrees C). In both cases drug signals exhibit monophasic thermal dependence (Et = 81 degrees C; Et2 = 95 degrees C). Evidence is presented showing that the ethidium dimer bisintercalates into poly(dA-dT) in high salt, based on the observation that i) dimer and monomer ring protons exhibit similar upfield shifts upon DNA binding, ii) upfield shifts of DNA sugar protons are twice as large with the dimer than with ethidium bromide. Comparison between native DNA fraction and bound drug fraction indicates that ethidium covers, n = 2.5-3 base pairs. The dimer bisintercalates and covers, n = 5.7 base pairs when the helix fraction is high but as the number of available sites decreases the binding mode changes and the drug monointercalates (n = 2.9).     

Properties of polyadenylate-associated ribonucleic acid from Saccharomyces cerevisiae ascospores.

Bulk ribonucleic acid (RNA) was isolated from mechanically disrupted ascospores of Saccharomyces cerevisiae. After two passes over an oligo (dT10) cellulose column, the portion which bound, called poly(A)(+), was characterized. It is heterodisperse in size with a mean molecular weight of approximately 4 X 10(5), but contains some species as large as 7 X 10(5). The base composition is similar to vegetative poly(A)(+) RNA. The polyadenylate segment is also heterogenous in size, ranging from 90 to 20 bases in length, with a peak at approximately 60 nucleotides in length. Pulse-labeling of asci with [3H-methyl]methionine yields two "caps," 7-methyl guanosine-5'-triphosphoryl-5'-adenosine (or guanosine) identical to that found in vegetative poly(A)(+) RNA. The poly(A)(+) RNA in spores is found in polyribosomes which are, on the average, smaller than vegetative ones. Long-term labeling studies indicate that the fraction of poly(A)(+) RNA in spores is similar to that in vegetative cells.     

Circular dichroism study of polyriboxanthylic acid.

We report in the present paper the circular dichroism spectra of poly(X) at different pH and temperature values. The spectra are characteristic of three stable forms of poly(x) in the pH range of protonation of xanthosine. An electrostatic barrier is proposed to account for the hysteresis and metastability observed in a certain pH range. Some results on oligo(X) at basic pH are also presented. Poly(X) at basic pH is investigated also by hydrodynamic techniques.     

Reduction of prostatic binding protein-messenger ribonucleic acid sequences in rat prostate by castration

Messenger RNA coding for the three subunits of prostatic binding protein was isolated from polysomal RNA of rat ventral prostate by oligo (dT)-cellulose affinity chromatography and purified by repeated sedimentations through sucrose gradients under denaturing conditions. The purified mRNA migrated as a 9S peak in sucrose gradient centrifugation and hybridized with its cDNA within 2 log Rot units. In a cell-free reticulocyte lysate system, the mRNA directed the synthesis of three polypeptides of 12000, 9000, and 8000 daltons. These translation products were identified as the subunits of prostatic binding protein by immunoreaction with antibodies to this protein. Quantitation of prostatic binding protein-mRNA sequences in normal and castrated rats by hybridization with the cDNA probe showed that 3-day castration reduced the prostatic binding protein-mRNA sequences to less than 2% of the normal level. Similar hybridization was performed by using the cDNA to determine the level of prostatic binding protein coding sequences in polysomal poly(A) RNA following castration. The results showed a first-order rate constant of 3.92 X 10-2 h-1 for reduction of prostatic binding protein-mRNA sequences in polysomes. The period of castration required to reduce the level of these sequences to 50% of the normal level was calculated to be 17.6 h.     

Studies of yeast phenylalanine-accepting transfer ribonucleic acid backbone structure in solution by phosphorus-31 nuclear magnetic resonance spectroscopy.

Approximately 17 diester phosphates from the backbone structure of yeast tRNAPhe give rise to phosphorus resonances, which are resolved in its 31P NMR spectrum. To localize these diester phosphates within the tRNA structure, 31P NMR spectra of several chemically or enzymatically modified yeast tRNAPhe species were recorded. To this end selective modifications were performed in the anticodon, the DHU, and the T psi C loop. Modifications, performed in different loop regions, give rise to perturbation of different characteristic 31P resonances. The 31P spectra were correlated with the corresponding 1H NMR spectra of the ring N hydrogen-bonded protons and interpreted in view of the X-ray results obtained on yeast tRNAPhe. It is concluded that the diester phosphate groups, which experience an unusual shift, can be accounted for in the X-ray structure in terms of hydrogen-bonded phosphates groups and diester phosphates with a diester geometry, deviating from the normal double-helical conformation.     

Specific modification of nucleic acids inside the cell with alkylating derivatives of oligonucleotides ethylated at internucleotide phosphates

The alkylating derivatives of (C6H5NH)2P[dTp(Et)]4U and [dTp(Et)]9 U with completely esterified internucleotide phosphates bearing reactive 2,3 -O-4(N-2-chloroethyl N-methylamino)-benazylidene moiety attached to 3 -end cis-diol group were prepared. These alkylating derivatives of non-ionisable oligonucleotide analogs were demonstrated to penetrate efficiently into Krebs ascites tumor cells and to alkylate nucleic acids inside the cells with a strong preference towards complementary poly(A)-fragments of mRNA.     

Isolation and characterization of poly(adenylic acid)-containing messenger ribonucleic acid from rat liver polysomes

Undegraded rat liver polysomes were obtained after homogenizing the tissue in a medium containing NH4Cl, heparine, and yeast tRNA. Purification of poly(A)-containing RNA from polysomal RNA was accomplished by affinity chromatography on oligo(dT)-cellulose columns. Poly(A)-containing RNA molecules were monitored by the formation of ribonuclease-resistant hybrids with [3H]poly(U). To improve the separation of messenger RNA and ribosomal RNA by oligo(dT)-cellulose it was found essential to dissociate the aggregates formed between both molecular species by heat treatment in the presence of dimethylsulfoxide (Me2SO) prior to chromatography. Sucrose gradient analysis under denaturing conditions showed that the preparations obtained were virtually free of ribosomal RNA. Poly(A)-containing RNA constituted approx. 2.2% of the total polysomal RNA and the number average size was 1500--1800 nucleotides, as judged by sedimentation analysis on sucrose density gradients containing Me2SO. Approximately 8.2% of the purified preparation obtained was able to anneal with [3H]poly(U); the number average nucleotide length of the poly(A) segment of the RNA population was calculated to be 133 adenylate residues. Based on these values, our preparations appear to be greater than 90% pure. The RNA fractions obtained after oligo(dT)-cellulose chromatography were used to direct the synthesis of liver polypeptides in a heterologous cell-free system derived from wheat-germ. The system was optimized with respect to monovalent and divalent cations, and presence of polyamines (spermine). More than 65% of the translational activity present in the unfractionated polysomal RNA was recovered in the final poly(A)-containing RNA fraction. However, about 25% of the activity was found to be associated with the unbound fraction which was essentially free of poly(A)-containing RNA. Immunoprecipitation analysis with a specific antiserum to rat serum albumin demonstrated that about 6--8% of the labeled synthetic products translated from the poly(A)-containing RNA sample corresponded to serum albumin. Analysis of the translation products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a heterogeneous distribution of molecular sizes ranging from 15 000 to greater than 70 000 daltons. Spermine not only increased the overall yield and extent of protein synthesis, but also resulted in higher yields of large protein products. Under optimal translation conditions a discrete peak representing about 7% of the total radioactivity was observed to migrate with rat serum albumin.     

High-resolution phosphorus nuclear magnetic resonance spectroscopy of transfer ribonucleic acids: multiple conformations in the anticodon loop

The temperature dependence of the 31P NMR spectra of yeast phenylalanine tRNA, E. coli tyrosine, glutamate (2), and formylmethionine tRNA is presented. The major difference between the 31P NMR spectra of the different acceptor tRNAs is in the main cluster region between -0.5 and -1.3 ppm. This confirms an earlier assignment of the main cluster region to the undistorted phosphate diesters in the hairpin loops and helical stems. In addition the 31P NMR spectra for all tRNAs reveal approximately 16 nonhelical diester signals spread over approximately 7 ppm besides the downfield terminal 3'-phosphate monoester. In the presence of 10 mM Mg2+ most scattered and main cluster signals do not shift between 22 and 66 degrees C, thus supporting our earlier hypothesis that 31P chemical shifts are sensitive to phosphate ester torsional and bond angles. At greater than 70 degrees C, all of the signals merge into a single random-coil conformation signal. A number of the scattered peaks are shifted (0.2-1.7 ppm) and broadened between 22 and 66 degrees C in the presence of Mg2+ and spermine as a result of a conformational transition in the anticodon loop. The 31P NMR spectrum of the dimer formed between yeast tRNAPhe and E. coli tRNA 2Glu is reported. This dimer simulates codon-anticodon interaction since the anticodon triplets of the two tRNAs are complementary. Evidence is presented that the anticodon-anticodon interaction alters the anticodon conformation and partially disrupts the tertiary structure of the tRNA.     

Magic-angle spinning (31)P NMR spectroscopy of condensed phosphates in parasitic protozoa: visualizing the invisible

We report the results of a solid-state (31)P nuclear magnetic resonance (NMR) spectroscopic investigation of the acidocalcisome organelles from Trypanosoma brucei (bloodstream form), Trypanosoma cruzi and Leishmania major (insect forms). The spectra are characterized by a broad envelope of spinning sidebands having isotropic chemical shifts at approximately 0, -7 and -21 ppm. These resonances are assigned to orthophosphate, terminal (alpha) phosphates of polyphosphates and bridging (beta) phosphates of polyphosphates, respectively. The average polyphosphate chain length is approximately 3.3 phosphates. Similar results were obtained with whole L. major promastigotes. (31)P NMR spectra of living L. major promastigotes recorded under conventional solution NMR conditions had spectral intensities reduced with respect to solution-state NMR spectra of acid extracts, consistent with the invisibility of the solid-state phosphates. These results show that all three parasites contain large stores of condensed phosphates which can be visualized by using magic-angle spinning NMR techniques.     

Role of 2',5'-oligo(adenylic acid) polymerase in the degradation of ribonucleic acid linked to double-stranded ribonucleic acid by extracts of interferon-treated cells

RNA covalently linked to double-stranded RNA (dsRNA) is preferentially degraded in extracts of interferon-treated HeLa cells [Nilsen, T. W., & Baglioni, C. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2600-2604]. The size of the dsRNA required for this preferential degradation has been determined by annealing poly(I) of known length to the poly(C) tract of encephalomyocarditis virus (EMCV) RNA or by annealing poly(U) to poly(A) of known length of vesicular stomatitis virus mRNA. The dsRNA must be longer than about 60 base pairs to observe the preferential degradation of RNA. Moreover, triple-stranded regions that do not activate synthesis of 2',5'-oligo(A) and ethidium bromide, which intercalates in dsRNA and blocks 2',5'-olido(A) polymerase activation, prevent this degradation. Ethidium also blocks the degradation of the replicative intermediate of EMCV by extracts of interferon-treated cells. These experiments indicate that synthesis of 2',5'-oligo(A) is required for the degradation of RNA linked to dsRNA. The 2',5'-oligo(A)-dependent endonuclease does not cleave single- or double-stranded DNA, nor does it cleave homopolyribonucleotides. The potential role of the 2',5'-oligo(A) polymerase/endonuclease system in the inhibition of viral RNA replication is discussed.