Action of Nucleases on Double‐Stranded RNA

Edy, V G; Székely, Mária; Loviny, Thérèse; Dreyer, Christine

doi:10.1111/j.1432-1033.1976.tb10051.x

Cited by 44 publications

(29 citation statements)

References 32 publications

(5 reference statements)

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“…Degradation was also observed at pH 7.2, but at a greater level of enzyme (data below). These results are not unexpected because it is known that this ribonuclease attacks double-stranded molecules (8,9,19).…”

Section: Rnase Treatment O/sindbis Virus Rn~4supporting

confidence: 52%

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The effect of ribonuclease on the replicative forms of Sindbis virus RNA

Martin

Riggsby

Beck

1979

Archives of Virology

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Three species of double-stranded RNA, designated RF I, RF II, and RF III in order of decreasing size (25), are produced by ribonuclease treatment of extracts of chicken embryo cells infected for 6 hours with Sindbis virus. Only one class of replicative form RNA is present in extracts not treated with ribonuclease; this class contains some molecules which can be enzymatically cleaved to produce the other two replicative forms. At a low level of enzyme (0.001 microgram/ml) the major species obtained was RF I, the replicative form of the genome. When the enzyme concentration was increased 10-, 100-, and 1000-fold, there was a progressive increase in the proportions of RF's II and III and a concomitant decrease in the proportion of RF I. The generation of RF's II and III by nuclease resulted in the ratio expected for these two species if they are produced by cleavage of RF I-like molecules. In preparations of isolated double-stranded RNA, only RF I and replicative intermediate RNA were present. Mild nuclease treatment of these preparations converted the replicative intermediates primarily to RF I. Higher enzyme levels generated greater proportions of RF II and RF III, but RF I-like molecules were the major source for these increased proportions. Treatment of the isolated naturally occurring replicative form with 0.01 microgram of ribonuclease per ml cleaved some molecules migrating as RF I during gel electrophoresis into molecules which migrated as RF II and RF III.

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Section: Rnase Treatment O/sindbis Virus Rn~4supporting

confidence: 52%

“…3e). ED¥ et al (8) have reported the random breakdown of the ds RNA of Penicillium chrysogenum virus into large fragments by treatment with 1%Nase under conditions which approximate those used in this study. EalKSOZ¢ and FI~A~KLI~ ~ (9) found that the ds ]%NA produced by phage 1% 17 was nicked by even mild 1%Nase treatment.…”

Section: Discussionmentioning

confidence: 87%

The effect of ribonuclease on the replicative forms of Sindbis virus RNA

Martin

Riggsby

Beck

1979

Archives of Virology

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“…poly(C) [37]. Also ribonuclease TI, to which doublestranded RNA is completely resistant [38], is a very acidic protein [39]. The idea that the action of a ribonuclease on double-stranded RNA may be related to the basic Tables 1 and 2), while sharing with ribonuclease BS-1, whale pancreatic ribonuclease and bovine ribonuclease A many catalytic properties.…”

Section: Guinea Pig a Pancreliicmentioning

confidence: 99%

Basic Charges on Mammalian R bonuclease Molecules and the Ability to Attack Double‐Stranded. RNA

Libonati

Furia

Beintema

1976

European Journal of Biochemistry

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A ribonuclease recently isolated from the pancreas of the lesser rorqual or pike whale (Baluenoptera acutorostrata), definitely more basic than bovine ribonuclease A, degrades very efficiently doublestranded RNA under conditions where the action of bovine pancreatic ribonuclease is minimal.On the contrary, a ribonuclease from the pancreas of reindeer, definitely less basic than bovine ribonuclease A, is also significantly less active than the bovine enzyme at degrading the doublehelical RNA.Two ribonucleases (from the pancreas of guinea pig, the A component, and red deer), with a net charge and a number of basic amino acids similar to those of bovine pancreatic ribonuclease, show an activity on double-stranded RNA which is indistinguishable from that of bovine ribonuclease A.Finally, a ribonuclease from the pancreas of rat, having a net charge equal to that of bovine ribonuclease A, but three basic residues more than the latter enzyme, shows a modest activity towards the double-helical RNA.These results are compatible with the idea that the action on secondary structures of RNA by ribonucleases non-specific for double-stranded RNA may be related to the number (and possibly the position) of basic charges on the protein molecules. Double-stranded RNA's are specifically degraded by ribonuclease 111 from Escherichiu coli [l -41, whereas the action on them of bovine pancreatic ribonuclease A is negligible, provided the ionic strength is high enough to maintain the secondary structure of the nucleic acids [5,6]. Resistance of RNA to digestion by ribonuclease A under standard conditions [7] is generally assumed, in fact, as one of the criteria to assess that the RNA has a stable secondary structure.Other nucleases have been described as capable of degrading double-stranded as well as single-stranded RNA [S, 91. Among them, bovine seminal ribonuclease (ribonuclease BS-l), a basic (PI = 10.3), dimeric protein [lo-121, in many respects similar to ribonuclease A [lo, 113, definitely degrades double-helical polyribonucleotides [13].Abbreviations. NaCl/Cit, 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7; poly(A), polyadenylate; poly(A) . poly(U) and poly(G) . poly(C), the complexes formed between polyadenylate and polyuridylate, and between polyguanylate and polycytidylate, respectively; poly(rUj . poly(dA), the complex formed by polyribouridylate and polydeoxyriboadenylate.Enzymes. Ribonuclease TI (EC 3.1.4.8); bovine pancreatic ribonuclease A (EC 3.1.4.22); ribonuclease 111 (EC 3.1.4.24). ~-Artificial dimerization [14-161 of bovine ribonuclease A makes this enzyme also capable of significantly depolymerizing double-stranded RNA's [13, 15-171. The dimeric nature of a ribonuclease active on double-stranded RNA, with the simultaneous availability of two active sites on its molecule, was consequently thought to be responsible for the activity towards double-helical polyribonucleotides [ 3 8,191. However, recent results (degradation of DNA . RNA hybrids by ribonuclease , and by aggregates of ribonuclease A [21], the main...

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“…4e). The sensitivity ofdsRNA to RNase is dependent on both salt and RNase concentration (Edy et al, 1976). On melting (100 °C, 2 min) followed by rapid cooling with ice water, the isolated dsRNA was completely degraded with RNase (not shown).…”

Section: Fractionation and Analysis Of Virus-induced Dsrnasmentioning

confidence: 99%

Replication of Turnip Rosette Virus RNA in Inoculated Turnip Protoplasts

Morris-Krsinich

Hull

1983

Journal of General Virology

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SUMMARYSynthesis of turnip rosette virus (TRosV)-induced RNA was examined in turnip protoplasts inoculated in vitro. TRosV RNA (mol. wt. 1.4 x 106; approx. 4.3 kb) was first detected 10 to 20 h post-inoculation by 32p labelling. When actinomycin D was added to protoplast cultures 6 h or less post-inoculation, labelling of virus RNA was less than in untreated cultures. Synthesis of TRosV RNA in infected protoplasts appeared to involve a double-stranded (ds)RNA; a replicative form (RF) of the virus RNA was isolated and characterized. The RF, mol. wt. 2.8 x 106 (approx. 4.3 kbp), was not degraded by treatment with RNase or DNase, contained 50~ sequences complementary to TRosV RNA (as determined by hybridization) and was denatured to singlestranded (ss)RNAs of mol. wt, ( x 10 -6) of 1"4, 0"7, 0"3 and 0"09 (4-3, 2"1, 0-9 and 0-27 kb respectively). Virus infection apparently induced the synthesis of a ssRNA of mol. wt. 0.3 x 106 which could be resolved from other RNA species in nucleic acid extracts from infected protoplasts. A minor dsRNA of 1.4 x 106 mol. wt. (2-1 kbp) was also apparently induced by virus infection. These virus-induced small RNA species may represent subgenomic messengers or their RF.

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Action of Nucleases on Double‐Stranded RNA

Cited by 44 publications

References 32 publications

The effect of ribonuclease on the replicative forms of Sindbis virus RNA

The effect of ribonuclease on the replicative forms of Sindbis virus RNA

Basic Charges on Mammalian R bonuclease Molecules and the Ability to Attack Double‐Stranded. RNA

Replication of Turnip Rosette Virus RNA in Inoculated Turnip Protoplasts

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