Archives of Virology

, Volume 140, Issue 8, pp 1373–1383 | Cite as

Experimental evaluation of the ribonuclease protection assay method for the assessment of genetic heterogeneity in populations of RNA viruses

  • M. A. Aranda
  • A. Fraile
  • F. Garcia-Arenal
  • J. M. Malpica
Original Papers


The ribonuclease (RNase) protection assay (RPA) was evaluated as a method to estimate genetic distances among sequence variants of RNA viruses. The patterns of fragments generated, under different RPA conditions, by three sets of RNA sequence variants of known nucleotide sequence, were analyzed. Both the effectiveness of cleavage (i.e. the probability of cleavage in a certain heteroduplex) and its degree (i.e. in all the molecules in the assay or in a part of them) varied largely according to the nature of the mismatch. Probability and degree of cleavage were also dependent on distant sequence context effects. No correlation could be established between context and cleavage, so that the pattern of fragments in RPA cannot be unequivocally predicted from sequence information. Accordingly, nucleotide sequence differences between two sequence variants cannot be directly derived from RPA data. For all three sequence sets linear relationships were found between the number of non-shared fragments in the RPAs of two variants and their nucleotide sequence differences. Nevertheless, both linearity and the linear regression parameters varied largely according to the sequence set and according to RPA conditions, in a non-predictable way. Thus, under experimental conditions, RPA may not be as appropriate a method to estimate genetic distances between RNA sequences as simulation under an ideal model suggested. Possible ways to diminish the gap between the ideal model and the experimental procedure are proposed.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Aranda MA, Fraile A, García-Arenal F (1993) Genetic variability and evolution of the satellite RNA of cucumber mosaic virus during natural epidemics. J Virol 67: 5896–5901Google Scholar
  2. 2.
    Bruening G, Beachy RN, Scalla R, Zaitlin M (1976) In vitro and in vivo translation of the ribonucleic acids of the cowpea strain of tobacco mosaic virus. Virology 71: 498–517Google Scholar
  3. 3.
    Cristina J, Moya A, Arbiza J, Russ J, Hortal M, Albó C, García-Barreno B, García O, Melero JA, Portela A (1991) Evolution of the G and P genes of human respiratory syncytial virus (subgroup A) studied by the RNase A mismatch cleavage method. Virology 184: 210–218Google Scholar
  4. 4.
    Dopazo J, Sobrino F, López-Galíndez C (1993) Estimates by computer simulation of genetic distances from comparison of RNase A mismatch cleavage patterns. J Virol Methods 45: 73–82Google Scholar
  5. 5.
    England TE, Bruce AG, Uhlenbeck OC (1980) Specific labelling of 3′ termini of RNA with T4 RNA ligase. Methods Ezymol 65: 65–74Google Scholar
  6. 6.
    Fichot O, Girard M (1990) An improved method for sequencing RNA templates. Nucleic Acids Res 16: 6162Google Scholar
  7. 7.
    Freier SM, Kierzek R, Jeaeger JA, Sugimoto N, Caruthers MH, Neilson T, Turner DH (1986) Improved free-energy parameters for predictions of RNA duplex stability. Proc Natl Acad Sci USA 83: 9373–9377Google Scholar
  8. 8.
    García-Arenal F, Zaitlin M, Palukaitis P (1987) Sequence analysis of six satellite RNAs of cucumber mosaic virus: primary sequence and secondary structure alterations do not correlate with differences in pathogenicity. Virology 158: 339–347Google Scholar
  9. 9.
    Grange DK, Gottesman GS, Lewis MB, Marini JC (1990) Detection of point mutations in type I collagen by RNase digestion of RNA/RNA hybrids. Nucleic Acids Res 18: 4227–4236Google Scholar
  10. 10.
    He L, Kierzek R, Santa Lucía J, Walter AE, Turner DH (1991) Neartest-neighbour parameters for G.U. mismatches. Biochemistry 30: 11124–11132Google Scholar
  11. 11.
    Holbrook SR, Cheong C, Tinoco I, Kim SH (1991) Crystal structure of an RNA double helix incorporating a track of non-Watson-Crick base pairs. Nature 353: 579–581Google Scholar
  12. 12.
    Krupp G, Gross MJ (1979) Rapid RNA sequencing: nucleases fromStaphylococcus aureus andNeurospora crassa discriminate between uridine and cytidine. Nucleic Acids Res 6: 3481–3490Google Scholar
  13. 13.
    Kurath G, Palukaitis P (1989) RNA sequence heterogeneity in natural populations of three satellite RNAs of cucumber mosaic virus. Virology 173: 231–240Google Scholar
  14. 14.
    Kurath G, Rey MEC, Dodds JA (1992) Analysis of genetic heterogeneity within the type strain of satellite tobacco mosaic virus reveals several variants and a strong bias for G to A substitution mutations. Virology 189: 233–244Google Scholar
  15. 15.
    Lot H, Marrou J, Quiot JB, Esvan C (1972) Contribution à l'étude du virus de la mosaïque du concombre (CMV). Méthode de purification rapide du virus. Ann Phytopathol 4: 25–38Google Scholar
  16. 16.
    Myers RM, Larin Z, Maniatis T (1985) Detection of single base substitutions by ribonuclease cleavage at mismatches in RNA:RNA duplexes. Science 230: 1242–1246Google Scholar
  17. 17.
    Owen J, Shintaku M, Aeschleman P, Ben Tahar S, Palukaitis P (1990) Nucleotide sequence and evolutionary relationships of cucumber mosaic virus (CMV) strains: CMV RNA3. J Gen Virol 71: 2243–2249Google Scholar
  18. 18.
    Palukaitis P, Roossinck MJ, García-Arenal F (1994) Applications of ribonuclease protection assay in plant virology. Methods Mol Genet 4: 237–250Google Scholar
  19. 19.
    Perucho M (1989) Detection of single-base substitutions with the RNase A mismatch cleavage method. Strat Mol Biol 2: 37–41Google Scholar
  20. 20.
    Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  21. 21.
    Smith HO, Bernstiel ML (1976) A simple method for DNA restriction site mapping. Nucleic Acids Res 3: 2387–2398Google Scholar
  22. 22.
    Solís I, García-Arenal F (1990) The complete nucleotide sequence of the genomic RNA of the tobamovirus tobacco mild green mosaic virus. Virology 177: 553–558Google Scholar
  23. 23.
    Sokal RR, Rohlf FJ (1981) Biometry, 2nd ed. Freeman, New YorkGoogle Scholar
  24. 24.
    Winter E, Yamamoto F, Almoguera C, Perucho M (1985) A method to detect and characterize point mutations in transcribed genes: amplification and overexpression of the mutant c-ki-ras allele in human tumor cells. Proc Natl Acad Sci USA 82: 7575–7579Google Scholar

Copyright information

© Springer-Verlag 1995

Authors and Affiliations

  • M. A. Aranda
    • 1
  • A. Fraile
    • 1
  • F. Garcia-Arenal
    • 1
  • J. M. Malpica
    • 2
  1. 1.Departmento de Patología Vegetal, E.T.S.I. AgrónomosCiudad UniversitariaMadridSpain
  2. 2.Area de Biología Moleculary Virología Vegetal, CIT-INIAMadridSpain

Personalised recommendations