Detection and Quantification of Modified Nucleotides in RNA Using Thin-Layer Chromatography

  • Henri Grosjean
  • Gérard Keith
  • Louis Droogmans
Part of the Methods in Molecular Biology book series (MIMB, volume 265)


Identification of a modified nucleotide and its localization within an RNA molecule is a difficult task. Only direct sequencing of purified RNA molecules and high-performance liquid chromatography mass spectrometry analysis of purified RNA fragments allow determination of both the type and location of a given modified nucleotide within an RNA of 50–150 nt in length. The objective of this chapter is to describe in detail a few simple procedures that we have found particularly suited for the detection, localization, and quantification of modified nucleotides within an RNA of known sequence. The methods can also be used to reveal the enzymatic activity of a particular RNA-modifying enzyme in vitro or in vivo. The procedures are based on the use of radiolabeled RNA (with [32P], [14C], or [3H]) or [32P]-postlabeled oligonucleotides and twodimensional thin-layer chromatography of labeled nucleotides on cellulose plates. This chapter provides useful maps of the migration characteristics of 70 modified nucleotides on thin-layer cellulose plates.

Key Words

RNA maturation posttranscriptional modification thin-layer chromatography sequencing modified nucleotides 2′-O-methylation nuclease RNase kinase phosphatase 


  1. 1.
    Rozenski, J., Crain, P. F., and McCloskey, J. A. (1999) The RNA modification database—1999 update. Nucleic Acids Res. 27, 196–197.PubMedCrossRefGoogle Scholar
  2. 2.
    Sprinzl, M., Horn, C., Brown, M., Ioudovitch, A., and Steinberg, S. (1998) Compilation of tRNA sequences and sequences of tRNA genes. Nucleic Acids Res. 26, 148–153.PubMedCrossRefGoogle Scholar
  3. 3.
    Limbach, P. A., Crain, F. C., and McCloskey, J. A. (1994) Summary: the modified nucleosides of RNA. Nucleic Acids Res. 12, 2183–2196.CrossRefGoogle Scholar
  4. 4.
    Brownlee, G. G. (1967) Determination of sequences in RNA, in Laboratory Techniques in Biochemistry and Molecular Biology (Work, T. S. and ork, E. E., eds.), North-Holland/American Elsevier, Amsterdam.Google Scholar
  5. 5.
    Sanger, F. and Brownlee, G. G. (1967) A two dimensional fractionation method for radioactive nucleotides, in Methods in Enzymology, vol. XII (Grossman, L. and Moldave, K., eds.), Academic, New York, pp. 361–381.Google Scholar
  6. 6.
    Szekely, M. and Sanger, F. (1969) Use of polynucleotide kinase in fingerprinting non-radioactive nucleic acids. J. Mol. Biol. 43, 607–617.PubMedCrossRefGoogle Scholar
  7. 7.
    Stanley, J. and Vassilenko, S. (1978) A different approach to RNA sequencing. Nature 274, 87–89.PubMedCrossRefGoogle Scholar
  8. 8.
    Silberklang, M. O., Gillum, A. M., and RajBhandary, U. L. (1979) Use of in vitro 32P labeling in the sequence analysis of non-radioactive tRNAs, in Methods in Enzymology, vol. LIX (Moldave, K. and Grossman, L., eds.), Academic, New York, pp. 58–109.Google Scholar
  9. 9.
    Kuchino, Y., Hanyu, N., and Nishimura, S. (1987) Analysis of modified nucleosides and nucleotide sequence of tRNA, in Methods in Enzymology, vol. 155 (Wu, R., ed.), Academic, New York, pp. 379–396.Google Scholar
  10. 10.
    Nishimura. S. and Kuchino, Y. (1983) Characterization of modified nucleosides in tRNA, in Methods of DNA and RNA Sequencing (Weissman, M. S., ed.), Praeger, New York, pp. 235–255.Google Scholar
  11. 11.
    Keith, G. (1990) Nucleic acid chromatographic isolation and sequence methods, in Chromatography and Modifications of Nucleosides, vol. 45A, Journal of Chromatography Library Series (Gehrke, C. W. and Kuo, K. C., eds.), Elsevier, The Netherlands, pp. A103–A141.Google Scholar
  12. 12.
    McCloskey, J. A. (1990) Constituents of nucleic acids: overview and strategy, in Methods in Enzymology, vol. 193 (McCloskey, J. A., ed.), Academic, New York, pp. 796–824.Google Scholar
  13. 13.
    Kowalak, J. A., Pomerantz, S. C., Crain, P. F., and McCloskey, J. A. (1993) A novel method for the determination of posttranscriptional modification in RNA by mass spectrometry. Nucleic Acids Res. 21, 4577–4585.PubMedCrossRefGoogle Scholar
  14. 14.
    Rozenski, J. and McCloskey, J. A. (1999) Determination of nearest neighbors in nucleic acids by mass spectrometry. Anal. Chem. 71, 454–459.CrossRefGoogle Scholar
  15. 15.
    Crain, P. F. (1998) Detection and structure analysis of modified nucleosides in RNA by mass spectrometry, in Modification and Editing of RNA (Grosjean, H. and Benne, R., eds.), ASM Press, Washington, DC, pp. 47–57.Google Scholar
  16. 16.
    Buck, M, Connick, M., and Ames, B. N. (1983) Complete analysis of tRNA-modified nucleosides by high-performance liquid chromatography: the 29 modified nucleosides of S. typhimurium and E. coli tRNA. Anal. Biochem. 129, 1–13.PubMedCrossRefGoogle Scholar
  17. 17.
    Gehrke, C. W. and Kuo, K. C. (1989) Ribonucleoside analysis by reversed-phase high-performance liquid chromatography. J. Chromatogr. 471, 3–36.PubMedCrossRefGoogle Scholar
  18. 18.
    Gehrke, C. W. and Kuo, K. C. (1990) Ribonucleoside analysis by reverse-phase high performance liquid chromatography, in Chromatography and Modifications of Nucleosides, vol. 45A, Journal of Chromatography Library Series (Gehrke, C. W. and Kuo, K. C., eds.), Elsevier, The Netherlands, pp. A3–A71.Google Scholar
  19. 19.
    Pomerantz, S. C. and McCloskey, J. A. (1990) Analysis of RNA hydrolyzates by liquid chromatography-mass spectrometry, in Methods in Enzymology, vol. 193 (McCloskey, J. A., ed.), Academic, New York, pp. 796–824.Google Scholar
  20. 20.
    Polson, A. G., Crain, P. F., Pomerantz, S. C., McCloskey, J. A., and Bass, B. L. (1991) The mechanism of adenosine to inosine conversion by double-stranded RNA unwinding/modifying activity: a high-performance liquid chromatographymass spectrometry analysis. Biochemistry 30, 11,507–11,514.PubMedCrossRefGoogle Scholar
  21. 21.
    Xue, H., Glasser, A. L., Desgres, J., and Grosjean, H. (1993) Modified nucleotides in Bacillus subtilis tRNA-trp hyperexpressed in a E. coli. Nucleic Acids Res. 21, 2479–2486.PubMedCrossRefGoogle Scholar
  22. 22.
    Takeda, N., Nakamura, M., Yoshizumi, H., and Tatematsu, A. (1994) Structural characterization of modified nucleosides in tRNA hydrolysates by frit-fast atom bombardment liquid chromatography/mass spectrometry. Biol. Mass Spectrom. 23, 465–475.PubMedCrossRefGoogle Scholar
  23. 23.
    Dalluge, J. J., Hashizume, T., and McCloskey, J. A. (1996) Quantitative measurement of dihydrouridine in RNA using isotope dilution liquid chromatography-mass spectrometry (LC/MS). Nucleic Acids Res. 24, 3242–3245.PubMedCrossRefGoogle Scholar
  24. 24.
    Tanigushi, H. and Hayashi, N. (1998) A liquid chromatography/electrospray mass spectrometric study on the posttranscriptional modification of tRNA. Nucleic Acids Res. 26, 1481–1486.CrossRefGoogle Scholar
  25. 24a.
    Kirpekar, F., Douthwaite, S., and Roepstorff, P. (2000) Mapping posttranscriptional modifications in 5S ribosomal RNA by MALDI mass spectometry. RNA 6, 296–306.PubMedCrossRefGoogle Scholar
  26. 24b.
    Madsen, C. T., Mengel-Jorgensen, J. M., Kirpekar, F., and Douthwaite, S. (2003) Identifying the methyltransferases for m5U747 and m5U1939 in 23S rRNA using MALKDI mass spectometry. Nucleic Acids Res. 31, 4738–4746.PubMedCrossRefGoogle Scholar
  27. 25.
    Keith, G. (1995) Mobilities of modified ribonucleotides on two-dimensional cellulose thin-layer chromatography. Biochimie 77, 142–144.PubMedCrossRefGoogle Scholar
  28. 26.
    Nishimura. S. (1979) Chromatographic mobilities of modified nucleotides, in Transfer RNA, Structure, Properties, and Recognition (Schimmel, P. R., Söll, D., and Abelson, J. N., eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 547–552.Google Scholar
  29. 27.
    Hashimoto, S., Sakai, M., and Muramatsu, M. (1975) 2′-O-methylated oligonucleotides in ribosomal 18S and 28S RNA of a mouse hepatoma, MH134. Biochemistry 14, 1956–1964.PubMedCrossRefGoogle Scholar
  30. 28.
    Holley, R. W., Apgar, J., and Merrill, S. H. (1961) Evidence for the liberation of a nuclease from human fingers. J. Biol. Chem. 236, PC42, PC43.PubMedGoogle Scholar
  31. 29.
    Ikemura, T. (1989) Purification of RNA molecules by gel techniques, in Methods in Enzymology, vol. 180 (Moldave, K. and Grossman, L., eds.), Academic, New York, pp. 14–25.Google Scholar
  32. 30.
    Tanner, K. (1990) Purifying RNA by column chromatography, in Methods in Enzymology, vol. 180 (Moldave, K. and Grossman, L., eds.), Academic, New York, pp. 25–29.Google Scholar
  33. 31.
    Cedergren, R. and Grosjean, H. (1987) RNA design by in vitro RNA recombination and synthesis. Biochem. Cell. Biol. 65, 677–692.PubMedCrossRefGoogle Scholar
  34. 32.
    Grosjean, H., Motorin, Y., and Morin, A. (1998) RNA-modifying and RNA-editing enzymes: methods for their detection, in Modification and Editing of RNA (Grosjean, H. and Benne, R., eds.), ASM Press, Washington, DC, pp. 21–46.Google Scholar
  35. 33.
    Moore, M. J. and Query, C. C. (1998) Uses of site-specifically modified RNAs constructed by RNA ligation, in RNA-Protein Interactions: A Practical Approach (Smith, C., ed.), IRL, Oxford, UK, pp. 75–108.Google Scholar
  36. 34.
    Yu, Y. T. and Steitz, J. A. (1997) A new strategy for introducing photoactivable 4-thiouridine (S4U) into specific positions in a long RNA molecule. RNA 3, 807–810.PubMedGoogle Scholar
  37. 35.
    Paul, M. S. and Bass, B. L. (1998) Inosine exists in mRNA at tissue-specific levels and is most abundant in brain mRNA. EMBO J. 17, 1120–1127.PubMedCrossRefGoogle Scholar
  38. 36.
    Warner, J. R. (1991) Labeling of RNA and phosphoproteins in iS. cerevisiae, in Methods in Enzymology, vol. 194 (Guthrie, C. and Fink, G. R., eds.), Academic, New York, pp. 423–428.Google Scholar
  39. 37.
    Landy, A., Abelson, J., Goodman, H. M., and Smith, J. D. (1967) Specific hybridization of tyrosine tRNA with DNA from transducing bacteriophage Phi80 carrying the amber suppressor gene suIII. J. Mol. Biol. 29, 457–471.PubMedCrossRefGoogle Scholar
  40. 38.
    Monier, R., Stephenson, M. L., and Zamecnick, P. C. (1960) The preparation and some properties of a low molecular weight RNA from Baker’s yeast. Biochem. Biophys. Acta 43, 1–8.PubMedCrossRefGoogle Scholar
  41. 39.
    Brubacker, L. H. and McCorquodale, D. J. (1963) The preparation of amino acid-tRNA from Escherichia coli by direct phenol extraction of intact cells. Biochem. Biophys. Acta. 76, 48–53.CrossRefGoogle Scholar
  42. 40.
    Tsurui, H., Kumazawa, Y., Sanokawa, R., Watanabe, Y., Kuroda, T., Wada, A., Watanabe, K., and Shirai, T. (1994) Batchwise purification of specific tRNAs by solid-phase DNA probe. Anal. Biochem. 221, 166–172.PubMedCrossRefGoogle Scholar
  43. 41.
    Mörl, M., Dörner, M., and Pääbo, S. (1994) Direct purification of tRNAs using oligonucleotides coupled to magnetic beads, in Advances in Biomagnetic Separation (Uhlén, M., Hornes, E., and Olsik, O., eds.). Eaton, Natik, MA, pp. 107–111.Google Scholar
  44. 42.
    Sakano, H., Shimura, Y. and Ozeki, H. (1974) Selective modification of nucleosides of tRNA precursors accumulated in a temperature sensitive mutant of E. coli. FEBS Lett. 48, 117–121.PubMedCrossRefGoogle Scholar
  45. 43.
    Nishikura, L. and De Robertis, E.M. (1981) RNA processing in microinjected Xenopus laevis oocytes: sequential addition of base modification in a spliced tRNA. J. Mol. Biol. 154, 405–420.CrossRefGoogle Scholar
  46. 44.
    Sullivan, M. A., Cannon, J. F., Webb, F. H., and Bock, R. M. (1985) Anti-suppressor mutation in E. coli defective in biosynthesis of 5-methylaminomethyl-2-thiouridine. J. Bacteriol. 161, 368–376.PubMedGoogle Scholar
  47. 45.
    Adanchi, Y., Yamao, F., Muto, A., and Osawa, S. (1989) Codon recognition patterns as deduced from sequences of the complete set of tRNA species in Mycoplasma capricolum. J. Mol. Biol. 209, 37–54.CrossRefGoogle Scholar
  48. 46.
    Laten, H. M., Cramer, J. H., and Rownd, R. H. (1983) Thiolated nucleotides in yeast transfer RNA. Biochem. Biophys. Acta 741, 1–6.PubMedGoogle Scholar
  49. 47.
    Cavaillé, J., Chetouani, F., and Bachellerie, J. P. (1999) The yeast S. cerevisiae YDL112w ORF encodes the putative 2′-O-ribose methyltransferase catalyzing the formation of Gm18 in tRNAs. RNA 5, 66–81.PubMedCrossRefGoogle Scholar
  50. 48.
    Pintard, L., Lecointe, F., Bujnicki, J. M., Bonnerot, C., Grosjean, H., and Lapeyre, B. (2002) Trm7p catalyses the formation of two 2′-O-methylriboses in yeast tRNA anticodon loop. EMBO J. 21, 1811–1820.PubMedCrossRefGoogle Scholar
  51. 49.
    Gupta, R. (1984) Halobacterium volcanii identification of 41 tRNAs covering all amino acids and the sequences of 33 class I tRNAs. J. Biol. Chem. 259, 9461–9471.PubMedGoogle Scholar
  52. 50.
    Zimmermann, R. A., Gait, M. J., and Moore, M. J. (1998) Incorporation of modified nucleotides into RNA for studies on RNA structure, function and intermolecular interactions, in Modification and Editing of RNA (Grosjean, H. and Benne, R., eds.), ASM Press, Washington, DC, pp. 59–84.Google Scholar
  53. 51.
    Bruce, A. G. and Uhlenbeck, O. C. (1982) Enzymatic replacement of the anticodon of yeast phenylalanine tRNA. Biochemistry 21, 855–861.PubMedCrossRefGoogle Scholar
  54. 52.
    Carbon, P., Haumont, E., Fournier, M., de Henau, S., and Grosjean, H. (1983) Site-directed in vitro replacement of nucleosides in the anticodon loop of tRNA: application to the study of structural requirements for queuine insertase activity. EMBO J. 2, 1093–1097.PubMedGoogle Scholar
  55. 53.
    Droogmans, L. and Grosjean, H. (1987) Enzymatic conversion of guanosine 3′-adjacent to the anticodon of yeast tRNA-phe to N 1-methylguanosine and the Wye nucleoside: dependence on the anticodon sequence. EMBO J. 6, 477–483.PubMedGoogle Scholar
  56. 54.
    Kretz, K. A., Trewyn, R. W., Keith, G., and Grosjean, H. (1990) Site directed replacement of nucleotides in the anticodon loop of tRNA: application to the study of inosine biosynthesis in yeast tRNA-ala, in Chromatography and Modifications of Nucleosides vol. 45B, Journal of Chromatography Library Series (Gehrke, C. W. and Kuo, K. C., eds.), Elsevier, The Netherlands, pp. B143–B171.CrossRefGoogle Scholar
  57. 55.
    Droogmans, L. and Grosjean, L. (1991) 2′-O-methylation and inosine formation in the wobble position of anticodon-substituted tRNA-phe in a homologous yeast in vitro system. Biochimie 73, 1021–1025.PubMedCrossRefGoogle Scholar
  58. 56.
    Ma, X., Zhao, X., and Yu, Y. T. (2003) Pseudouridylation (Psi) of U2 snRNA in S. cerevisiae is catalyzed by an RNA-independent mechanism. EMBO J. 22, 1889–1897.PubMedCrossRefGoogle Scholar
  59. 57.
    Jackman, J. E., Montange, R. K., Malik, H. S., and Phizicky, E. M. (2003) Identification of the yeast gene encoding the tRNA m1G methyltransferase responsible for modification at position 9. RNA 9, 574–585.PubMedCrossRefGoogle Scholar
  60. 58.
    Grosjean, H, Droogmans, L., Giegé, R., and Uhlenbeck, O. C. (1990) Guanosine modifications in runoff transcripts of synthetic tRNA-Phe microinjected into Xenopus laevis oocytes. Biochim. Biophys. Acta. 1050, 267–273.PubMedGoogle Scholar
  61. 59.
    Grosjean, H., Edqvist, J., Sträby, K. B., and Giegé, R. (1996) Enzymatic formation of modified nucleosides in tRNA: dependence on tRNA architecture. J. Mol. Biol. 255, 67–85.PubMedCrossRefGoogle Scholar
  62. 60.
    Jiang, H.-Q., Motorin, Y., Jin, Y.-X., and Grosjean, H. (1997) Pleiotropic effects of intron removal on base modification pattern of yeast tRNA-Phe: an in vitro study. Nucleic Acids Res. 25, 2694–2701.PubMedCrossRefGoogle Scholar
  63. 61.
    Morin, A., Auxilien, S., Senger, B., Tewari, R., and Grosjean, H. (1998) Structural requirements for enzymatic formation of threonylcarbamoyladenosine (t6A) in tRNA: an in vivo study with Xenopus laevis oocytes. RNA 4, 24–37.PubMedGoogle Scholar
  64. 62.
    Constantinesco, F., Motorin, Y., and Grosjean, H. (1999) Transfer RNA modification enzymes from Pyrococcus furiosus detection of the enzymatic activities in vitro. Nucleic Acids Res. 27, 1308–1315.PubMedCrossRefGoogle Scholar
  65. 63.
    Motorin, Y. and Grosjean, H. (1999) Multisite-specific tRNA-m5C-methyl-transferase (Trm4) in yeast S. cerevisiae: identification of the gene and substrate specificity of the enzyme. RNA 5, 1105–1118.PubMedCrossRefGoogle Scholar
  66. 64.
    Alexandrov, A., Martzen, M. R., and Phizicky, E. M. (2002) Two proteins that form a complex are required for 7-methylguanosine modification of yeast tRNA. RNA 8, 1253–1266.PubMedCrossRefGoogle Scholar
  67. 65.
    Boorstein, W. R. and Craig, E. A. (1989) Primer extension analysis of RNA, in Methods in Enzymology, vol. 180 (Dahlberg, J. E. and Abelson, J. N., eds.), Academic, New York, pp. 347–369.Google Scholar
  68. 66.
    Hagenbüchle, O., Santer, M., and Steitz, J. A. (1978) Conservation of the primary structure at the 3′ end of 18S rRNA from eukaryotic cells. Cell 13, 551–563.PubMedCrossRefGoogle Scholar
  69. 67.
    Wittig, B. and Wittig, S. (1978) Reverse transcription of tRNA. Nucleic Acids Res. 5, 1165–1178.PubMedCrossRefGoogle Scholar
  70. 68.
    Youvan, D. C. and Hearst, J. E. (1979) Reverse transcriptase pauses at N 2-methyl-guanine during in vitro transcription of E. coli 16S ribosomal RNA. Proc. Natl. Acad. Sci. USA 76, 3751–3754.PubMedCrossRefGoogle Scholar
  71. 69.
    Youvan, D. C. and Hearst, J. E. (1981) A sequence from Drosophila melanogaster 18S rRNA bearing the conserved hypermodified nucleoside amPsi: analysis by reverse transcription and high performance liquid chromatography. Nucleic Acids Res. 9, 1723–1741.PubMedCrossRefGoogle Scholar
  72. 70.
    Maden, B. E. H., Corbett, M. E., Heeney, P. A., Pugh, K., and Ajuh, P. M. (1995) Classical and novel approaches to the detection and localization of the numerous modified nucleotides in eukaryotic ribosomal RNA. Biochimie 77, 22–29.PubMedCrossRefGoogle Scholar
  73. 71.
    Liu, J., Zhou, W., and Doetsch, P. W. (1995) RNA polymerase bypass at sites of dihydrouracil: implications for transcriptional mutagenesis. Mol. Cell. Biol. 15, 6729–6735.PubMedGoogle Scholar
  74. 72.
    Auxilien, S., Keith, G., Le Grice, F. J., and Darlix, J.-L. (1999) Role of post-transcriptional modifications of primer tRNA-Lys,3 in the fidelity and efficacy of plus strand DNA transfer during HIV-1 reverse transcription. J. Biol. Chem. 274, 4412–4420.PubMedCrossRefGoogle Scholar
  75. 73.
    Pintard, L., Bujnicki, J. M., Lapeyre, B., and Bonnerot, C. (2002) MRM2 encodes a novel yeast mitochondrial 21S rRNA methyltransferase. EMBO J. 21, 1139–1147.PubMedCrossRefGoogle Scholar
  76. 74.
    Rhee, Y., Valentine, M. R., and Termini, J. (1995) Oxidative base damage in RNA detected by reverse transcriptase. Nucleic Acids Res. 23, 3275–3282.PubMedCrossRefGoogle Scholar
  77. 75.
    Ho, N. W. Y. and Gilham, P. T. (1971) Reaction of pseudouridine and inosine with N-cyclohexyl-N′-beta-(4-methylmorpholinium)ethylcarbodiimide. Biochemistry 10, 3651–3657.PubMedCrossRefGoogle Scholar
  78. 76.
    Bakin, A. V. and Ofengand, J. (1993) Four newly located pseudouridylate residues in E. coli 23S ribosomal RNA are all at the peptidyl center: analysis by the application of a new sequencing technique. Biochemistry 32, 9754–9762.PubMedCrossRefGoogle Scholar
  79. 77.
    Bakin, A. V. and Ofengand, J. (1998) Mapping of pseudouridine residues in RNA to nucleotide resolution, in Methods in Molecular Biology, vol. 77: Protein Synthesis: Methods and Protocols (Martin, R., ed.), Humana, Totowa, NJ, pp. 297–309.Google Scholar
  80. 77a.
    Ofengand, J., Del Campo, M., and Kaya, Y. (2001) Mapping pseudouridines in RNA molecules. Methods 25, 365–373.PubMedCrossRefGoogle Scholar
  81. 78.
    Wintermeyer, W. and Zachau, H. G. (1970) A specific chemical chain scission of tRNA at 7-methylguanosine. FEBS Lett. 11, 160–164.PubMedCrossRefGoogle Scholar
  82. 79.
    Wintermeyer, W. and Zachau, H. G. (1974) Replacement of odd bases in tRNA by fluorescent dyes, in Methods in Enzymology, vol. XX (Grossman, L. and Moldave, K., eds.), Academic, New York, pp. 667–673.Google Scholar
  83. 80.
    Thiebe, R. and Zachau, H. G. (1971) Half-molecules from phenylalanine tRNA’s, in Methods in Enzymology, vol. XX (Moldave, K. and Grossman, L., eds.), Academic, New York, pp. 178–182.Google Scholar
  84. 81.
    Beltchev, B. and Grunberg-Manago, M. (1970) Preparation of pG-fragment from yeast tRNA-Phe by chemical scission at the dihydrouracil and inhibition of yeast tRNA-Phe charging by this fragment when combined with the −CCA half of this tRNA. FEBS Lett. 12, 24–27.PubMedCrossRefGoogle Scholar
  85. 82.
    Cerutti, P., Holt, J. W., and Miller, N. (1968) Detection and determination of 5,6-dihydrouridine and 4-thiouridine in transfer RNA from different sources. J. Mol. Biol. 34, 505–518.PubMedCrossRefGoogle Scholar
  86. 83.
    Bird, A. (1980) DNA methylation and the CpG frequency in the animal DNA. Nucleic Acids Res. 8, 1499–1505.PubMedCrossRefGoogle Scholar
  87. 84.
    Lindahl, T. and Nyberg, B. (1974) Heat-induced deamination of cytosine residues in deoxyribonucleic acid. Biochemistry 13, 3405–3410.PubMedCrossRefGoogle Scholar
  88. 85.
    Lindahl, T. ansd Nyberg, B. (1972) Rate of depurination of native deoxyribonucleic acid. Biochemistry 11, 3610–3618.PubMedCrossRefGoogle Scholar
  89. 86.
    Keith, G. and Dirheimer, G. (1995) Postlabeling: a sensitive method for studying DNA adducts and their role in carcinogenesis. Curr. Opin. Biotechnol. 6, 3–11.PubMedCrossRefGoogle Scholar
  90. 87.
    Dirheimer, G., Pfohl-Leszkowicz, A., and Keith, G. (1999) Methods of DNA-adduct detection: some applications to detect environmental carcinogen exposure, in Trends in Environmental Mutagenesis (Sobti, R. C., Obe, G., and Quillardet, A., eds.), Tausco Book Distributors, New Dehli, pp. 23–46.Google Scholar
  91. 88.
    Duranton, B., Keith, G., Gossé, F., Bergmann, C., Schleiffer, R., and Raul, F. (1998) Concomitant changes in polyamine pools and DNA methylation during growth inhibition of human colonic cancer cells. Exp. Cell Res. 243, 319–325.PubMedCrossRefGoogle Scholar
  92. 89.
    Glasser, A. L., Desgres, J., Heitzler, J., Gehrke, C. W., and Keith, G. (1991) O-Ribosyl-phosphate purine as a constant modified nucleotide located at position 64 in cytoplasmic initiator tRNA-Met of yeasts. Nucleic Acids Res. 19, 5199–5203.PubMedCrossRefGoogle Scholar
  93. 90.
    Gehrke, C. W., Desgres, J., Keith, G., Gerhardt, K. O., Agris, P. F., Gracz, H., Tempesta, M. S., and Kuo, K. C. (1990) Structural elucidation of nucleosides in nucleic acids, in Chromatography and Modifications of Nucleoside, vol. 45A, Journal of Chromatography Library Series (Gehrke, C. W. and Kuo, K. C., eds.), Elsevier, The Netherlands, pp. A159–A223.Google Scholar
  94. 91.
    Hiramaru, M., Ushida, T., and Egami, F. (1966) Ribonuclease preparation for the base analysis of polyribonucleotides. Anal. Biochem. 17, 135–142.PubMedCrossRefGoogle Scholar
  95. 92.
    Cameron, V. and Uhlenbeck, O. C. (1977) 3′-Phosphatase activity in T4 polynucleotide kinase. Biochemistry 16, 5120–5126.PubMedCrossRefGoogle Scholar
  96. 93.
    Watanabe, K. (1980) Reactions of 2-thioribothymidine and 4-thiouridine with hydrogen peroxide in transfer RNA from Thermus thermophilus and Escherichia coli as studied by circular dichroism. Biochemistry 19, 5542–5549.PubMedCrossRefGoogle Scholar
  97. 94.
    Feldmann, H. and Falter, H. (1971) Transfer ribonucleic acid from Mycoplasma laidlawii A. Eur. J. Biochem. 18, 573–581.PubMedCrossRefGoogle Scholar
  98. 95.
    Hall, R. H. (1971) The Modified Nucleosides in Nucleic Acids. Columbia University Press, New York.Google Scholar
  99. 96.
    Randerath, K., Gupta, R. C., and Randerath, E. (1980) 3H and 32P derivative methods for base composition and sequence analysis of RNA, in Methods in Enzymology, vol. 65 (Grossman, L. and Moldave, K., eds.), Academic, New York, pp. 638–680.Google Scholar
  100. 97.
    Gupta, R. C., Randerath, E., and Randerath, K. (1976) A double-labeling procedure for sequence analysis of picomole amounts of nonradioactive RNA fragments. Nucleic Acids Res. 3, 2895–2914.PubMedGoogle Scholar
  101. 98.
    Gupta, R. C., Randerath, E., and Randerath, K. (1976) An improved separation procedure for nucleoside monophosphates on polyethyleneimine-(PEI)-cellulose thin layers. Nucleic Acids Res. 3, 2915–2922.PubMedGoogle Scholar
  102. 99.
    Gupta, R. C. and Randerath, K. (1979) Rapid readthrough technique for sequencing of RNAs containing modified nucleotides. Nucleic Acids Res. 6, 3443–3458.PubMedCrossRefGoogle Scholar
  103. 100.
    Randerath, K. and Randerath, E. (1983) Selected postlabeling procedures for base composition and sequence analysis of nucleic acids, in Methods of DNA and RNA Sequencing (Weissman, M. S., ed.), Praeger, New York, pp. 169–233.Google Scholar
  104. 101.
    Rogg, H., Brambilla, R., Keith, G., and Staehelin, M. (1976) An improved method for the separation and quantitation of the modified nucleosides of transfer RNA. Nucleic Acids Res. 3, 285–295.PubMedGoogle Scholar
  105. 102.
    Suzuki, T., Suzuki, T., Wada, T., Saigo, K., and Watanabe, K. (2002) Taurine as a constituent of mitochondrial tRNAs: new insights into the functions of taurine and human mitochondrial diseases. EMBO J. 21, 6581–6589.PubMedCrossRefGoogle Scholar

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© Humana Press Inc. 2004

Authors and Affiliations

  • Henri Grosjean
    • 1
  • Gérard Keith
    • 2
  • Louis Droogmans
    • 3
  1. 1.Laboratoire d’Enzymologie et Biochimie Structurales du CNRSGif-sur-YvetteFrance
  2. 2.Institut de Biologie Moléculaire et Cellulaire du CNRSStrasbourg CedexFrance
  3. 3.Laboratoire de Microbiologie Institut de Recherches Microbiologiques J. M. WiameUniversité Libre de BruxellesBruxellesBelgium

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