Chapter 2 : RNA-Modifying and RNA-Editing Enzymes: Methods for Their Identification

MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.

Preview this chapter:
Zoom in

RNA-Modifying and RNA-Editing Enzymes: Methods for Their Identification, Page 1 of 2

| /docserver/preview/fulltext/10.1128/9781555818296/9781555811334_Chap02-1.gif /docserver/preview/fulltext/10.1128/9781555818296/9781555811334_Chap02-2.gif


This chapter reviews the methods that have been successfully used to date for the identification of RNA modification and editing enzymes. The development of recombinant DNA and RNA techniques and techniques for chemical synthesis of DNA and RNA offers adequate alternatives for obtaining synthetic or semisynthetic unmodified or partially modified RNA substrates suitable for analyzing enzymatic posttranscriptional RNA modifications both in vivo and in vitro. Stepwise automated chemical synthesis of DNA oligonucleotides is widely used for producing starting material for gene construction, primers for PCR, DNA splints, or DNA probes for hybridization and deoxyribozymes. Two distinct approaches are generally used to gain the information on the precise location of modification or editing sites in RNA molecules. The first is based on direct sequencing of pure RNA and is applied mostly for the analysis of rather short RNA molecules, while the other is based on the use of reverse transcription with unfractionated RNA mixtures and specific oligonucleotide primers. Microinjection of antisense RNA, with the aim of blocking translation of selected mRNAs in the oocyte or fertilized frog eggs, revealed the existence of what was first identified as an unwinding RNA protein. With the recent access to complete genome sequences, it has become possible to search for homologous genes in the different genomes, to clone them into suitable vectors after amplification by PCR, and to express them in appropriate host cells.

Citation: Grosjean H, Motorin Y, Morin A. 1998. RNA-Modifying and RNA-Editing Enzymes: Methods for Their Identification, p 21-46. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch2
Highlighted Text: Show | Hide
Loading full text...

Full text loading...


Image of Figure 1
Figure 1

Schematic procedures for construction of DNA templates that can be used for in vitro transcription into RNA using specific DNA-dependant RNA polymerases. On the left side, the starting material corresponds to pure chemically synthesized oligodeoxynucleotides, while on the right, the starting material corresponds to natural RNA genes present in isolated genomic DNA. The different steps of the procedure are indicated in boxes. DNA is represented by bars. A thick bar stands for the DNA portion corresponding to a promoter (P) of bacteriophage T7 or SP6 RNA polymerase. The asterisk stands for the restriction site needed for the linearization of the plasmid, before the transcription. The resulting “runoff” RNA transcript is indicated by a curved gray line. It can be radiolabeled internally on one of the four nucleotides with either P,H, or any other isotope by adding the corresponding radiolabeled nucleotide triphosphate into the transcription mixture. Hydroxyl and phosphate terminal groups are indicated only when these are essential. Intermediate purification steps by electrophoresis on agarose or polyacrylamide gels are not indicated.

Citation: Grosjean H, Motorin Y, Morin A. 1998. RNA-Modifying and RNA-Editing Enzymes: Methods for Their Identification, p 21-46. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch2
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of Figure 2
Figure 2

Site-specific cleavage of RNA. RNA fragments are prepared from long natural RNA by hybridization selection and addressed cleavages by RNase T1 (G specific). Short synthetic DNA, long complementary DNA (cDNA) obtained by reverse transcription of a natural RNA, or single-stranded phage DNA (ssDNA) may be used to protect the desired region of the targeted RNA from nuclease digestion. Modified nucleotides present in the natural RNA are illustrated by a black dot and a black square. If present in the RNA portion to be protected, they should allow formation of a perfect Watson-Crick base pair. “Mild conditions” means that a low ratio of RNase T1 to the amount of substrate to be digested is used; also, the digestion time is carefully controlled. After removal of the RNase T1 by phenolization, the resulting RNA fragments are purified by gel electrophoresis or any other purification procedure. The purified fragments have a 5′-hydroxyl group and a 3′-phosphate, which are not suitable for subsequent RNA ligation (see Fig. 5 and 6 ).

Citation: Grosjean H, Motorin Y, Morin A. 1998. RNA-Modifying and RNA-Editing Enzymes: Methods for Their Identification, p 21-46. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch2
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of Figure 3
Figure 3

Isolation of large RNA fragments. RNA is cleaved within an RNα-DNA hybrid by RNase Η or catalytic DNA enzyme. With complementary DNA 6 to 20 nucleotides long, multiple cuts (shown by arrows) arise in the hybrid region (left part of the figure). A site-specific single cut in RNA can be produced only when a short cDNA (4-mers) is flanked by two complementary 2'-O-methylated RNAs of 6 to 10 nucleotides on each side (central part of the figure). The DNA enzyme (DNAzyme) shown in the right part of the figure corresponds to a catalytic domain of 15 synthetic deoxynucleotides, flanked by two RNA-complementary domains of 7 to 8 deoxynucleotides on each side. The DNAzyme binds to the target RNA through Watson-Crick base pairs. The substrate is cleaved at a phosphodiester bond located between an unpaired purine and a paired pyrimidine residue. If modified nucleotides (illustrated by a black dot and a black square) are present in the target RNA, they should allow formation of a perfect Watson-Crick base pair in the RNA/DNA hybrid.

Citation: Grosjean H, Motorin Y, Morin A. 1998. RNA-Modifying and RNA-Editing Enzymes: Methods for Their Identification, p 21-46. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch2
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of Figure 4
Figure 4

Simple one-step procedure for isolating of RNA or RNA fragments from bulk RNA by the affinity purification technique. A DNA (synthetic or natural, represented by a thick bar) bearing a tail complementary to the target RNA is covalently linked via an appropriate spacer (represented by a zigzag line) to a solid support (usually agarose beads, paramagnetic beads or diol-silica beads). Alternatively, synthetic DNA biotinylated at its 5′ end is immobilized on avidin-coated agarose beads (which could also be paramagnetic). Bulk cellular RNA or fragmented RNA bearing the complementary region of the DNA tail is hybridized to the bound DNA. The beads are then collected by centrifugation or with a magnet and washed several times. The trapped RNA is recovered by using appropriate experimental conditions (use of low salt concentration, high temperature, proteinase K). After concentration, the isolated RNA is further purified by gel electrophoresis or by any other technique such as anion exchange or adsorption chromatography.

Citation: Grosjean H, Motorin Y, Morin A. 1998. RNA-Modifying and RNA-Editing Enzymes: Methods for Their Identification, p 21-46. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch2
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of Figure 5
Figure 5

In vitro recombination of RNA fragments (natural or synthetic). The joining reactions can be catalyzed either by T4 DNA ligase or by T4 RNA ligase. With DNA ligase (A), the RNA acceptor and the RNA donor have to be bridged with a DNA splint (black thick line) complementary to the two RNA extremities, thus forming a sort of nicked duplex. With RNA ligase (B), the RNA donor and the RNA acceptor have to be self-complementary in the vicinity of the ligation sites, thus forming a stem-loop structure. RNA recombination can also occur with RNA partners that cannot form such stem-loop structures, but the yield of the ligation reaction is usually dramatically low. For both ligation reactions, the 3′ end of the acceptor RNA should have free 2'- and 3′-hydroxyl groups, while the 5′ end of the donor RNA molecule has to be phosphorylated. If this 5′-phosphate is radiolabeled with P, the resulting recombinant RNA will contain a site-specific radiolabeled phosphate precisely at the ligation site. The black dot stands for naturally occurring modified or edited nucleotides.

Citation: Grosjean H, Motorin Y, Morin A. 1998. RNA-Modifying and RNA-Editing Enzymes: Methods for Their Identification, p 21-46. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch2
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of Figure 6
Figure 6

Strategies for site-specific replacement of a nucleotide, Ζ (modified or not), by another nucleotide, Y (modified or not), in a naturally occurring RNA. Site-specific cleavage (initial step) as well as in vitro recombination of fragments (last step) is performed by one of the methods outlined above ( Fig. 2 , 3 , and 5 ). Terminal nucleotide Ζ of the 5′-half RNA fragment can be quantitatively removed after periodate oxidation of the 2'-3′-glycol end followed by a β-elimination of the nucleoside induced by either aniline or lysine. The resulting 3 '-phosphate of the RNA fragment, now lacking nucleotide ZMP, is removed by phosphatase. The subsequent ligation with a large excess of nucleotide Y (in the form of 3′-5′-diphosphate) is catalyzed by T4 RNA ligase. The 5′-P of the newly incorporated donor (Y) dinucleotide phosphate can be radiolabeled with 32P (symbolized by an asterisk). Phosphatase treatment of this newly synthesized RNA molecule allows a new cycle of ligation reaction with an appropriate donor RNA molecule. The global yield of this stepwise synthesis of chimeric RNA is rather low (at best a few percent of the starting material). Alternative methods for site-specific introduction of modified nucleotides in RNA are detailed in Chapter 4 by Zimmermann et al. (see also Moore and Query, 1997).

Citation: Grosjean H, Motorin Y, Morin A. 1998. RNA-Modifying and RNA-Editing Enzymes: Methods for Their Identification, p 21-46. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch2
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of Figure 7
Figure 7

Schematic of a procedure for direct RNA sequencing of an RNA fragment containing modified nucleotides. The RNA is first cleaved randomly (single cut per molecule) under mild conditions. Each fragment is then radiolabeled with P at the 5′ end. The resulting 5′-radiolabeled fragments are separated by electrophoresis on a polyacrylamide gel. Hydrolysis of each fragment into mononucleotides is performed in situ with RNase T2. Contact transfer of the resulting nucleotides is performed on a PEI-cellulose plate. After the chromatography, the plate is autoradiographed. Identification of each radiolabeled terminal 3′,5′-dinucleotide phosphate on the plate is obtained from its position as compared with those of published reference maps (method of Gupta and Randerath, 1979). The presence of 2'-O-methylated nucleosides in RNA is signaled by “gaps” in the ladder on the gel. Alternatively, the RNA fragment from each band of the gel can be eluted and completely digested by nuclease P1, and the resulting 5′-nucleotide monophosphate can be analyzed by 2D thin-layer chromatography. Identification of each radiolabeled spot on the plate is performed by comparison with those of published reference maps ( ). The example illustrates the sequencing of the tRNA region corresponding to the ΤΨ-loop. This method could also be used to detect base-conversion-type RNA editing processes.

Citation: Grosjean H, Motorin Y, Morin A. 1998. RNA-Modifying and RNA-Editing Enzymes: Methods for Their Identification, p 21-46. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch2
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of Figure 8
Figure 8

Experimental protocol for the determination of the base composition of an RNA fragment, including modified nucleotides (designated X in the example). The series of steps ends up in 2D thin-layer chromatographic analysis of the 5′-P-nucleotide monophosphate (qualitative test). Identification of each radiolabeled spot on the plate is performed by comparison with those in published reference maps ( ). An alternative approach is based on quantitative analysis of the nucleoside composition by high-pressure liquid chromatography (HPLC) coupled to a diode array spectrophotometer (spectral analysis) and/or mass spectrometry (for details see Chapter 3 by Crain).

Citation: Grosjean H, Motorin Y, Morin A. 1998. RNA-Modifying and RNA-Editing Enzymes: Methods for Their Identification, p 21-46. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch2
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of Figure 9
Figure 9

Nearest-neighbor analysis of modified nucleotides in RNA. The RNA fragment has to be internally labeled with P at one of the four nucleotides (in this example, 5′-P of CMP). The 2D thin-layer analysis of the nuclease P1 hydrolysate (that retains the 5′-phosphate) allows the identification of the modified nucleotides corresponding to the parent radiolabeled nucleotide (in the example of the figure, a 5′-CMP derivative posttranscriptionally modified on the base or sugar represented by C*). The chromatographic analysis of the nucleotides obtained after complete hydrolysis of the same radiolabeled RNA but with RNase T2 (leaving the 3′-phosphate) allows all of the modified nucleotides that are 3′-adjacent to a CMP in the original RNA chain (nearest neighbor) to be revealed.

Citation: Grosjean H, Motorin Y, Morin A. 1998. RNA-Modifying and RNA-Editing Enzymes: Methods for Their Identification, p 21-46. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch2
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of Figure 10
Figure 10

Identification of a C-to-U editing site by the primer extension assay. Here a 5′-P-labeled DNA primer (thick black line) is prepared so that it hybridizes close to the editing site, leaving no C residue between the 3′ end of the primer and the first putative edited C. Reverse transcription is performed in the presence of dATP, dTTP, dCTP, and ddGTP. A “strong stop” will appear on the sequencing gel at unedited C, except if an edited U is present ( ). The same analysis can be performed for testing α-to-I editing or modification sites in RNA. Indeed, inosine will be read as G during transcription. From such analysis, the ratio of edited to nonedited molecules at a given position in the RNA population can be evaluated.

Citation: Grosjean H, Motorin Y, Morin A. 1998. RNA-Modifying and RNA-Editing Enzymes: Methods for Their Identification, p 21-46. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch2
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of Figure 11
Figure 11

Detection of modified nucleotides in RNA by combined chemical and reverse transcription techniques. The cartoon illustrates different potential situations. In one case (left part) RNA contains a naturally occurring bulky residues. It will cause a strong stop upon reverse transcription that is easily detected on the sequencing gel after primer extension. In vitro chemical alteration of RNA [for example, with l-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate (CMCT)] can produce bulky residues (middle part of the figure) that will also generate strong stops on the sequencing gel. However, if the in vitro chemical conditions are optimized for producing random (partial) chemical “hypermodification,” several positions of the same RNA molecule will give the strong stop phenomenon. The right part of the figure illustrates the case where naturally occurring nucleotide modifications (like 2'-O-methylation of the ribose moiety) or an in vitro random chemical alteration (as with glyoxal on G) can render RNA resistant to enzymatic or hydrolytic cleavage at specific sites. These sites will be revealed on the sequencing gel by “gaps” in the radiolabeled RNA “ladder” after primer extension. This type of analysis gives only qualitative information, and cannot be used to quantify the relative amount of a modified nucleotide at a given position of an RNA population. The main advantages are that the RNA does not have to be pure, and that a long RNA can be explored with different primers complementary to different regions of the RNA molecule.

Citation: Grosjean H, Motorin Y, Morin A. 1998. RNA-Modifying and RNA-Editing Enzymes: Methods for Their Identification, p 21-46. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch2
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of Figure 12
Figure 12

Alternative protocols for the identification of enzyme activities corresponding to selected RNA modification or editing phenomena. For certain types of analysis, the RNA used as substrates has to be radiolabeled with P. This can be done either during in vitro or in vivo transcription of synthetic or natural genes, or during the recombination of RNA fragments. Details of the preparation of different substrates and RNA modification/editing analysis are described in the previous figures. HPLC, high-performance liquid chromatography; MS, mass spectrometry.

Citation: Grosjean H, Motorin Y, Morin A. 1998. RNA-Modifying and RNA-Editing Enzymes: Methods for Their Identification, p 21-46. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch2
Permissions and Reprints Request Permissions
Download as Powerpoint


1. Agris, P. F.,, A. Malkiewicz,, A. Kraszewski,, K. Everett,, B. Nawrot,, E. Sochacka,, J. Jankowska,, and R. Guenther. 1995. Site-selected introduction of modified purine and pyrimidine ribonucleosides into RNA by automated phosphoramidite chemistry. Biochimie 77: 125 134.
2. Aphasizhev, R.,, B. Senger,, and F. Fasiolo. 1997. Importance of structural features for tRNAMet identity. RNA 3: 489 497.
3. Araya, A.,, V. Blanc,, D. Begu,, F. Crabier,, A. Mouras,, and S. Lit-vak. 1995. RNA editing in wheat mitochondria. Biochimie 77: 87 91.
4. Aström, S. U.,, and A. S. Byström. 1994. Rit1, a tRNA backbone-modifying enzyme that mediates initiator and elongator tRNA discrimination. Cell 79: 536 546.
5. Auxilien, S.,, P. F. Crain,, R. W. Trewyn,, and H. Grosjean. 1996. Mechanism, specificity and general properties of the yeast enzyme catalysing the formation of inosine 34 in the anticodon of transfer RNA. J. Mol. Biol. 262: 437 458.
6. Barnes, W. M. 1992. The fidelity of Taq polymerase catalizing PCR is improved by an N-terminal deletion. Gene 112: 29 35.
7. Bass, B. L.,, and H. Weintraub. 1987. A developmental^ regulated activity that unwinds RNA duplexes. Cell 48: 607 613.
8. Bates, G. W. 1995. Electroporation of plant protoplasts and tissues. Methods Cell Biol. 50: 363 373.
9. Becker, H. F.,, Y. Motorin,, R. J. Planta,, and H. Grosjean. 1997a. The yeast gene YNL292w encodes a pseudouridine synthase (Pus4) catalyzing the formation of Ψ55 in both mitochondrial and cytoplasmic tRNAs. Nucleic Acids Res. 25: 4493 4499.
10. Becker, H. F.,, Y. Motorin,, M. Sissler,, C. Florentz,, and H. Grosjean. 1997b. Major identity determinants for enzymatic formation of ribothymidine and pseudouridine in the TΨ-loop of yeast tRNAs. J. Mol. Biol. 274: 505 518.
11. Benne, R. 1996. RNA editing: how a message is changed. Curr. Opin. Genet. Dev. 6: 221 231.
12. Björk, G. R., 1995a. Biosynthesis and function of modified nucleosides, p. 165 206. In D. Söll,, and U. RajBhandary, (ed.), tRNA: Structure, Biosynthesis and Function, ASM Press, Washington.
13. Björk, G. R. 1995b. Genetic dissection of synthesis and function of modified nucleosides in bacterial transfer RNA. Progr. Nucl. Acid Res. Mol. Biol. 50: 263 338.
14. Bokar, J. A.,, M. E. Shambaugh,, D. Polayes,, A. G. Matera,, and F. M. Rottman. 1995. Purification and cDNA cloning of the AdoMet-binding subunit of the human mRNA (N6-adenosine)-methyltransferase. RNA 3: 1233 1247.
15. Boorstein, W. R.,, and E. A. Craig. 1989. Primer extension analysis of RNA. Methods Enzymol. 180: 347 369.
16. Bossi, L.,, and M. S. Ciampi. 1983. The expression of prokaryotic tRNA genes in frog oocytes. Nucleic Acids Res. 11: 3207 3226.
17. Boyer, J.-C.,, B. Zaccomer,, and A.-L. Haenni. 1993. Electrotrans-fection of turnip yellow mosaic virus RNA into Brassica leaf protoplasts and detection of viral RNA products with a nonradioactive probe. J. Gen. Virol. 74: 1911 1917.
18. Branch, A. D.,, B. J. Bonenfeld,, and H. D. Robertson. 1989. RNA fingerprinting. Methods Enzymol. 180: 130 154.
19. Bratty, J.,, T. F. Wu,, K. Nicoghosian,, K. K. Ogilvie,, J. P. Perreault,, G. Keith,, and R. Cedergren. 1990. Characterization of a chemically synthesized RNA having the sequence of the yeast initiator tRNAMet. FEBS Lett. 269: 60 64.
20. Bruce, A. G.,, and O. C. Uhlenbeck. 1982. Enzymatic replacement of the anticodon of yeast phenylalanine transfer ribonucleic acid. Biochemistry 21: 855 861.
21. Buck, M.,, M. Connick,, and B. N. Ames. 1983. Complete analysis of tRNA-modified nucleosides by high-performance liquid chromatography: the 29 modified nucleosides of Salmonella typhimurium and Escherichia coli tRNA. Anal. Biochem. 129: 1 13.
22. Carbon, P.,, E. Haumont,, M. Fournier,, S. de Henau,, and H. Grosjean. 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.
23. Casey, J. L.,, K. F. Bergmann,, T. L. Brown,, and J. L. Gerin. 1992. Structural requirements for RNA editing in hepatitis delta virus: evidence for a uridine-to-cytidine editing mechanism. Proc. Natl. Acad. Sci. USA 89: 7149 7153.
24. Cazenave, C.,, and O. C. Uhlenbeck. 1994. RNA template-directed RNA synthesis by T7 RNA polymerase. Proc. Natl. Acad. Sci. USA 91: 6972 6976.
25. Cedergren, R.,, and H. Grosjean. 1987. RNA design by in vitro RNA recombination and synthesis. Biochem. Cell. Biol. 65: 677 692.
26. Celis, J. E.,, A. Graessmann,, and A. Loyter (ed.). 1986. Microinjection and Organelle Transplantation Techniques. Academic Press, London, United Kingdom.
27. Ciampi, M. S.,, F. Arena,, and R. Cortese. 1977. Biosynthesis of pseudouridine in the in vitro transcribed tRNATyr precursor. FEBS Lett. 77: 75 82.
28. Colman, A., 1984. Translation of eukaryotic messenger RNA in Xenopus oocytes. In D. Rickwood, and B. D. Hames (ed.), Transcription and Translation: a Practical Approach. IRL Press, Oxford, United Kingdom.
29. Diamond, A.,, and B. Dudock. 1983. Methods of RNA sequence analysis. Methods Enzymol. 100: 431 453.
30. Donnis-Keller, H.,, A. M. Maxam,, and W. Gilbert. 1977. Mapping adenines, guanines, and pyrimidines in RNA. Nucleic Acids Res. 4: 2527 2538.
31. Doudna, J. A.,, C. Grosshans,, A. Gooding,, and C. E. Kundrot. 1993. Crystallization of ribozymes and small RNA motifs by a sparse matrix approach. Proc. Natl. Acad. Sci. USA 90: 7829 7833.
32. Draper, D. E.,, S. A. White,, and J. M. Kean. 1988. Preparation of specific ribosomal RNA fragments. Methods Enzymol. 164: 221 237.
33. Dreher, T. W.,, J. J. Bujarski,, and T. C. Hall. 1984. Mutant viral RNAs synthesized in vitro show altered aminoacylation and replicase template activities. Nature 311: 171 175.
34. Driscoll, D. M.,, and E. Casanova. 1990. Characterization of the apolipoprotein B mRNA editing activity in enterocyte extracts. J. Biol. Chem. 265: 21401 21403.
35. Driscoll, D. M.,, J. K. Wynne,, S. C. Wallis,, and J. Scott. 1989. An in vitro system for editing of apolipoprotein B mRNA. Cell 58: 519 525.
36. Droogmans, L.,, and H. Grosjean. 1987. Enzymatic conversion of guanosine 3' adjacent to the anticodon of yeast tRNAPhe to N1-methylguanosine and the Wye nucleoside: dependence on the anticodon sequence. EMBO J. 6: 477 483.
37. Droogmans, L.,, and H. Grosjean. 1991. 2'-O-Methylation and inosine formation in the wobble position of anticodon-substituted transfer RNAPhe in a homologous yeast in vitro system. Biochimie 73: 1021 1025.
38. Droogmans, L.,, E. Haumont,, S. de Henau,, and H. Grosjean. 1986. Enzymatic 2'-O-methylation of the wobble nucleoside of eukaryotic tRNAPhe: specificity depends on structural elements outside the anticodon loop. EMBO J. 5: 1105 1109.
39. Eckstein, F.,, and O. Heidenreich. 1994. Synthesis of modified RNA: approaches and applications. FASEB J. 7: 90 96.
40. Edqvist, J.,, H. Grosjean,, and K. B. Straby. 1992. Identity elements for N2-dimethylation of guanosine-26 in yeast tRNAs. Nucleic Acids Res. 20: 6575 6581.
41. Ehresmann, C.,, F. Baudin,, M. Mougel,, P. Romby,, J.-P. Ebel,, and B. Ehresmann. 1987. Probing the structure of RNAs in solution. Nucleic Acids Res. 15: 9109 9128.
42. Fosse, P.,, M. Mougel,, G. Keith,, E. Westhof,, B. Ehresmann,, and C. Ehresmann. 1998. Modified nucleotides of tRNAPro restrict interactions in the binary primer/template complex of M-MuLV. J. Mol. Biol. 275: 731 746.
43. Foster, W.,, and E. Neumann,. 1989. Gene transfer by electropor-ation, p. 299 318. In E. Neumann,, A. E. Sovers,, and C. A. Jordan (ed.), Electroporation and Electrofusion in Cell Biology. Plenum Press, New York, N.Y.
44. Fournier, M.,, E. Haumont,, S. de Henau,, J. Gangloff,, and H. Grosjean. 1983. Posttranscriptional modification of the wobble nucleotide in anticodon-substituted yeast tRNAArgII after microinjection into Xenopus laevis oocytes. Nucleic Acids Res. 11: 707 718.
45. Gasparutto, D.,, T. Livache,, H. Bazin,, A. M. Duplaa,, A. Guy,, A. Khorlin,, D. Molko,, A. Roget,, and R. Teoule. 1992. Chemical synthesis of a biologically active natural tRNA with its minor bases. Nucleic Acids Res. 20: 5159 5166.
46. Gehrke, C. W.,, and K. C. Kuo,. 1990. Ribonucleoside analysis by reversed-phase high performance liquid chromatography, p. A3 A71. In C. W. Gehrke, and K. C. Kuo (ed.), Chromatography and Modifications of Nucleosides, vol. 45 A. Elsevier Press, Amsterdam, The Netherlands.
47. Giege, R.,, C. Florentz,, A. Garcia,, H. Grosjean,, V. Perret,, J. Puglisi,, A. Theobald-Dietrich,, and J.-P. Ebel. 1990a. Exploring the aminoacylation function of transfer RNA by macromolecular engineering approaches. Involvement of conformational features in the charging process of yeast tRNAAsp. Biochimie 72: 453 461.
48. Giege, R.,, J. Rudinger,, T. Dreher,, V. Perret,, E. Westhof,, C. Florentz,, and J.-P. Ebel. 1990b. Search of essential parameters for the aminoacylation of viral tRNA-like molecules. Comparison with canonical transfer RNAs. Biochim. Biophys. Acta 1050: 179 85.
49. Giege, R.,, J. D. Puglisi,, and C. Florentz. 1993. tRNA structure and aminoacylation efficiency. Prog. Nucleic Acid Res. Mol. Biol. 45: 129 206.
50. Goodwin, J. T.,, W. A. Stanick,, and G. D. Glick. 1994. Improved solid-phase synthesis of long oligoribonucleotides: application to tRNAPhe and tRNAGly. J. Org. Chem. 59: 7941 7943.
51. Gorlich, D.,, and I. W. Mattaj. 1996. Nucleoplasms transport. Science 271: 1513 1518.
52. Grabowski, P. J.,, and P. H. Sharp. 1986. Affinity chromatography of splicing complex U2, U5 and U4+U6 small nuclear ribonucleoprotein particles in the spliceosome. Science 233: 1294 1298.
53. Graessmann, M.,, and A. Graessmann. 1983. Microinjection of tissue culture cells. Methods Enzymol. 101: 482 492.
54. Grosjean, H.,, and E. Kubli,. 1986. Functional aspects of tRNA microinjected into Xenopus laevis oocytes: results and perspectives, p. 304 326. In J. E. Celis,, A. Graessmann,, and A. Loyter (ed.), Microinjection and Organelle Transplantation Techniques. Academic Press, London, United Kingdom.
55. Grosjean, H.,, E. Haumont,, L. Droogmans,, P. Carbon,, M. Fournier,, S. de Henau,, T. Doi,, G. Keith,, J. Gangloff,, K. Kretz,, and R. Trewyn. 1987. A novel approach to the biosynthesis of modified nucleosides in the anticodon loops of eukaryotic tRNAs, p. 355 378. In K. Bruzik and W. Stec (ed.), Biophosphates and Their Analogues: Synthesis, Structure, Metabolism and Activity. Elsevier Science Publishers, Amsterdam, The Netherlands.
56. Grosjean, H.,, L. Droogmans,, R. Giege,, and O. C. Uhlenbeck. 1990. Guanosine modifications in runoff transcripts of synthetic transfer RNAPhe genes microinjected into Xenopus oocytes. Biochim. Biophys. Acta 1050: 267 273.
57. Grosjean, H.,, F. Constantinesco,, D. Foiret,, and N. Benachenhou. 1995a. A novel enzymatic pathway leading to 1-methylinosine modification in Haloferax volcanii tRNA. Nucleic Acids Res. 23: 4312 4319.
58. Grosjean, H.,, M. Sprinzl,, and S. Steinberg. 1995b. Posttranscrip-tionally modified nucleosides in transfer RNA: their locations and frequencies. Biochimie 77: 139 141.
59. Grosjean, H.,, S. Auxilien,, F. Constantinesco,, C. Simon,, Y. Corda,, H. F. Becker,, D. Foiret,, A. Morin,, Y. X. Jin,, M. Fournier,, and J. L. Fourrey. 1996a. Enzymatic conversion of adenosine to inosine and to N-l-methylinosine in transfer RNAs: a review. Biochimie 78: 488 501.
60. Grosjean, H.,, J. Edqvist,, K. B. Straby,, and R. Giege. 1996b. Enzymatic formation of modified nucleosides in tRNA: dependence on tRNA architecture. J. Mol. Biol. 255: 67 85.
61. Gupta, R. C, and K. Randerath. 1979. Rapid print-readout technique for sequencing of RNAs containing modified nucleotides. Nucleic Acids Res. 6: 3443 3458.
62. Gustafsson, C.,, R. Reid,, P. J. Greene,, and D. V. Santi. 1996. Identification of new RNA modifying enzymes by iterative genome search using known modifying enzymes as probes. Nucleic Acids Res. 24: 3756 3762.
63. Hagenbuchle, O.,, M. Santer,, and J. A. Steitz. 1978. Conservation of the primary structure at the 3' end of 18S rRNA from eucaryotic cells. Cell 13: 551 563.
64. Hahn, C. S.,, E. G. Strauss,, and J. H. Strauss. 1989. Dideoxy sequencing of RNA using reverse transcriptase. Methods Enzymol. 180: 121 130.
65. Hall, K. B.,, J. R. Sampson,, O. C. Uhlenbeck,, and A. G. Redfield. 1989. Structure of an unmodified tRNA molecule. Biochemistry 28: 5794 5801.
66. Harada, F.,, M. Matsubara,, and N. Kato. 1984. Stable tRNA precursors in HeLa cells. Nucleic Acids Res. 12: 9263 9269.
67. Haumont, E.,, M. Fournier,, S. de Henau,, and H. Grosjean. 1984. Enzymatic conversion of adenosine to inosine in the wobble position of yeast tRNAAsp: the dependence on the anticodon sequence. Nucleic Acids Res. 12: 2705 2715.
68. Haumont, E.,, L. Droogmans,, and H. Grosjean. 1987. Enzymatic formation of queuosine and of glycosyl queuosine in yeast tRNAs microinjected into Xenopus laevis oocytes. The effect of the anticodon loop sequence. Eur. J. Biochem. 168: 219 225.
69. Hayase, Y.,, M. Jahn,, M. J. Rogers,, L. A. Sylvers,, M. Koizumi,, H. Inoue,, E. Ohtsuka,, and D. Söll. 1992. Recognition of bases in E. coli tRNAGln by glutaminyl-tRNA synthetase: a complete identity set. EMBO J. 11: 4159 4165.
70. Helm, M.,, H. Brule,, F. Degoul,, C. Cepanec,, J.-P. Leroux,, R. Giege,, and C. Florentz. 1997. The presence of a modified nucleoside is required for the cloverleaf folding of a human mitochondrial tRNA. 17th International tRNA Workshop, Kazusa Akademia Center, Japan, p. 3 13.
71. Ho, N.,, and P. Gilham. 1971. Reaction of pseudouridine and inosine with N-cyclohexyl-N'-beta-(4-methylmorpholinium) ethylcarbodiimide. Biochemistry 10: 3651 3657.
72. Holley, R. W.,, J. Apgar,, G. A. Everett,, J. T. Madison,, M. Marquise,, S. H. Merril,, J. R. Penswick,, and R. Zamir. 1965. Structure of a ribonucleic acid. Science 147: 1462 1465.
73. Hopper, A. K. 1990. Genetic methods for study of trans-acting genes in processing of precursors to yeast cytoplasmic tRNAs. Methods Enzymol. 181: 400 421.
74. Hopper, A. K.,, F. Banks,, and V. Evangelidis. 1978. A yeast mutant which accumulates precursor tRNAs. Cell 14: 211 219.
75. Horton, R. M.,, and L. R. Pease,. 1991. Recombination and mutagenesis of DNA sequences using PCR, p. 217 247. In M. J. McPherson (ed.), Directed Mutagenesis: a Practical Approach. IRL Press, Oxford, United Kingdom.
76. Inoue, H.,, I. Hayase,, A. Imura,, K. Iwai,, and E. Ohtsuka. 1987. Sequence-dependent hydrolysis or RNA using modified oligonucleotide splints and RNase H. FEBS Lett. 215: 327 330.
77. Iwase, R.,, M. Maeda,, T. Fujiwara,, M. Sekine,, T. Hata,, and K. I. Miura. 1992. Molecular design of a eukaryotic messenger RNA and its chemical synthesis. Nucleic Acids Res. 20: 1643 1648.
78. Izaurralde, E.,, J. Lewis,, C. Gamberi,, A. Jarmolowski,, C. McGuigan,, and I. Mattaj. 1995. A cap-binding protein complex mediating U snRNA export. Nature 376: 709 712.
79. Jiang, H.-Q.,, Y. A. Motorin,, Y.-X. Jin,, and H. Grosjean. 1997. Pleiotropic effects of intron removal on base modifications pattern of yeast tRNAPhe: an in vitro study. Nucleic Acids Res. 25: 2694 2701.
80. Johnson, L.,, H. Hayashi,, and D. Söll. 1970. Isolation and properties of a transfer ribonucleic acid deficient in ribothymidine. Biochemistry 9: 2823 2831.
81. Joyce, G. F. 1994. In vitro evolution of nucleic acids. Curr. Opin. Struct. Biol. 4: 331 336.
82. Kaufmann, G.,, and U. Z. Littauer. 1975. Covalent joining of Phenylalanine transfer ribonucleic acid half-molecules by T4 RNA ligase. Proc. Natl. Acad. Sci. USA 71: 3741 3745.
83. Kay, B. K.,, and H. B. Peng (ed.). 1991. Methods in Cell Biology, vol. 36. Xenopus laevis: Practical Uses in Cell and Molecular Biology. Academic Press, New York, N.Y.
84. Keith, G. 1995. Mobilities of modified ribonucleotides on two-dimensional cellulose thin-layer chromatography. Biochimie 77: 142 144.
85. Knight, D. E.,, and M. C. Scrutton. 1986. Gaining access to the cytosol: the technique and some applications of electropermeabilization. Biochem. J. 234: 497 506.
86. Komine, Y.,, T. Adachi,, H. Inokuchi,, and H. Ozeki. 1990. Genomic organization and physical mapping of the transfer RNA genes in Escherichia coli K12. J. Mol. Biol. 212: 579 598.
87. Koonin, E. V. 1996. Pseudouridine synthases: four families of enzymes containing a putative uridine-binding motif also conserved in dUTPases and dCTP deaminases. Nucleic Acids Res. 24: 2411 2415.
88. Kostyuk, D. A.,, S. M. Dragan,, D. L. Lyakhov,, V. O. Rechinsky,, V. L. Tunitskaya,, B. K. Chernov,, and S. N. Kochetkov. 1995. Mutants of T7 RNA polymerase that are able to synthesize both RNA and DNA. FEBS Lett. 369: 165 168.
89. Kretz, K. A.,, R. W. Trewyn,, G. Keith,, and H. Grosjean,. 1990. Site directed replacement of nucleotides in the anticodon loop of tRNA: application to the study of inosine biosynthesis in yeast tRNAAla, p. B144 B171. In C. W. Gehrke, and K. C. T. Kuo (ed.), Chromatography and Modification of Nucleosides Part B: Biological Roles and Function of Modification. J. Chrom. Library, vol. 45B. Elsevier Science Publishing, Amsterdam, The Netherlands.
90. Krzyzosiak, W.,, R. Denman,, K. Nurse,, W. Hellmann,, M. Boublik,, C. Gehrke,, P. Agris,, and J. Ofengand. 1987. In vitro synthesis of 16S ribosomal RNA containing single base changes and assembly into a functional 30S ribosome. Biochemistry 26: 2353 2364.
91. Kunkel, T. A.,, J. D. Robert,, and R. A. Zakour. 1987. Rapid and efficient site-specific mutagenesis without Phenotypic selection. Methods Enzymol. 154: 367 382.
92. Lafontaine, D.,, J. Delcour,, A. L. Glasser,, J. Desgres,, and J. Vandenhaute. 1994. The DIM1 gene responsible for the conserved m62Am62A dimethylation in the 3'-terminal loop of 18 S rRNA is essential in yeast. J. Mol. Biol. 241: 492 497.
93. Lafontaine, D.,, J. Vandenhaute,, and D. Tollervey. 1995. The 18S rRNA dimethylase Dimlp is required for pre-ribosomal RNA processing in yeast. Genes and Devel. 9: 2470 2481.
94. Lafontaine, D. L. J.,, C. Bousquet-Antonelli,, Y. Henry,, M. Caizergues-Ferrer,, and D. Tollervey. 1998. The box H+ACA snoRNAs carry Cbf5p, the putative rRNA pseudouridine synthase. Genes Dev. 12: 527 537.
95. Lapham, J.,, and D. M. Crothers. 1996. RNase H cleavage for processing of in vitro transcribed RNA for NMR studies and RNA ligation. RNA 2: 289 296.
96. Lapham, J.,, Y.-T. Yu,, M.-D. Shu,, J. A. Steitz,, and D. M. Crothers. 1997. The position of site-directed cleavage of RNA using RNAse H and 2'-O-methyl oligonucleotides is dependent from the enzyme source. RNA 3: 950 951.
97. Lecointe, F.,, G. Simos,, A. Sauer,, E. C. Hurt,, Y. Motorin,, and H. Grosjean. 1998. Identification of yeast protein Deg 1 as Pseudouridine synthase (Pus 3) catalysing the formation of Ψ38 and Ψ39 in tRNA anticodon loop. J. Biol. Chem. 273: 1316 1323.
98. Liu, J.,, W. Zhou,, and P. W. Doetsch. 1995. RNA polymerase bypass at sites of dihydrouracil: implications for transcriptional mutagenesis. Mol. Cell. Biol. 15: 6729 6735.
99. Lowary, P.,, J. Sampson,, J. Milligan,, D. Groebe,, and O. C. Uhlenbeck,. 1986. A better way to make RNA for physical studies, p. 69 76. In P. H. van Knippenberg, and C. W. Hilbers (ed.), Structure and Dynamics of RNA, vol. 110. Plenum Press, New York, N.Y.
100. Lundberg, K. S.,, D. D. Shoemaker,, M. W. Adams,, J. M. Short,, J. A. Sorge,, and E. J. Mathur. 1991. High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus. Gene 108: 1 6.
101. Maden, B. E. H.,, M. E. Corbett,, P. A. Heeney,, K. Pugh,, and P. M. Ajuh. 1995. Classical and novel approaches to the detection and localization of the numerous modified nucleotides in eukaryotic ribosomal RNA. Biochimie 77: 22 29.
102. McCloskey, J. A. 1991. Structural characterization of natural nucleosides by mass spectrometry. Acc. Chem. Res. 24: 81 88.
103. Melton, D. A.,, P. A. Krieg,, M. R. Rebagliati,, T. Maniatis,, K. Zinn,, and M. R. Green. 1984. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 12: 7035 7056.
104. Meyhack, B.,, B. Pace,, O. C. Uhlenbeck,, and N. Pace. 1978. Use of T4-RNA ligase to construct model substrate for a ribosomal RNA maturation endonuclease. Proc. Natl. Acad. Sci. USA 75: 3045 3049.
105. Mialhe, E.,, and L. H. Miller. 1994. Biolistic techniques for transfection of mosquito embryos ( Anopheles gambiae). Bio-Techniques 16: 924 931.
106. Miele, E. A.,, D. R. Mills,, and F. R. Kramer. 1983. Autocatalytic replication of a recombinant RNA. J. Mol. Biol. 171: 281 295.
107. Milligan, J. F.,, and O. C. Uhlenbeck. 1989. Synthesis of small RNAs using T7 polymerase. Methods Enzymol. 180: 51 62.
108. Moore, M.,, and P. Sharp. 1992. Site-specific modification of pre-mRNA: the 2'OH groups at the splice sites. Science 256: 992 997.
109. Moore, M. J.,, and C. C. Query,. 1998. Uses of specifically modified RNAs constructed by RNA ligation, p. 75 108. In C. Smith (ed.), RNA-Protein Interactions: a Practical Approach. IRL Press, Oxford, United Kingdom.
110. Morin, A.,, A. Belayew,, J. A. Martial,, C. Tougard,, and A. Tixier-Vidal. 1996a. Expression and secretion of rat prolactin in transfected pituitary cells in culture. Mol. Cell Endocrinol. 117: 59 73.
111. Morin, A.,, D. Foiret,, and H. Grosjean. 1996b. Variation of tRNAs modifying enzymes activities during differentiation of normal cells and in tumoral cells. 6th International Congress on Cell Biology, December 7-11, San Francisco, California, 493a.
112. Morin, A.,, S. Auxilien,, B. Senger,, R. Tewari,, and H. Grosjean. 1998. Structural requirements for enzymatic formation of threonylcarbamoyladenosine (t6A) in tRNA: an in vivo study with Xenopus laevis oocytes. RNA 4: 24 37.
113. Morin, A.,, C. Simon,, D. Foiret,, and H. Grosjean. Unpublished data.
114. Möri, M.,, M. Dörner,, and S. Pääbo,. 1994. Direct purification of tRNAs using oligonucleotides coupled to magnetic beads, p. 107 111. In M. Uhlén,, E. Homes,, and O. Olsvik (ed.), Advances in Biomagnetic Separation. Eaton Publishers, Natick, Mass.
115. Morse, D. P.,, and B. L. Bass. 1997. Detection of inosine in messenger RNA by inosine-specific cleavage. Biochemistry 36: 8429 8434.
116. Motorin, Y.,, G. Bee,, R. Tewari,, and H. Grosjean. 1997. Transfer RNA recognition by the Escherichia coli Δ2-isopentenyl-pyrophosphate:tRNAΔ2-isopentenyl transferase: dependence on the anticodon arm structure. RNA 3: 721 733.
117. Motorin, Y.,, C. Simon,, D. Foiret,, G. Keith,, G. Simos,, E. Hurt,, and H. Grosjean. Unpublished data.
118. Mueller, S. O.,, and R. K. Slany. 1995. Structural analysis of the interaction of the tRNA modifying enzymes Tgt and QueA with a substrate tRNA. FEBS Lett. 361: 259 264.
119. Mullenbach, G. T.,, H. O. Kammen,, and E. E. Penhoet. 1976. A heterologous system for detecting eukaryotic enzymes which synthesize pseudouridine in transfer ribonucleic acids. J. Biol. Chem. 251: 4570 4578.
120. Mullis, K. B.,, and F. A. Faloona. 1987. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 155: 335 350.
121. Nakamura, H.,, Y. Oda,, S. Iwai,, H. Inoue,, E. Ohtsuka,, S. Kanaya,, S. Kimura,, C. Katsuda,, K. Katayanagi,, K. Morikawa,, H. Miyashiro,, and M. Ikehara. 1991. How does RNase H recognize a DNA*RNA hybrid? Proc. Natl. Acad. Sci. USA 88: 11535 11539.
122. Narayan, P.,, and F. Rottman. 1988. An in vitro system for accurate methylation of internal adenosine residues in messenger RNA. Science 242: 1159 1162.
123. Nau, F. 1976. The methylation of tRNA. Biochimie 58: 629 645.
124. Negrutskii, B. S.,, and M. P. Deutscher. 1991. Channeling of aminoacyl-tRNA for protein synthesis in vivo. Biochemistry 30: 4991 4995.
125. Negrutskii, B. S.,, and M. P. Deutscher. 1992. A sequestered pool of aminoacyl-transfer RNA in mammalian cells. Proc. Natl. Acad. Sci. USA 89: 3601 3604.
126. Nishikura, L.,, and E. M. De Robertis. 1981. RNA processing in microinjected Xenopus oocytes. Sequential addition of base modification in a spliced transfer RNA. J. Mol. Biol. 154: 405 420.
127. Ofengand, J.,, and A. Bakin. 1997. Mapping to nucleotide resolution of pseudouridine residues in large subunit ribosomal RNAs from representative eukaryotes, prokaryotes, archaebacteria, mitochondria and chloroplasts. J. Mol. Biol. 266: 246 268.
128. Ogilvie, K. K.,, N. Usman,, K. Nicoghosian,, and R. J. Cedergren. 1988. Total chemical synthesis of a 77-nucleotide-long RNA sequence having methionine-acceptance activity. Proc. Natl. Acad. Sci. USA 85: 5764 5768.
129. Ohtsuka, E.,, S. Nishikawa,, M. Ikehara,, and S. Takemura. 1976. Reconstitution of chemically synthesized ribooligonucleotides with naturally occurring tRNA fragments. Eur. J. Biochem. 66: 251 255.
130. Ohtsuka, E.,, S. Tanaka,, T. Tanaka,, T. Miyake,, A. F. Markham,, E. Nakagawa,, T. Wakabayashi,, Y. Taniyama,, S. Nishikawa,, R. Fukumoto,, H. Uemura,, T. Doi,, T. Tokunaga,, and M. Ikehara. 1981. Total synthesis of a RNA molecule with sequence identical to that of Escherichia coli formylmethionine tRNA. Proc. Natl. Acad. Sci. USA 78: 5493 5497.
131. Ohtsuka, E.,, T. Doi,, R. Futumoto,, H. Matsugi,, and M. Ikehara. 1983. Modification of the anticodon triplet of E. coli tRNAfMet by replacement with trimers complementary to non-sense codons UAG and UAA. Nucleic Acids Res. 11: 3863 3872.
132. Patton, J. R. 1991. Pseudouridine modification of U5 RNA in ribonucleoprotein particles assembled in vitro. Mol. Cell. Biol. 11: 5998 6006.
133. Paul, M. S.,, and B. L. Bass. 1997. Sensitive methods for the detection of inosine in messenger RNA. Second RNA Meeting, 21-26 May, Banff, Canada, p. 408.
134. Peattie, D. A.,, and W. Gilbert. 1980. Chemical probes for higher-order structure in RNA. Proc. Natl. Acad. Sci. USA 77: 4679 4682.
135. Pegg, A. E. 1972. Methylation of yeast aspartic acid transfer RNA by rat liver extracts. FEBS Lett. 22: 339 342.
136. Pley, H. W.,, K. M. Flaherty,, and D. B. McKay. 1994. Three-dimensional structure of a hammerhead ribozyme. Nature 372: 68 74.
137. Poison, A. G.,, P. F. Crain,, S. C. Pomerantz,, J. A. McCloskey,, and B. L. Bass. 1991. The mechanism of adenosine to inosine conversion by the double-stranded RNA unwinding/modifying activity—a high-performance liquid chromatography-mass spectrometry analysis. Biochemistry 30: 11507 11514.
138. Pomerantz, S. C.,, and J. A. McCloskey. 1990. Analysis of RNA hydrolyzates by LC/MS. Methods Enzymol. 193: 796 824.
139. Potter, H. 1988. Electroporation in biology: methods applications and instrumentation. Anal. Biochem. 174: 361 373.
140. Qian, Q.,, and G. R. Björk. 1997. Structural requirements for the formation of 1-methylguanosine in vivo in tRNAPro(GGG) of Salmonella typhimurium. J. Mol. Biol. 266: 283 297.
141. Randerath, E.,, and K. Randerath,. 1983. Selected postlabeling procedures for base composition and sequence analysis of nucleic acids, p. 169-233. In S. M. Weissman (ed.), Methods of DNA and RNA sequencing. Praeger Publishers, New York, N.Y.
142. Rebagliati, M. R.,, and D. A. Melton. 1987. Antisense RNA injections in fertilized frog eggs reveal an RNA duplex unwinding activity. Cell 48: 599 605.
143. Reyes, V. M.,, and J. Abelson. 1987. A synthetic substrate for tRNA splicing. Anal. Biochem. 166: 90 106.
144. Rhee, Y.,, M. R. Valentine,, and J. Termini. 1995. Oxidative base damage in RNA detected by reverse transcriptase. Nucleic Acids Res. 23: 3275 3282.
145. Rogg, H.,, R. Brambilla,, G. Keith,, and M. Staehelin. 1976. An improved method for the separation and quantitation of the modified nucleosides of transfer RNA. Nucleic Acids Res. 3: 285 295.
146. Sampson, J.,, F. Sullivan,, L. Behlen,, A. DiRenzo,, and O. C. Uhlenbeck. 1987. Characterization of two RNA-catalyzed RNA cleavage reactions. Cold Spring Harbor Symp. Quant. Biol. 52: 267 275.
147. Samuelsson, T.,, T. Boren,, T. I. Johansen,, and F. Lustig. 1988. Properties of a transfer RNA lacking modified nucleosides. J. Biol. Chem. 263: 13692 13699.
148. Santoro, S. W.,, and G. F. Joyce. 1997. A general purpose RNA-cleaving DNA enzyme. Proc. Natl. Acad. Sci. USA 94: 4262 4266.
149. Sayers, J.,, C. Krekel,, and F. Eckstein. 1992. Rapid high-efficiency site-directed mutagenesis by the phosphorothioate approach. BioTechniques 13: 592 596.
150. Scadden, A. D. J.,, and C. W. J. Smith. 1997. A ribonuclease specific for inosine containing RNA: a potential role in antiviral defence? EMBO J. 16: 2140 2149.
151. Schaefer, K. P.,, S. Altman,, and D. Soli. 1973. Nucleotide modification in vitro of the precursor of transfer RNA of Escherichia coli. Proc. Natl. Acad. Sci. USA 70: 3626 3630.
152. Scott, W. G.,, J. T. Finch,, R. Grenfell,, J. F. T. Smith,, M. J. Gait,, and A. Klug. 1995. Rapid crystallization of chemically synthesized hammerhead RNAs using a double screening procedure. J. Mol. Biol. 250: 327 332.
153. Seiwert, S. D.,, and K. Stuart. 1994. RNA editing: transfer of genetic information from gRNA to precursor mRNA in vitro. Science 266: 114 117.
154. Silberklang, M.,, A. M. Gillum,, and U. L. RajBhandary. 1979. Use of in vitro i2P labelling in the sequence analysis of nonradioactive tRNAs. Methods Enzymol. 59: 58 109.
155. Simos, G.,, H. Tekotte,, H. Grosjean,, A. Segref,, K. Sharma,, D. Tollervey,, and E. C. Hurt. 1996. Nuclear pore proteins are involved in the biogenesis of functional tRNA. EMBO J. 15: 2270 2284.
156. Slany, R. K.,, and H. Kersten. 1994. Genes, enzymes and coenzymes of queuosine biosynthesis in procaryotes. Biochimie 76: 1178 1182.
157. Smith, J. E.,, B. S. Cooperman,, and P. Mitchell. 1992. Methylation sites in Escherichia coli ribosomal RNA: Localization and identification of four new sites of methylation in 23 S rRNA. Biochemistry 31: 10825 10834.
158. Sprinzl, M.,, C. Horn,, M. Brown,, A. Ioudovitch,, and S. Steinberg. 1998. Compilation of tRNA sequences and sequences of tRNA genes. Nucleic Acids Res. 26: 148 153.
159. Stanley, J.,, and S. Vassilenko. 1978. A different approach to RNA sequencing. Nature 274: 87 89.
160. Steinberg, S.,, and R. Cedergren. 1995. A correlation between N2-dimethylguanosine presence and alternate tRNA conformers. RNA 1: 886 891.
161. Stern, S.,, D. Moazed,, and H. Noller. 1988. Structural analysis of RNA using chemical and enzymatic probing monitored by primer extension. Methods Enzymol. 164: 481 489.
162. Streeter, D. G.,, and B. G. Lane. 1970. Studies of the biogenesis of N2-dimethylguanylate. I. Generation of N2-dimethylguanylate when bulk Escherichia coli transfer RNA is used as a substrate for wheat embryo methyltransferases. Biochim. Biophys. Acta 199: 394 404.
163. Sylvers, L. A.,, K. C. Rogers,, M. Shimizu,, E. Ohtsuka,, and D. Soll. 1993. A 2-thiouridine derivative in tRNAGlu is a positive determinant for aminoacylation by Escherichia coli glutamyl-tRNA synthetase. Biochemistry 32: 3836 3841.
164. Szkukalek, E.,, A. Myslinski,, A. Mougin,, R. Luhrmann,, and C. Branlant. 1995. Phylogenetic conservation of modified nucleotides in the terminal loop 1 of the spliceosomal U5 snRNA. Biochimie 77: 16 21.
165. Szostak, J. W.,, and A. D. Ellington,. 1993. In vitro selection of functional RNA sequences, p. 511 533. In R. F. Gesteland, and J. F. Atkins (ed.), The RNA World. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
166. Szweykowska-Kulinska, Z.,, B. Senger,, G. Keith,, F. Fasiolo,, and H. Grosjean. 1994. Intron-dependent formation of pseudo-uridines in the anticodon of Saccharomyces cerevisiae minor tRNAIle. EMBO J. 13: 4636 4644.
167. Tarassov, I. A.,, and N. S. Entelis. 1992. Mitochondrially imported cytoplasmic transfer RNALys(CUU) of Saccharomyces cerevisiae: in vivo and in vitro targetting systems. Nucleic Acids Res. 20: 1277 1281.
168. Tsurui, H.,, Y. Kumazawa,, R. Sanokawa,, Y. Watanabe,, T. Ku-roda,, A. Wada,, K. Watanabe,, and T. Shirai. 1994. Batchwise purification of specific tRNAs by a solid-phase DNA probe. Anal. Biochem. 221: 166 172.
169. Tycowski, K. T.,, C. M. Smith,, M. D. Shu,, and J. A. Steitz. 1996. A small nucleolar RNA requirement for site-specific ribose methylation of rRNA in Xenopus. Proc. Natl. Acad. Sci. USA 93: 14480 14485.
170. Uhlenbeck, O. C.,, and V. Cameron. 1977. Equimolar addition of oligoribonucleotides with T4-RNA ligase. Nucleic Acids Res. 4: 85 98.
171. Uhlenbeck, O. C. 1995. Keeping RNA happy. RNA 1: 4 6.
172. Usman, N.,, and R. Cedergren. 1992. Exploiting the chemical synthesis of RNA. Trends Biochem. Sci. 17: 334 339.
173. Usman, N.,, M. Egli,, and A. Rich. 1992. Large scale chemical synthesis, purification and crystallization of RNA-DNA chimeras. Nucleic Acids Res. 20: 6695 6699.
174. van den Hoff, M. J. B.,, W. T. Labruyere,, A. F. M. Moorman,, and W. H. Lamers. 1990. The osmolarity of the electroporation medium affects the transient expression of genes. Nucleic Acids Res. 18: 6464.
175. van den Hoff, M. J. B.,, A. F. M. Moorman,, and W. H. Lamers. 1992. Electroporation in 'intracellular' buffer increases cell survival. Nucleic Acids Res. 20: 2902.
176. Wang, Y. 1984. A total synthesis of yeast alanine transfer RNA. Acc. Chem. Res. 17: 393 397.
177. Wildenauer, D.,, H. J. Gross,, and D. Riesner. 1974. Enzymatic methylations: III. Cadaverine-induced conformational changes of E. coli tRNAfMet as evidenced by the availability of a specific adenosine and specific cytidine residue for methylation. Nucleic Acids Res. 1: 1165 1182.
178. Wittig, B.,, and S. Wittig. 1978. Reverse transcription of tRNA. Nucleic Acids Res. 5: 1165 1178.
179. Wower, J.,, K. V. Rosen,, S. S. Hixson,, and R. A. Zimmermann. 1994. Recombinant photoreactive tRNA molecules as probes for cross-linking studies. Biochimie 76: 1235 1246.
180. Wright, M. C.,, and G. F. Joyce. 1997. Continuous in vitro evolution of catalytic function. Science 276: 614 617.
181. Wrzesinski, J.,, K. Nurse,, A. Bakin,, B. G. Lane,, and J. Ofengand. 1995. A dual-specificity pseudouridine synthase: an Escherichia coli synthase purified and cloned on the basis of its specificity for Ψ746 in 23S RNA is also specific for Ψ32 in tRNAPhe. RNA 1: 437 448.
182. Xue, H.,, A.-L. Glasser,, J. Desgres,, and H. Grosjean. 1993. Modified nucleotides in Bacillus subtilis tRNATrp hyperexpressed in Escherichia coli. Nucleic Acids Res. 21: 2479 2486.
183. Yin, Y. H.,, and C. W. Carter. 1996. Incomplete factorial and response surface methods in experimental design: yield optimization of tRNATrp from in vitro T7 RNA polymerase transcription. Nucleic Acids Res. 24: 1279 1286.
184. Yisraeli, J. K.,, and D. A. Melton. 1989. Synthesis of long, capped transcripts in vitro by SP6 and T7 RNA polymerases. Methods Enzymol. 180: 42 50.
185. Yokobori, S.,, and S. Paabo. 1995. tRNA editing in metazoans. Nature 377: 490.
186. Youvan, D. C, and J. E. Hearst. 1979. Reverse transcriptase pauses at N2-methyIguanine during in vitro transcription of Escherichia coli 16S ribosomal RNA. Proc. Natl. Acad. Sci. USA 76: 3751 3754.
187. Youvan, D. C, and J. E. Hearst. 1981. A sequence from Dro-sophila melanogaster 18S rRNA bearing the conserved hyper-modified nucleoside amΨ: analysis by reverse transcription and high-performance liquid chromatography. Nucleic Acids Res. 9: 1723 1741.
188. Yu, W.,, and W. Schuster. 1995. Evidence for a site-specific cytidine deamination reaction involved in C to U RNA editing of plant mitochondria. J. Biol. Chem. 270: 18227 18233.
189. Yu, Y. T.,, and J. A. Steitz. 1997. A new strategy for introducing photoactivatable 4-thiouridine (s4U) into specific positions in a long RNA molecule. RNA 3: 807 810.
190. Yu, Y.-T.,, M.-D. Shu,, and J. A. Steitz. 1997. A new method for detecting sites of 2'-O-methylation in RNA molecules. RNA 3: 324 331.
191. Zagorska, L.,, J. Van Duin,, H. Noller,, B. Pace,, K. Johnson,, and N. Pace. 1984. The conserved 5s rRNA complement to tRNA is not required for translation of natural mRNA. J. Biol. Chem. 259: 2798 2802.
192. Zeevi, M.,, and V. Daniel. 1976. Aminoacylation and nucleoside modification of in vitro synthesised transfer RNA. Nature 260: 72 74.
193. Zhao, L.-J.,, Q. X. Zhang,, and R. Padmanabhan. 1993. Polymerase chain reaction-based point mutagenesis protocol. Methods Enzymol. 217: 218 227.
194. Zheng, H.,, T. B. Fu,, D. Lazinski,, and J. Taylor. 1992. Editing on the genomic RNA of human hepatitis delta virus. J. Virol. 66: 4693 4697.
195. Zhou, W.,, D. Reines,, and P. W. Doetsch. 1995. T7 polymerase bypass of large gaps on the template strand reveals a critical role of the non-template strand in elongation. Cell 82: 577 585.

This is a required field
Please enter a valid email address
Please check the format of the address you have entered.
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error