The Nucleotide Sequence of tRNkVal of Drosophila melanogaster CHLOROACETALDEHYDE MODIFICATION AS AN AID TO RNA SEQUENCING*

The nucleotide sequence of tRNAV* from Drosophila melanogaster was determined It is probable that residue C 49 is modified to m6C. The use of tRNA modified with chloroacetaldehyde to overcome secondary structure problems in sequencing is described. Modification of the with chloroacetaldehyde allowed the sequence to be resolved. A 50% solution (v/v) of chloroacetaldehyde was prepared by heating 25 pl of 97% chloroacetaldehyde dimethyl acetal (Aldrich) with an equal volume of 0.2 M HCI in a sealed glass tube at 100 "C for 30 min. End-labeled tRNA plus carrier tRNA (2-4 pg) was dissolved in 15 p1 of 1 M sodium acetate buffer, pH 4.0,1 mM EDTA containing 2 p1 of the chloroacetaldehye solution. The solution was sealed in a glass capillary tube and heated to 100 "C for 2 min, then chilled on ice. The contents of the tube were expelled into 100 pl of 0.1 M sodium acetate buffer, pH 4.0, containing 0.1 mM EDTA and the solution incubated at 80 "C for 20 min. The RNA was precipitated with ethanol and its sequence determined by the gel read-off method.


* This work was supported by grants from the Medical Research
Council of Canada and the British Columbia Health Care Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ Recipient of a Medical Research Council of Canada Studentship and an H. R. MacMillan Family Fellowship. and 1.2 units of tRNA nucleotidyl transferase for 20 min at 32 "C. The reaction was stopped by adding 10 pl of 7 M urea containing 0.5% xylene cyanol and bromophenol blue. The mixture was applied to the sample slot in a 20% polyacrylamide gel (16 x 17 X 0.15 cm) containing 7 M urea. After electrophoresis, the terminally labeled tRNA was located on the gel by autoradiography and eluted from the gel slice by soaking in 0.5 ml of 0.1 M sodium acetate, 0.1% sodium dodecyl sulfate for 1-2 days at 4 "C. The labeled tRNA and 10 pg of added carrier tRNA was precipitated from the eluate with 3 volumes of ethanol and was used for sequence analysis. Alternatively, tRNA?' was labeled at the 3' end with [5'-3zP]pCp using T4 RNA ligase as described by England and Uhlenbeck (5).
Labeling the 5' end of tRN&""' was inefficient unless the tRNA was first cut in the anticodon loop by limited digestion with RNase Up to give half-molecules. The 10-pl reaction mixture containing 2.5 pg of tRN&""', 10 m~ ammonium acetate buffer, pH 4.5, and 0.01 units of RNase Up was incubated at 4 "C for 1 hr, heated to 100 "C for ated and labeled with [y3'P]ATP and T, polynucleotide kinase as 1 mi n, then chilled on ice. The tRNA fragments were dephosphoryldescribed in Ref. 6. The kinase reaction was stopped by adding 10 pl of 7 M urea containing 0.5% xylene cyanol and bromophenol blue and was applied to the sample slot in a denaturing 20% polyacrylamide gel (35 X 15 X 0.05 cm) After electrophoresis, labeled tRNA fragments were located on the gel by autoradiography. Fragments approximately 35 nucleotides long were eluted from gel slices and used for sequence analysis.

Sequence Analysis
The isolation of Drosophila melanogaster tRNA?' and its nucleoside analysis have been described previously (2). Sequencing was done by a combination of methods. Most of the sequence was obtained using the Stanley and Vassilenko method (7) modified in the following ways. The RNA was partially hydrolyzed in 5 m~ MOPS' buffer, pH 7.2, containing 0.1 m~ EDTA at 100 "C for 3 min. Alkaline hydrolysis of the labeled RNA fragments was replaced by digestion with 0.05 units of RNase TZ in 10 pl of 10 mM ammonium acetate buffer, pH 4.6, containing 2 mM EDTA for 16 h at 37 "C. The labeled nucleoside-5',3' bisphosphates (pNp) were identified by comparing their mobilities on PEI-cellulose TLC plates (Polygram Cel 300 PEI, Machery-Nagel) in solvent A (0.8 M (NH4)2S04, 10 m~ EDTA) and on cellulose TLC plates (E. Merck or Eastman Kodak) in solvent B (isobutyric acid, concentrated NROH, 1 m~ EDTA; 66:1:33) with published values (6,8). Modified pNps were incubated with 1 pg of nuclease PI for 16 h at 25 "C and the resulting nucleoside 5'-phosphates were identified by chromatography on cellulose TLC plates in solvent B and solvent C (0.1 M sodium phosphate buffer, pH 6.8, (NH4)2S04, 1-propanol; 100:60:2, v/w/v). The sequence near the 5' and 3' ends of the tRNA was determined by two-dimensional homochromatography as described by Silberklang et al. (6) except that the RNA was hydrolyzed in 98% formamide at 100 "C for 1 h. Sequencing by partial, base specific, enzymatic cleavage of end-labeled RNA (9-11) (gel read-off method) was used to fd the gaps in the sequence and c o n f m results obtained by other methods.
The occurrence of five consecutive Cp residues (C 47-C 51) (in the variable loop and T stem) could not be satisfactorily demonstrated by either the Stanley and Vassilenko or the gel read-off methods, presumably because of strong secondary structure in this region. Modification of the RNA with chloroacetaldehyde allowed the sequence to be resolved. A 50% solution (v/v) of chloroacetaldehyde was prepared by heating 25 pl of 97% chloroacetaldehyde dimethyl acetal (Aldrich) with an equal volume of 0.2 M HCI in a sealed glass tube at 100 "C for 30 min. End-labeled tRNA plus carrier tRNA (2-4 pg) was dissolved in 15 p1 of 1 M sodium acetate buffer, pH 4.0,1 mM EDTA containing 2 p1 of the chloroacetaldehye solution. The solution was sealed in a glass capillary tube and heated to 100 "C for 2 min, then chilled on ice. The contents of the tube were expelled into 100 pl of 0.1 M sodium acetate buffer, pH 4.0, containing 0.1 mM EDTA and the solution incubated at 80 "C for 20 min. The RNA was precipitated with ethanol and its sequence determined by the gel read-off method.

RESULTS AND DISCUSSION
The sequence of tRN&"' , determined in this study, is shown in cloverleaf form in Fig. 1. It contains 76 nucleotide residues and has all the invariant and other strongly conserved nucleotides expected in a cytoplasmic tRNA (12). The residue at position 4 was observed to be resistant to the formamideinduced hydrolysis used in sequencing the 5' end of the molecule by two-dimensional homochromatography. Ribosemethylated nucleotides are present at position 4 of all sequenced eukaryotic glycine and proline tRNAs (13) and tRN&y of D. melanogaster.* Of the two ribose-methylated nucleosides detected in the nucleoside analysis (2) the 2"Omethylcytidine had been found at position 32 by the Stanley and Vassilenko method. Therefore, the 2'-0-methyuridine residue was assigned to position 4.
Determination of the nucleotide sequence by the Stanley and Vassilenko method indicated that a modified nucleotide was present at position 9. The mobility of this nucleotide on PEI-cellulose or cellulose TLC plates in a number of solvent systems did not match published mobility values for various modified nucleotides in the same systems but did suggest that the unknown nucleotide contained a modified guanosine residue. On the basis of the nucleoside analysis (2), l-methylguanosine (m'G) was assigned to position 9. This assignment is consistent with the observation that m'G is found only at position 9 in previously sequenced eukaryotic tRNAs (13).
About 50% of the uridine residues at position 20 are modified to 3-(3-amino-3-carboxypropyl)uridine (acp3U) (Fig. 30). Authentic acp3U-Yphosphate, isolated from a nuclease PI digest of crude Escherichia coli tRNA and characterized by its UV absorption spectrum, positive ninhydrin reaction, and the chromatographic properties of the nucleoside (14), was used as a standard for identification of the modified nucleotide. The presence of acp3U in tRN&' " is consistent with the work of White (15) who has shown that Drosophila tRNAVa' reacts with cyanogen bromide and the N-hydroxysuccinimide ester of naphthoxyacetic acid, reagents thought to react with the amino group of acp3U in tRNAs. In prokaryotes and plant chloroplasts, acp3U is found exclusively 3' to the 7-methylguanosine in the variable arm of several tRNAs (13) while in eukaryotes acp3U has been found only at position 20 of rat liver tRNAA"" (16).
The series of five cytidine residues at positions 47-51 could not be determined by either the gel read-off or Stanley and Vassilenko methods. Electrophoresis of partial enzymatic or formamide digests of tRN&' "' on denaturing polyacrylamide gels exhibited both strong band compression and incomplete enzymatic cleavage in the variable loop and T stem regions.
In yeast tRNAPhe, these regions are involved in strong secondary and tertiary interactions with other parts of the molecule (17). Similar interactions in tRNA?' may be particularly strong because of the high G-C content of these regions. The * D. L. Cribbs, unpublished results. observed band compression indicates that the products of such RNase cleavage as does occur fold back on themselves and migrate anomalously during polyacrylamide gel electrophoresis.
At pH 4.0, chloroacetaldehyde reacts with nonbase-paired cytidine and adenosine residues in nucleic acids to form etheno derivatives (18-21) that cannot form Watson-Crick base pairs. Thus, if tRNA were denatured and then modified with chloroacetaldehyde, it should lose much of its secondary structure. Fig. 2 compares the results of the gel read-off method applied to tRNA?' with and without modification of the tRNA with chloroacetaldehyde. The severe band compression and inadequate enzymatic cleavage observed when intact tRN&""' was the substrate (Fig. 2 A ) was relieved when the RNA was modified in this way before sequencing (Fig. 2B). The ribonucleases used for sequencing display the same substrate specificity towards modified RNA as to the unmodified form. This is surprising considering the strict requirement of RNase A for an unsubstituted nitrogen at position 3 of pyrimidines in its substrate (22). Thus, the ethenocytidine residues produced by modification with chloroacetaldehyde would not be expected to be sites for RNase A cleavage. Tolman et al. (23) modified dinucleoside monophosphates containing cytidine with chloroacetaldehyde and examined the sensitivity of the etheno derivatives to hydrolysis by RNase A. They found ethenocytidylyl uridine (&pU) and cCpcA to be completely resistant to hydrolysis while cCpG and cCpeC showed slight hydrolysis after prolonged RNase A treatment. It is possible, therefore, that RNase A recognizes the modified cytidine residues but it is more likely that modification was not complete and that unmodified cytidine residues were the sites of RNase A cleavage.
The nucleoside analysis (2) suggests that tRNA:" contains two 5-methylcytidine (m5C) residues per molecule. One of these was located at position 38; the other is assigned to a probable position at residue 48 or 49 since m5C has been found in this region of other eukaryotic valine tRNAs (13). The bands corresponding to C-49 in Fig. 2B move anomalously fast compared to the surrounding cytidine bands. This suggests modification of C-49.
The T loops of mammalian tRNAsic) and tRNAzYo of human placenta are unique among sequenced cytoplasmic tRNAs in having a uridine residue at position 54 and an adenosine at position 60 (24-27). These two nucleosides could base pair to produce a tRNA with a six-base pair T stem and a five-nucleotide T loop. Jank et al. (28) have provided some evidence favoring such a structure. Mammalian tRNAZc) is also unusual in its coding properties. According to the wobble hypothesis (29) an IAC anticodon should decode GUA, GUC, and GUU codons but not GUG. Ribosome-binding experiments show that mammalian tRNA& binds to ribosomes in the presence of all four valine triplets but most strongly with GUG (27). Unlike the mammalian tRNA, Drosophila tRN&' "' has a typical T loop with rT at position 54 and C at position 60. tRNA?' binds strongly to ribosomes in response to GUC, GUA, and GUU as predicted by the wobble hypothesis and only weakly to GUG (2). Comparison of the two tRNA structures suggests that the anomalous coding properties of the mammalian tRNA$c) may be related to its unusual T loop structure. The sequence of tRN&' " matches that of the two valine tRNA genes of the recombinant plasmid pDt55. The tRN&' "'like genes in pDt92, pDtl20, and pDtl4 (l), originally identitied by hybridization with tRN&"" (30), differ from the RNA sequence determined here at four sites. The three tRN&' "'like genes all have dT, dC, dG, and dA at sites corresponding to C16, U29, A41, and G57, respectively, in the RNA. Fig. 3 shows autoradiographs of RNA sequencing TLC plates for the sites where the tRNA and tRNA-like genes differ. At none of the four sites is there evidence for heterogeneity in the RNA. Sequence heterogeneity has been noted in tRNAZc) of mouse myeloma cells (25). In 15-35% of this tRNA, the $27-A43 base pair is replaced by a C-G base pair. Preliminary sequence data (not presented) on D. melanogaster tRNA;:' and tRNA:$' show that the sequence of the tRNA,V"'-like genes does not correspond to these tRNAs.
Comparison of the sequences of tRNAs from mammals and insects determined to date (13) shows a great deal of homology between equivalent tRNAs of different animal phyla. If differences due to post-transcriptional modifications are excluded, these homologies range from 100% for tRNAiY" of Drosophila and rabbit liver to 92% fortRNAP of mammals and Drosophila. Drosophila tRNA?' differs from mammalian tRNA&) at 10 sites (87% homology), making it less like its Autoradiograms of developed PEI-cellulose "LC plates showing sites at which the tRNAV" sequence differs from the tRN&V"'-like genes in plasmids pDt92, pDtl20, and pDtl4. The [5'-"P]pNps resulting from the Stanley and Vassilenko sequencing technique ("Materials and Methods") were separated on PEI-cellulose thin layers developed in solvent A. The TLC plates were autoradiographed and the pNps identified by their mobilities relative to pup. Positions at which the RNA sequence differs from the tRNG""'-like gene sequences are marked (A). At these sites the t R N e I -l i k e genes contain A, dA instead of G57; B, dG instead of A41; C, dC instead of U29 and D, d T instead of C16. Sequence heterogeneity is detected only at position 20 (D) where a uridine residue is only partially modified to acp3U. mammalian counterpart than any insect tRNA sequenced to date. Interestingly, the tRNe'-like genes described in the accompanying paper (1) differ from mammalian tRNA&) at only seven sites (91% homology). tRN&' "' k 81% homologous to yeast tRNA$!o. This strong homology is similar to that between the initiator methionine tRNAs of the two species (82%) and contrasts with the lower degree of homology for the other tRNAs for which seauence data are available (tRNAgh,, 68%; tRNA&), 74%; tRNA&,76%) (13). The