Nucleic Acids Research: VOLUME 39 ISSUE 3 2011

The major valine acceptor tRNAj( from rabbit liver was purified and its nucleotide sequence determined by in vitro [32p] _ labeling with T4 phage induced polynucleotide kinase and fingerprinting techniques. Its primary structure was found to be identical with the major valine tRNA from mouse myeloma cells. According to the wobble hypothesis this tRNA, which exclusively has an IAC anticodon, should decode the valine codons GUU, GUC and GUA only. However, this tRNA recognizes all four valine codons with a surprising preference for GUG. It is unknown whether this is due to the lack of Ag7 modification next to the 3'end of the anticodon IAC. The nature of the inosine-guanosine interaction remains to be clarified. INTRODUCTION Val The primary structure of tRNA, from mouse myeloma cells has been elucidated by Piper and Clark (1,2). These authors noted, besides other remarkable features, (a) an unmodified A in position 37 next to the 3'end of the anticodon, (b) the presence of U V C in place of the common T Y C sequence, (c) a unique adenosine in position 60, opposite to U_., and hence (d) the possibility of an extended T V C stem due to additional Ag^: U-. and A_Q:4* cc loop IV base pairs. These unique properties D4 oy oo ya2 ya2 of the major mouse myeloma tRNA, prompted us to purify the Val corresponding tRNA, from rabbit liver, to compare their sequences and to study this tRNA's loop IV structure (3) and coding properties. According to the "wobble" hypothesis (4), the tRNA* anticodon IAC should decode the codons GUU, GUC and GUA. MATERIALS and METHODS RNAase T 2 (E.C. 3.1.4.23) and RNAase T x (E.C. 3.1.4.8) were from Sankyo, Tokyo; RNAase A (E.C. 3.1.4.22), alkaline phosphatase from E_.coli (E.C. 3.1.3.1) and yeast hexokinase (E.C. 2.7.1.1) were from Boehringer Mannheim GmbH. Venom 1999 © Information Retrieval Limited 1 Falconberg Court London W1V5FG England Nucleic Acids Research phosphodiesterase (K.C. 3.1.4.1) was from Worthington Biochemical Corp. and cellulose acetate strips (3 x 55 cm) were from Schleicher and Schiill; DE-81 paper (46 x 57 cm) was from Whatman. [32 l Pj-ATP was prepared as described by Glynn and Chappell (5) and had a specific activity of about 50 Ci/mmole. Polynucleotide kinase was prepared from T4 phage infected E.coli by extraction of the cells, streptomycin precipitation, autolysis, ammoniumsulfate fractionation, DEAE-cellulose, phosphocellulose and hydroxyapatite fractionation as described by Richardson (6), followed by a pH-gradient elution from a phosphocellulose column (7). The final enzyme fractions were dialyzed against 25 mM KC1 10 mM mercaptoethanol 20 mM potassium phosphate, pH 7.0 in 50 % glycerol, and stored at -20° C. Crude rabbit liver tRNA was prepared as described by Rogg et al. (8). Purification of rabbit liver valine tRNA was performed by column chromatography as shown in Fig. 1. The nucleotide composition of purified liver valine tRNA was Val determined by digestion of 8 A 2 6 Q units tRNA.̂ with 16 units RNAase T2 in 100 (xl 0.1 M potassium acetate, pH 4.7, 5 h 37° C.