Isolation and sequence analysis of two major leucine transfer ribonucleic acids (anticodon Mm-A-A) from a Morris hepatoma 5123D rat tumor

Abstract

The nucleotide sequences of 2 major tRNALeu species (anticodon Mm-A-A) isolated from Morris hepatoma 5123D were determined by a combination of a newly developed thin-layer readout sequencing method and additional 3H- and 32P-labeled derivative methods entailing chromatographic fingerprinting and base-specific enzymatic cleavages. The nucleotide sequence of the 2 hepatoma tRNAMm-A-ALeu species, one of which has U and the other of which has A in position 50 at the tip of the long extra arm, is pG-U-C-A-G-m2G-A-U-G-(m2)G-C-(ac4)C-G-A-G-U-G-G-D-C-.psi.-A-A-G-G-C-m22G-C-C-A-G-A-C-U-Mm-A-A-m1G*-.psi.-.psi.-C-U-G-G-L-(.psi.)U-C-C-G-U- or A-A-U-G-G-A-G-m5C-G-U-G-G-G-T-.psi.-C-G-m1A-A-U-C-C-C-A-C-U-U-C-U-G-A-C-A-C-C-AOH. These are the first leucine tRNA sequences from higher eukaryotes that have been determined. Noteworthy features of the mammalian leucine tRNA are the presence of .psi. in the .beta. region of the D loop and the occurrence of 3 unknown hypermodified nucleosides (Mm, m1G* and L) in positions 35, 38 and 45, respectively. m1G* was converted to m1G by treatment with alkali. Sequencing gels indicated that the parent base of the 2''-O-methylated nucleoside Mm may be a pyrimidine, probably a C derivative, as indicated by the chromatographic behavior of nucleotides containing Mm. The presence of a pyrimidine in the wobble position would be consistent with the anticodon sequence Mm-A-A and the leucine codons U-U-G and U-U-A. The occurrence of a hypermodified nucleoside, L, in the first position of the long extra arm appears unusual; thus far the only modified nucleoside found in this position is Um in eukaryotic serine tRNA. Since all tRNA with a long extra arm sequenced to date have a pyrimidine in this position, L is likely to be a pyrimidine, probably a U derivative, as inferred from chromatographic data.