Cloning, sequencing and expression of a cDNA encoding mammalian valyl-tRNA synthetase

Abstract

A fragment of the cDNA encoding a rat valyl-tRNA synthetase (Trs Val)-like protein was cloned from a rat cDNA library in λgt11 using an oligodeoxyribonucleotide (oligo) probe. Three independent plaque clones containing the human Trs Val cDNA were then isolated from a λgt10 human erythroleukemia cDNA library using the rat cDNA fragment as the hybridization probe. Sequence analyses of the cDNA fragments provided a 3.2-kb sequence with an open reading frame that contained the ‘HIGH’ synthetase signature sequence and the tRNA 3′-end-binding motif, KMSKS, and putative Val-binding motif, EWCISRQ. The sequence was extended to the 3′ end of the cDNA by the polymerase chain reaction using an internal primer and an oligo(dT) adapter. The deduced 1051-amino-acid sequence shares 65% identity with yeast Trs Val, and contains a highly basic N-terminal region, a newly evolved protease-sensitive region in sequence close to the C terminus, and several sites for protein kinase C phosphorylation. A 3-kb cDNA fragment was sub-cloned into plasmid pSVL and expressed in COS-7 cells; up to a sevenfold increase in Trs Val activity was obtained. These results confirm the cloning and sequencing of a human Trs Val-encoding cDNA.