CLONING, SEQUENCING AND BACTERIAL EXPRESSION OF HUMAN GLYCINE TRANSFER-RNA SYNTHETASE

The human glycine tRNA synthetase gene (GlyRS) has been cloned and seq uenced. The 2462 bp cDNA for this gene contains a large open reading f rame (ORF) encoding 685 amino acids with predicted M(r) = 77 507 Da. T he protein sequence has similar to 60% identity with B.moriGlyRS and 4 5% identity with S.cerevisiae GlyRS and contains motifs 2 and 3 charac teristic of Class II tRNA synthetases. A second ORF encoding 47 amino acids is found upstream of the large ORF. Translation of this ORF may precede the expression of GlyRS as a possible regulatory mechanism. Th e enzyme was expressed in E.coli as a fusion protein with a 13 kDa bio tinylated tag with an apparent M(r) = 90 kDa. The fusion protein was i mmunoprecipitated from crude bacterial extract with human EJ serum, wh ich contains autoantibodies directed against GlyRS, and with rabbit po lyclonal serum raised against a synthetic peptide derived from the pre dicted amino acid sequence of human GlyRS. Bacterial extract containin g the fusion protein catalyses the aminoacylation of bovine tRNA with [C-14]-gly at 10-fold increased level above normal bacterial extract a nd confirms that the cDNA encodes human GlyRS.