IDENTIFICATION OF YHR019 IN SACCHAROMYCES-CEREVISIAE CHROMOSOME-VIII AS THE GENE FOR THE CYTOSOLIC ASPARAGINYL TRANSFER-RNA SYNTHETASE

Exploiting the asparagine auxotrophy of the Saccharomyces cerevisiae m utant strain 8556a, we have isolated the gene for the cytosolic aspara ginyl-tRNA synthetase (AsnRS) of S, cerevisiae, by functional compleme ntation of the mutation affecting this strain. The isolated gene could be identified to the open reading frame YHR019, called DED81, located on chromosome VIII. The mutant gene from he 8556a strain, asnrs(-)-1, was amplified from genomic DNA by PCR. This gene contains a point mut ation, leading to the replacement of a glycine residue by a serine in a region of the protein probably important for the asparaginyl-adenyla te recognition. The protein encoded by YHR019 is very similar to cytos olic AsnRS from other eukaryotic sources. In a phylogenetic analysis b ased on AsnRS sequences from various organisms, the eukaryotic sequenc es were clustered. Expression of YHR019 in Escherichia coli demonstrat ed that a yeast AsnRS activity was produced. The recombinant enzyme wa s purified to homogeneity in three chromatography steps. We showed tha t the recombinant S. cerevisiae AsnRS was able to charge unfractionate d yeast tRNA, but not E. coli tRNA, with asparagine. (C) 1998 John Wil ey Sr Sons, Ltd.