THE ASP1 GENE OF SACCHAROMYCES-CEREVISIAE, ENCODING THE INTRACELLULARISOZYME OF L-ASPARAGINASE

Saccharomyces cerevisiae produces two L-asparaginases (ASPs), intracel lular ASP I and cell-wall ASP II. In this report, the ASP-I-encoding g ene, ASP1, has been identified by homology cloning based on the struct ures of ASPs from other organisms. Its deduced protein product has a s ubunit M(r) of 41414, and shows substantial sequence homology to the b acterial amidohydrolase family. The product of the S. cerevisiae ASP3 gene, a further member of this family, encoding the nitrogen catabolit e-regulated cell-wall ASP II, has 46% overall sequence identity to ASP 1. Duplication of ancestral asparaginase genes, resulting in separate intra- and extracellular isozymes, appears to have occurred independen tly in the prokaryotic and eukaryotic lineages. Exact physical mapping of the new cloned ASP1 gene locates it 73% of the distance from the l eft telomere of chromosome IV, at a position precisely matching the kn own genetic map location of ASP1. This, along with the structural feat ures of the clone, confirms that ASP1 is the structural gene encoding cytoplasmic ASP I in S. cerevisiae. Sequence analysis of the ethylmeth anesulfonate-induced asp1-12 allele of strain XE101-1A revealed a C--> T transition altering Ala(176) to Val. This residue lies within a high ly conserved region, and the result suggests a critical function for A la(176) in ASP function. Expression of ASP1 and other recombinant ASPs may allow access to improved products for use in the chemotherapy of leukaemia.