CLONING OF THE ASN1 AND ASN2 GENES ENCODING ASPARAGINE SYNTHETASES INSACCHAROMYCES-CEREVISIAE - DIFFERENTIAL REGULATION BY THE CCAAT-BOX-BINDING FACTOR

Two new yeast genes, named ASN1 and ASN2, were isolated by complementa tion of the growth defect of an asparagine auxotrophic mutant. Genetic al analysis indicates that these two genes are allelic to the asnA and asnB loci described previously. Simultaneous disruption of both genes leads to a total asparagine auxotrophy, while disruption of asn1 or a sn2 alone has no effect on growth under tested conditions. Nucleotide sequences of ASN1 and ASN2 revealed striking similarities with genes e ncoding asparagine synthetase (AS) from other organisms. Regulation of ASN1 and ASN2 expression was studied using lacZ fusions and both gene s were found to be several times less expressed in the absence of the transcription activator Gcn4p. The HAP complex, another transcription factor that binds to CCAAT-box sequences, was shown to specifically af fect ASN1 expression. Hap2p and Hap3p subunits of the HAP complex are required for optimal expression of ASN1, while the Hap4p regulatory su bunit, which is required for regulation by the carbon source, plays a minor role in this process. Consistent with the weak effect of Hap4p, the carbon source does not significantly affect expression of ASN1. Ou r results show that the role of the HAP complex is not limited to acti vation of genes required for respiratory metabolism.