Primary structure and regulation of a glucoamylase-encoding gene (STA2) in Saccharomyces diastaticus

We have determined the complete nucleotide (nt) sequence of a 5070-bp DNA fragment containing a glucoamylase-encoding gene (STA2) from Saccharomyces diastaticus. The 5' transcription start points for STA1, STA2 and STA3 were determined by primer extension of their respective mRNAs using reverse transcriptase. The sequence data show one major open reading frame (ORF) of 767 amino acids encoding GAII with a calculated Mr of 82,514. The 5' region in the ORF contains two ATG sequences within 30 nt of each other. The upstream region of STA2 was amplified by the polymerase chain reaction (PCR) and fused to the Escherichia coli lacZ gene. Some of the PCR products contained mutations in ATG1 and/or ATG2. Results indicated that both ATG1 and ATG2 encode functional translation start codons, but ATG2 was shown to encode the stronger initiator. The upstream region of STA2 contains a canonical sequence that is homologous to known sites of repression by the MATa/MAT alpha-encoded repressor. Also, consensus RAP1 (Repressor-Activator Protein 1)-binding sites are located in the 5' upstream region and within the coding region of STA2.