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 glucoamylaseencoding 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 M_r of 82514. 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/MATα -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.