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DNA from the STA1 (extracellular glucoamylase) gene of Saccharomyces diastaticus was used as a probe to enable the cloning by colony hybridization of three DNA fragments from Saccharomyces cerevisiae; these were designated S1, S2, and SGA (intracellular, sporulation-specific glucoamylase gene). To examine the evolutionary relationship among these sequences at the nucleotide level, we sequenced S2, S1, SGA and compared them with STA1. These data and RNA blot analysis revealed that the following regions of STA1 were highly conserved in S2, S1, and SGA: upstream regulatory sequences responsible for transcription, a signal sequence for protein secretion, a threonine- and serine-rich domain, and a catalytic domain for glucoamylase activity. These results suggest that an ancestral STA gene was generated relatively recently in an evolutionary time scale by the sequential fusions of S2, S1, and SGA, with S1 functioning as a connector for S2 and SGA. We describe a model for the involvement of short nucleotide sequences flanking the junctions in the gene fusions.