Multiple changes underlie allelic divergence of CUP2 between Saccharomyces species.

Under the model of micromutationism, phenotypic divergence between species is caused by accumulation of many small-effect changes. While mapping the causal changes to single nucleotide resolution could be difficult for diverged species, genetic dissection via chimeric constructs allows us to evaluate whether a large-effect gene is composed of many small-effect nucleotide changes. In a previously described non-complementation screen, we found allele difference of CUP2, a copper-binding transcription factor, underlie divergence in copper resistance between Saccharomyces cerevisiae and S. uvarum. Here, we tested whether the allele effect of CUP2 was caused by multiple nucleotide changes. By analyzing chimeric constructs containing four separate regions in the CUP2 gene, including its distal promoter, proximal promoter, DNA binding domain and transcriptional activation domain, we found that all four regions of the S. cerevisiae allele conferred copper resistance, with the proximal promoter showing the largest effect, and that both additive and epistatic effects are likely involved. These findings support a model of multiple changes underlying evolution and suggest an important role of both protein coding and cis-regulatory changes in evolution.