To determine whether the mutation in a red yeast cell is in Ade1 or Ade2, the knowledge of complementation was used. The idea of complementation is that when two mutant haploid cells mate and produce a diploid, the ability of the diploid to produce functional, non-mutant proteins depends on whether the parent mutations were in the same gene or different genes. If the mutations were in the same gene, the diploid would inherit two dysfunctional alleles, and would therefore also be a mutant phenotype. However, if the mutations were in different genes, then the diploid would have one mutant allele and one functional allele for each of the mutant genes. The functional gene would be able to produce a functional product, and the diploid organism would not show the mutant phenotype. In this experiment, if the mutations of the haploid parents were both in Ade1 or Ade2, the diploid offspring would not have a functional copy of either enzyme, and thus still be red. If one mutation was in Ade1 and the other was in Ade2, the mutations would complement, and the diploid yeast would appear to be wild-type. Through complementation analysis, the unknown mutant gene in a red yeast colony can be determined.
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