In the laboratory, however, we will exploit the mutagenic properties of UV light to increase rates of mutation in yeast. Yeast cells will be plated on media supple mented with 5-FOA and treated with UV light. 5-FOA resistant colonies that grow will then be tested to determine if they require uracil for growth, which would be expected if they carried mutations in the URA3 mutants. 5-FOA resistance and uracil auxotrophy, however, are not sufficient to conclude that the mutations reside in URA3 . While both phenotypes are consistent with those expected for URA3 mutants, it is possible that mutations in other genes in the uracil biosynthetic pathway could be affected.
Complementation tests are one means to determine which gene is altered in the mutant strains.
As was conducted in Exercise 2, the 5-FOA resistant mutants will be crossed to marker strains that carry known mutations in uracil biosynthetic genes. The resulting diploids will be tested for complementation to determine which carry mutations in URA3 . You will first monitor the affect of UV light on cell viability by plating several hundred yeast cells onto four individual petri plates of rich media. These plates will be exposed to increasing doses of UV light and the number of surviving cells will be scored. You will also plate a large number of yeast cells onto a petri plate containing media supplemented with 5-FOA and uracil.