Cloning the Mating Type Loci in Yeast
How could the existence of reference copies of mating-type information at HML and HMR and an expression copy at MAT be proven physically? The most straightforward proof is simply to clone DNA copies from the three loci and use Southern transfers to show that the DNA occupying the MAT locus comes from HML or HMR and changes with a shift in mating type.
Cloning the mating-type loci required both a convenient genetic selection for the presence of mating-type genes and a suitable vector. Due to the low efficiency of yeast transformation, unreasonably large
Figure 16.3 Scheme for cloning yeast mating type genes.
quantities of yeast DNA, restriction enzymes, and ligase would have been required if investigators had tried directly to transform yeast. Instead, the yeast DNA was ligated into a bacterial-yeast shuttle vector, transformed into bacteria, amplified to suitable levels, extracted, and this was used to transform the yeast (Fig. 16.3).
The genetic selection for mating-type DNA was straightforward. The source DNA derived from yeast capable of mating, that is, having a functional MAT locus. The amplified plasmid DNA was transformed into a yeast strain possessing a defect in the MAT locus causing sterility. Therefore, after the transformation, the yeast having received a plasmid carrying the MAT locus would become capable of mating and could easily be detected. The plasmid could then be isolated from the yeast and transferred into E. coli for amplification.