Transfer of Mating Type Gene Copies to an Expression Site

Transfer of Mating Type Gene Copies to an Expression Site

Once the MATα mating-type clone had been obtained by Hicks, Strath-ern, and Klar, and by Nasmyth and Tatchell, Southern transfer experiments testing DNA transposition to the MAT locus were possible. Yeast DNA was cut with various restriction enzymes and was separated according to size by electrophoresis. The locations of the α-specific or a-specific sequences were determined by hybridization using the clonedα orasequences as probe.

We might expect that the α mating-type locus would have no homology to the a mating-type locus. Then, in the DNA from a MATα cell there would be two different DNA fragments with homology to the MATα locus, one from MATα itself, and one from HML or HMR. From a MATa cell we would expect only one fragment with homology to the α mating type sequence. Surprisingly, a different answer emerged. The α se-quence also possesses partial homology to a mating-type sequences. Therefore three different sizes of restriction fragments were observed in the DNA from MATa or MATα cells probed with either a or α sequences (Fig. 16.4). These are sequences at HML, HMR, and MAT.

Figure 16.4 Cleavage of DNA containing theHML, HMR,andMATloci by arestriction enzyme which does not cleave within the loci..

The fact that the a-specific and α-specific sequences were partially homologous, but of different size permitted a direct and simple demon- stration of the basis of mating-type conversion. The genetic data indi-cated that sequences from HML or HMR were copied into MAT, where they were expressed. Because the a mating-type sequence is about 100 nucleotides shorter than the α mating type, the identity of the sequence occupying the MAT locus can be directly determined on Southern transfers (Fig. 16.5). The experiments showed, as expected, that mating type a cells possessed the a sequences at MAT, and that mating type α cells possessed the α sequences at MAT. The Southern transfer of a diploid yeast that possesses a MATa/MATα genotype yields two bands originating from the MAT locus that differ in size by 100 base pairs. The identity of the shorter and longer mating-type segments was determined by isolating spores, growing, and testing both genetically as well as performing a transfer. The a-type cultures possess only the shorter MAT sequence and the α-type cultures possess only the longer MAT sequence.

Figure 16.5 Southern transfershowing that MATa sequences are about 100 base pairs shorter than MATαsequences.