Figure 3. Episomal complementation of the BC002639 gene under yeast promoter control:
(a) Tetrad dissection of the YOR159c::kanMX/YOR159c strain in presence of the human complementation construct on YPD medium (left). Spores in the 2^nd and 8^th tetrad show a 4:0 segregation between viable and unviable spores. Replica plating of these colonies on YPD geneticin plates show a 2:2 segregation, confirming the functional episomal complementation in the spores containing the KanMX4-deletion cassette (right).
(b) Confirmation of the haplotype of the 4:0 segregated spores of tetrads 2 (lanes 1 to 4) and 8 (lanes 6 to 9) from a (left). Was done by a standard Mat-locus PCR. For both tetrads, a 2:2 segregation between MATa­- (544 bp) and MATα- (404 bp) spores can be observed. forming 544 bp and 404 bp fragment respectively. Interestingly, all complemented haploid deletion mutants have a MATa-‘mating’-type. Subsequent PCR analysis using T7 and T3 primers confirmed the presence of the plasmid with a correct insert (right). The expected length of the amplicons (1261 bp) for the growing spores of tetrad two (lanes 1-2) and eight (lanes 4-5) are estimated relative to a λPstI ladder (lane 3).