Figure 4. Molecular analysis of genome rearrangements after Ku80c depletion.

(A) Detection of DNA fragments with excised or non-excised IES 51G4404 from the surface antigen G⁵¹ gene. Total genomic DNA was extracted during autogamy of 51ΔA cells subjected to RNAi against ICL7 (control) or KU80c. To compare the two time-courses, similar autogamy stages were numbered from 1 to 6, based on the observation of DAPI-stained cells (Figure S1C). PstI-hydrolyzed total genomic DNA was run on 1% agarose gels. Southern blots were hybridized with the Gmac probe (in grey), which hybridizes to the flanking MAC DNA downstream of the IES. (B) Detection of fragmented or non-fragmented DNA downstream of the G⁵¹ gene by Southern blot hybridization of PstI-digested total genomic DNA (same samples as in A) run on 0.8% agarose gels. The subtelomeric tel51G probe is shown in grey. The white box in the bottom right diagram represents telomeric repeats. (C) PCR detection of de novo IES excision junctions during autogamy of 51ΔA cells, in an ICL7 (control) or a KU80c RNAi. Note that a few autogamous cells were present at time-point 1 in the control RNAi (see Figure S1C). (D) PCR detection of IES circle junctions during autogamy in an ICL7 (control) or a KU80c RNAi. Triangles in C and D represent PCR primers (see Table S1).