Figure 7.

Ectopic initiation occurs within short DNA fragments. (A–C) Three different origins (A, ori-rDNA; B, oriX009; C, ori1068) were ligated into pINA214; the resulting plasmids were digested with BstXI and integrated into the LEU2 locus. The resulting genomic restriction map is shown, with the position of the sites used to digest the DNA for 2D gel analysis. The gels were blotted and hybridized with a pBR322 probe, indicated as black boxes. (D) The same experiment was performed with pINA214 as a control: hybridization of the 2D gel showed the absence of initiation at the LEU2 locus. Notice that the diagonal signal visible on these images is present on most chromosomal 2D gels performed in Yarrowia and may correspond to degradation products.