Figure 6.

Non-tDNA barrier elements do not support HMR cohesion. (A) Colocalization of HMR circles containing a CHA1p barrier following M excision. Strains RDY152 (wt), RDY180 [Δtt(agu)c∷loxP], and RDY206 [Δtt(agu)c∷CHA1p] were examined in rich media supplemented with 4 mM serine. (B) Colocalization of HMR circles bearing lexA operators following M excision. Strains RDY152 (wt) and RDY242 [Δtt(agu)c∷6lexOPs] were transformed with a plasmid expressing lexA (pLexA) or empty vector (pRS413). Plasmids were maintained by overnight growth in SC-trp,his + raffinose prior to replacing media with rich media containing raffinose and nocodazole. (C) Organization of the HMR region in RDY226 with GIT1 replaced by the K. lactis URA3 (klURA3). X marks the HMR-proximal tDNA that was substituted with heterologous sequences. (D) Barrier function of RDY226 (wt) and tDNA replacement strains RDY249 [Δtt(agu)c∷loxP], RDY251 [Δtt(agu)c∷CHA1p] and RDY263 [Δtt(agu)c∷6lexOPs]. Tenfold serial dilutions of each strain were spotted in rows on selective media containing or lacking 0.1% 5-FOA. All strains are prototrophic for tryptophan and grow on SC-trp media containing 4 mM serine. Plasmids pLexA and empty vector were maintained by SC-his selection.