A Single Heterochromatin Boundary Element Imposes Position-Independent Antisilencing Activity in Saccharomyces cerevisiae Minichromosomes
Figure 2
Boundary elements STAR and TEF2-UASrpg block the activity of the E and I silencers irrespective of their orientations.
A: Strain YPH499 (trp-, ura-) can grow on CSM, but is unable to grow on selective media conditions [TRP-; TRP-/URA-; TRP-/5-FOA+]. B: Minichromosome containing the TRP1 marker gene is able to grow on CSM; TRP-; unable to grown on TRP-/URA- (due to lack of URA3) and is 5-FOA resistant. C: Minichromosome containing both the TRP1 marker and the URA3 reporter genes is able to grow on CSM; TRP-; TRP-/URA- and is 5-FOA sensitive (due to the presence of the functional URA3 product). D: Minichromosome containing the HML-E and I silencer elements is able to silence the URA3 reporter gene and the cells are able to grow on CSM; TRP-; unable to grown on TRP-/URA- (due to lack of URA3) and is 5-FOA resistant. E–L: Two boundary elements (shown by arrows) STAR (S) and TEF2-UASrpg (T) were examined in different orientations in the presence of both the HML – E and I silencer elements. The URA3 reporter gene expression status (on or off) was assayed by the growth phenotypes of S. cerevisiae cells containing various minichromosome constructs tested in different selective media by serial dilutions. M: Southern blot of linearized minichromosomal and genomic DNA probed with radiolabeled TRP1-ARS1 containing fragment. Five independent clones transformed with minichromosome construct containing TEF2-UASrpg (left panel) and STAR (right panel). N: Graphic representation of minichromosome copy numbers quantified by scanning of the Southern blots (such as shown in Figure 2M) and normalized to the genomic TRP1-ARS1 signal. Error bars represent Standard Deviations.