< Back to Article

A Natural Polymorphism in rDNA Replication Origins Links Origin Activation with Calorie Restriction and Lifespan

Figure 5

Difference in rDNA origin activity in the RM and BY rDNA loci.

(A) Sequence comparison of ACS1 in the BY and RM rARSs. The sequence polymorphism in the RM rARS ACS1 is indicated by an arrow. (B) Replication origin sequences from a variety of sources were cloned into a vector containing the KanMX6 gene. The recombinant plasmids were then transformed into the BY strain. After a day of growth on YPD medium, the transformants were replica-plated onto YPD plates with G418 to select for cells with the recombinant plasmid. A plasmid with no ARS and a plasmid with the strong chromosomal origin ARS1 serve as the negative and positive controls, respectively. Plasmids containing the rARS from the BY rDNA locus, either as a 110 bp fragment or a 2.2 kb fragment encompassing the entire NTS region, transformed the BY strain at high frequency producing >1000 transformants/microgram of plasmid DNA. Plasmids containing the rARS from the RM rDNA locus, either as a 110 bp or a 2.2 kb fragment encompassing the entire NTS region, transformed much more poorly with <200 transformants per transformation. The colony sizes of these transformants were also more variable. (C) Chromosomes separated on a CHEF gel stained with ethidium bromide (left) or blotted and probed for rDNA (right) verify that chromosome XII is approximately the same size in the engineered RM and BY strains. The size of chromosome XII relative to other chromosomes of known size on the CHEF gel suggest that both strains have rDNA loci that contain ∼15 copies of the rDNA repeat. (D) Schematic diagram illustrating the replication intermediates that can be resolved by two-dimensional (2D) gel electrophoresis. To estimate relative origin efficiencies, the abundance of intermediates along the bubble arc is compared to the intermediates in the ascending arm of the Y arc (dotted and dashed regions, respectively). A bracket denotes the positions of paused replication forks at the 5S rRNA gene. (E) The 4.7 kb NheI fragments containing the rDNA ARS from a BY strain with ∼15 copies of either BY or RM rDNA repeats (in a fob1Δ background) are examined by Southern blotting of a 2D gel. The ratio of bubble∶Y was defined as 1.0 for the RM rDNA strain. In comparison, the normalized bubble∶Y ratio for the BY strain is 3.4.

Figure 5