A Natural Polymorphism in rDNA Replication Origins Links Origin Activation with Calorie Restriction and Lifespan
(A) The relative abundance of ERCs in the backcrossed RM and BY strains with either BY or RM rDNA was determined in populations of old and young cells. DNA (uncut) was separated on a 0.8% agarose gel and a Southern blot was probed for rDNA. Chromosomal rDNA (Genomic rDNA) runs with limiting mobility; ERCs of varying repeat numbers and topological properties generate multiple distinct bands. (B) To measure relative differences in rDNA copy number, genomic DNA from BY and RM strains was digested with BglII and a Southern blot was sequentially probed for the single copy MCM2 gene and an rDNA fragment. (There is a restriction polymorphism near MCM2 that distinguishes the two strains.) Assuming BY has 150 rDNA repeats, RM has ∼90. (C) Three independent clones from the BY strain, the RM strain and the BY strain with the RM rDNA were examined by CHEF gel electrophoresis to determine the size of chromosome XII and thereby confirm the size difference of the rDNA locus. A photograph of the ethidium bromide stained gel is shown.