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

Figure 6

rDNA and genome replication.

(A) Replication origins cloned into the KanMX6 vector were transformed into the BY strain and the BY strain with the RM rDNA as described in Figure 5. Transformation efficiency of the BY rDNA ARS was comparable in the two strains. Weak ARSs that produce few transformants in the BY background (2.2 kb RM NTS, 110 bp RM rARS, and L. waltii ARSVII-929) show a 5 to 10-fold increase in the frequency of transformation when the BY strain with the RM rDNA is used as a host. (B) A proposed model to explain the effect of the source of the rDNA locus on high-frequency ARS transformation and chromosomal origin firing. The rDNA locus competes with plasmid and genomic origins of replication (for example, origins on chromosome II) for an unknown limiting replication factor. (C) 2D gel analysis of genomic origins ARS605, ARS1413, and ARS1 in the BY strain with either BY rDNA or RM rDNA. Enzyme digests, fragment sizes and probes for these three origins can be found in Experimental Procedures. Relative origin efficiencies (bubble∶Y ratios) are normalized to the efficiency observed in the BY strain with BY rDNA. (D) Five-fold serial dilutions of isogenic BY orc2-1 temperature sensitive strains with either BY or RM rDNA grown at room temperature and 26°C, the semi-permissive temperature for orc2-1. RM rDNA locus improves viability of the orc2-1 mutant.

Figure 6