FIG. 9.

Changing guanosine at position 1575 to adenosine has no discernible effect on cell growth. (A) Schematic of strategy to introduce mutant ribosomes into yeast. 5-Fluoroorotic acid (5FOA)-containing media selects against the URA3 vector, forcing cells to utilize the rDNA locus introduced on the LEU2 vector. WT, wild type. (B) LEU2 plasmids expressing wild-type rRNA (pAJ718) (WT), G1575A mutant (pAJ2157), or no rRNA (pRS425) (vector) were transformed into strain AJY1185 and tested for growth on 5FOA-containing medium. (C) A 2.7-kb fragment spanning the G1575A mutation of the plasmid-borne rDNA locus was amplified by PCR using primers AJO214 and AJO1051. The PCR product was treated with EcoRI and analyzed on an agarose gel. The brightest band of the 1-kb ladder is 3 kb.