Fig. 5. A shift-back protocol facilitates the detection of ribosome biogenesis defects. (A) 5' ITS1 was localized in xrn1, nic96-1 xrn1, and kap104-16 xrn1 after shifting to 37°C for 3 hours (b,f,j, respectively) or shifting back from 37°C to 25°C for 30 minutes (d,h,l). Chromosomal DNA was labeled with DAPI (a,c,e,g,i,k). (B) pre-rRNA processing in wild-type (lanes 1-6) and nic96-1 (lanes 7-12) cells shifted to 37°C or shifted-back to 25°C. Cells were pulse labeled for 3 minutes and chased for 0,3 or 10 minutes. RNA was extracted and run on a formaldehyde-agarose gel. Sizes of the rRNAs are indicated.