1. Analysis of the 55S peak appearing after repressing the gene for 40S r-protein uS4.
Analysis of the 55S peak appearing after repressing the gene for 40S r-protein uS4. Pgal-uS4 was grown in YEP-galactose medium, and aliquots were harvested at 0 h and 8 h after the shift to glucose medium. (A, B) Equal A260 units of whole cell extracts were fractionated on sucrose gradients and equal portions of the fractions sedimenting as 40S or larger particles were fractionated on an agarose gel, transferred to a membrane, and stained with methylene blue. (C) Equal portions of fractions from identical positions in a set of sucrose gradients from a biological replicate experiment were loaded into alternate slots of an agarose gel, transferred to a membrane, and probed with a mixture of equal radioactivity of end-labeled oligonucleotides O1680 and O1660 that hybridize to ITS1 (distal to the A2 processing site) and ITS2 (Fig 3H). (D) The membrane was then stripped of radioactivity and probed with an oligonucleotide specific to the 5′ end of 25S rRNA. The positions of the A260 peaks in the gradients are indicated below the images of the membrane in (C, D). (E) Lanes marked with black dots in panel (D) were excised electronically from the image in (D) and optimized for viewing of the individual lanes. (F–G) Northern blots of RNA from the 55S and 60S fractions of a sucrose gradient of samples harvested 8 h after addition of glucose and probed with radioactive oligonucleotides hybridizing to the 5′ (panel F) or the 3′ (panel G) ends of 25S rRNA. Broken red lines to the left of the relevant lanes indicate rRNA degradation products.
Brian Gregory et al. LSA 2019;2:e
© 2019 Gregory et al.