Figure 4
Levels of Ty1 retromobility, RNA, and Gag: GFP protein in six rhfΔ mutants with defects in ribosome biogenesis. (A) The frequency of His+ prototroph formation (retrotransposition) in wild-type strain JC3807 (WT) and congenic rhfΔ derivatives harboring a chromosomal Ty1his3AI element. The frequency reported for the dbp7Δ strain is the maximum possible frequency determined as if one His + colony had formed in each independent culture tested. Error bars: standard error. (B) Northern blot analyses of Ty1 RNA (top panel) and PYK1 RNA (bottom panel) in each strain, using 32P-labeled riboprobes. The ratio of 32P activity in the Ty1 band to 32P activity in the PYK1 band was determined by phosphorimaging. Ty1/PYK1 RNA ratios for each strain normalized to that of the wild-type strain are provided below each lane. (C) The average level of Ty1 RNA in total RNA from three biological replicates of each strain relative to the wild-type strain was determined by qPCR analysis (left panel). The spt3Δ strain is a negative control. The average level of RNA derived from the Ty1(gag::GFP)-3566 chromosomal element in total RNA from three biological replicates of the wild type strain and the congenic bud21Δ derivative was measured by qPCR analysis. Error bars: standard error. (D) Western blot analyses of total cell lysate with anti-VLP antibody, which recognizes unprocessed p49-Gag and processed p45-Gag (top panel), and anti-alpha-tubulin antibody (bottom panel) as a loading control. (E) Histogram of the mean Gag:GFP fusion protein activity, as measured by flow cytometry, in rhfΔ strains relative to the wild-type strain. Each strain harbors the chromosomal Ty1(gag::GFP)-3566 element. Error bars: standard error.