Figure 4.

U1A expressing cells can be recovered from a mixture with IRP-1 expressing cells by TRAP. (A) Cells cotransformed with plasmid YCp22F-U1Awt.GFP and either YCpU1A or YCpIRP-1 were mixed in 1:10⁴ ratio and grown overnight in galactose-containing medium (M). Cells displaying low fluorescence levels (F1−) were isolated by FACS, regrown, and sorted for a second time (F2−). Total cell extracts were incubated with 1 ng of U1A probe and analyzed by native PAGE. (B) Dot plot profile of cells from F2−. Cells were analyzed by flow cytometry, plotted according to forward scatter (size) vs. fluorescence intensity and sorted for a third time (F3) into regions R2-R4 according to their fluorescence. (C) Extracts from cells sorted twice (F2−, lane 1) and three times (F3, lanes 2–5) were incubated with 1 ng of U1A probe and analyzed by native PAGE. (D) Extracts from single cell clones of culture R4 were analyzed in a gel retardation assay (lanes 1–10).