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. 1998 Feb 3;95(3):951–956. doi: 10.1073/pnas.95.3.951

Figure 5.

Figure 5

Cloning of IRP-1 by TRAP. (A) Recovery of IRP-1 expressing cells from a transformation with mixed plasmid DNA. RS453 cells carrying plasmid YCpIREwt-GFP were transformed with a DNA mixture containing plasmids YCpIRP-1 and YCpU1A in a ratio of 1:500,000. Transformants were pooled and grown in galactose medium. Cells with low fluorescence were sequentially sorted twice. Total extracts were analyzed in a gel retardation assay with 1 ng of IRE probe (lanes 1–3). The extract of cells sorted twice shows IRE binding activity (lane 3). IRE binding activity is below detection limit in the extract from pooled transformants (lane 1), or cells sorted once (lane 2). Cells from F2− were sorted for a third time (F3) into populations R2-R5. The IRE binding activity of extracts from cultures R2-R5 was analyzed (lanes 4–7). The highest activity is present in extract R4. (B) Fluorescence analysis and sorting of cells from F2− into regions R2-R4 according to their fluorescence.