Figure 5.

The PBE is required for PRE silencing in vivo. (A) Binding of PHO/PCC to the iab-7 PRE is abrogated when the PBEs are mutated (for sequences, see Fig. 4A), as revealed by DNaseI footprinting. (B-D) Mutations in the PBE impair PRE-directed PSS. PSS of mini-white by the minimal 260-bp wild-type iab-7 PRE or a mutant, PBE-less iab-7 PRE (PBE^mt iab-7 PRE) was compared. Multiple independent lines were established harboring the mini-white transgene under control of either the wild-type or PBE^mt iab-7 PRE. For each line, the eye color of homozygotes for the insertion was compared with that of heterozygotes. Representative examples are shown: (B) Homozygotes have a much darker eye color than their heterozygous siblings (P[w⁺]/P[w⁺]P[w⁺]/+), revealing the absence of PSS. (C) Homozygotes have much lighter eyes than heterozygotes ((P[w⁺]/P[w⁺][w⁺]/+), showing strong PSS. (D) The eye color of homozygotes is equal (P[w⁺]/P[w⁺] = P[w⁺]/+) to that of heterozygotes, revealing impaired PSS. (E) The number of lines exhibiting no PSS, impaired PSS, or strong PSS were scored and depicted in a graphical representation with the number of lines displaying a PSS phenotype expressed as percentage of the total number of lines analyzed on the Y-axis and the phenotype on the X-axis. Because the percentages refer to the numbers of independent lines, error bars reflecting the SEM are not applicable. χ² test of statistical significance for bivariate tabular analysis confirmed the high significance of the difference in PSS frequency between lines harboring either the wild-type or PBE^mt iab-7 PREs (χ² = 12.3, p < 0.01).