(A,B) β-galactosidase activity of independent transgenic lines carrying a derivative of the FBE1-yp2-LacZ transgene. The name and structure of the insertion is shown on the right, including the 3′ 157 bp 1A-2 fragment (P[F-1A-2(157)-yp2]), the 79 bp fragment with only two Su(Hw) BSs (P[F-1A-2(79)-yp2]), the 3′ 78 bp fragment combined with a Su(Hw) BR containing three copies of site 3 from the gypsy insulator (P[F-3R:3 1A-2(78)-yp2]), the 1A-2 78 bp fragment (P[F-1A-2(78)-yp2]) and four copies of the 3′ 1A-2 78 bp fragment (P[F-1A-2(78)×4-yp2]). Error bars indicate the standard deviation (n = 3). The vertical dashed line on the left represents the aau value for flies carrying P[F-gyp-yp2], while the vertical dashed line on the right represents the aau value for flies carrying P[F-yp2]. In B, the X-axis scale is increased three fold. Symbols are as described in Figure 1.

A. The region separating the y and ac genes contains a cluster of two strong Su(Hw) BS (black triangle, 1A-2) and two weak Su(Hw) BS (white triangle, 1A-2′). 1A-2′ is located 85 bp upstream of the most upstream yar transcription start site (bent arrow). The alternative splicing pattern is shown: thin lines represent introns and blue rectangles indicate exons. The ac DCE (black oval) resides within yar. The limits of the original 520 bp 1A-2 insulator are shown as a bracketed line marked 520. The extent of the regions deleted in the y^Δ1A-2 (Δ1A-2) and y^{Δ1A-2/Δ1A-2′} (Δ1A-2/1A-2′) flies are shown, where the bracketed regions were removed. B. The sequence of the Su(Hw) BSs in 1A-2 (top), 1A-2′ (middle), and the synthetic gypsy insulator (3R:3) (bottom), with the numbers indicating the distance of separation between BSs. The nucleotides different from the genomic Su(Hw) consensus BS [14] are underlined.

A. Ethidium bromide stained PCR products obtained from semi-quantitative PCR to evaluate ac, y, yar and Ras 64B RNA levels in wild type (Canton S), y^Δ1A-2 (Δ1A-2) and y^{Δ1A-2/Δ1A-2′} (Δ1A-2/Δ1A-2′). Ras64B is constitutively expressed and serves as a control. The minus (−) RT lanes control for genomic DNA contamination. Different stages of embryonic and mixed pupal RNA were analyzed. B. Quantitative real time PCR (Q-PCR) was used to determine levels of yar mRNA accumulation from RNAs isolated during development from wild type and mutant lines. Individual transcript levels defined by Q-PCR were normalized to Ras64B for amount of input cDNA (ΔC_T). A larger ΔC_T indicates a reduction in RNA. Error bars indicate standard deviation of values obtained from analyses of three independently isolated RNAs. Significant changes in RNA accumulation relative to wild type are as indicated (*, P = <0.01; †, P<0.001, Student's two-tailed t-test).

A. Top: The cuticle pigmentation yellow (y) gene contains two exons (yellow rectangles) and four tissue specific enhancers (ovals marked W for wing, B for body, Br for bristle and Tc for tarsal claw). The proneural achaete (ac) gene contains one exon (black rectangle). The tissue-specific enhancer in the upstream regulatory region is shown (oval marked DCE for dorsocentral enhancer). 1A-2 Su(Hw) BR is located downstream of the y gene, separating this gene from the ac regulatory region. Bottom: Structure of the FBE1-yp2-LacZ transgene used to define the sequences of 1A-2 required for enhancer blocking. Subregions of 1A-2 were inserted between the FBE1 enhancer and yp2 promoter. Effects of transcriptional activation were determined through enzymatic assay that tested β-galactosidase activity. The mini-white (w) gene was used for identification of transgenic flies in germ line transformation. B. β-galactosidase activity associated with transgenic lines carrying transposons derived from FBE1-yp2-LacZ. Each bar represents the average activity units (aau) for independent insertion lines corresponding to the indicated transposon (right). When pertinent, a cartoon is shown that represents the structure of sequences included in the FBE1-yp2-LacZ transgene. The gypsy Su(Hw) BS are shown as black rectangles, the 1A-2 Su(Hw) BS are shown as black triangles, a novel 1A-2 element is shown as an oval carrying 78 bp. Assays were completed on extracts isolated from females representing at least three independent crosses. Error bars indicate standard deviation. The vertical dashed line on the left represents the aau value for flies carrying P[F-gyp-yp2], while the vertical dashed line on the right represents the aau value for flies carrying P[F-yp2].

Northern analysis of five µg of polyA⁺ RNA isolated from staged embryo collections. The blot was probed with a yar cDNA, and two exposures are shown: 3 hours (left) and 24 hours (right). The blot was stripped and reprobed with DNA sequences corresponding to y, ac and the constitutively active RpL32 gene that served as a loading control.

A. Transgenic flies were generated that carried the mutant y¹ allele (structure shown where C indicates the mutation of the translation ATG start) at the endogenous X chromosome location (left) and the P[y^Δ1A-2 target] transposon on a different chromosome (right) that carries a y gene lacking the wing enhancer, but encodes a wild type RNA (A indicates the presence of the correct translation ATG start). In this transposon, the y gene, flanked by FRT sites (white arrows) and I-SceI sites, is within a P transposon (inverted black triangles). Other symbols representing the y, yar and ac genes are as described in Figure 1. Transgenic flies y¹ w¹¹¹⁸, P[y^Δ1A-2 target] had a light wing color and orange eyes. B. FLP and I-SceI enzymes catalyzed replacement of the y¹ allele at the endogenous locus, with y^Δ1A-2w, in which the 1A-2 insulator is substituted by the w^hs gene (raised triangle) inserted between loxP sites (black arrowheads on raised triangle). The recombinant y^Δ1A-2w flies had dark wings and red eyes. C. Cre recombinase deleted the w^hs gene, leaving behind a single loxP site to form y^Δ1A-2. In the case of the y^Δ1A-2, the remaining loxP site was mutated, forming a new EcoRV site (RV), whereas in the similarly derived y^{Δ1A-2/Δ1-A2′} flies a wild type loxP site remained. The bar under the y gene indicates the probe used in the Southern analyses (see Figure S1).