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1.
F <span style="font-variant: small-caps" class="small-caps">igure</span>  3.—

F igure 3.—. From: The Enhancer-Blocking Activity of the Fab-7 Boundary From the Drosophila Bithorax Complex Requires GAGA-Factor-Binding Sites.

Map of enhancer-blocking transgenes. (Top) The wEN:mini-white transgene. (Bottom) The ftz:hsp70-LacZ transgene. Boundary element (B) is inserted between the enhancer and the promoter. ftz enhancers are UPS (stripes) and NE (CNS expression).

Susan Schweinsberg, et al. Genetics. 2004 November;168(3):1371-1384.
2.
F <span style="font-variant: small-caps" class="small-caps">igure</span>  1.—

F igure 1.—. From: The Enhancer-Blocking Activity of the Fab-7 Boundary From the Drosophila Bithorax Complex Requires GAGA-Factor-Binding Sites.

Hypersensitive sites associated with the Fab-7 boundary. (Top) A map of the Fab-7 boundary and flanking sequences. Boxes indicate DNase I hypersensitive sites (HS). Large ovals represent GAGAG sequences and are numbered from proximal to distal. Small ovals represent GAGAA sequences. Fragments F12, F34, and F56 are drawn below. (Bottom) A portion of the sequences of these three fragments. GAGAG sequences are indicated by boldface type. The point mutations that were generated in these GAGAG sequences are shown in italics.

Susan Schweinsberg, et al. Genetics. 2004 November;168(3):1371-1384.
3.
F <span style="font-variant: small-caps" class="small-caps">igure</span>  4.—

F igure 4.—. From: The Enhancer-Blocking Activity of the Fab-7 Boundary From the Drosophila Bithorax Complex Requires GAGA-Factor-Binding Sites.

GAGA site mutations in the wEN:mini-white assay. Each panel shows the eye color of a representative blocking line (left) compared to the eye color of a random DNA control or a nonblocking transgenic line (right). To the right of each panel the corresponding percentage of enhancer-blocking lines for the construct depicted in the panel is indicated. (Top) Wild-type 1.2-kb Fab-7. (Middle) Fab-7 GAGAG1–5. (Bottom) Fab-7 GAGAG3–4. (Not shown) Fab-7 GAGAG1–2: Eye colors of enhancer-blocking lines carrying a 1.2-kb Fab-7 fragment with mutations in GAGA sites 1–2 is comparable to the 1.2-kb wild-type control shown at the top.

Susan Schweinsberg, et al. Genetics. 2004 November;168(3):1371-1384.
4.
F <span style="font-variant: small-caps" class="small-caps">igure</span>  6.—

F igure 6.—. From: The Enhancer-Blocking Activity of the Fab-7 Boundary From the Drosophila Bithorax Complex Requires GAGA-Factor-Binding Sites.

Boundary activity of the Fab-7 deletion mutant P6.1 is sensitive to a reduction in the gene dose of the GAGA factor. The sequence organization of the starting bluetail transposon and the three imprecise excision derivatives P14.1, P6.1, and P18.1 are indicated by the maps. Either wild-type females (+/+) or TrlR57/+ females (done with both TrlR67/Bal and TrlR67/+ females) as indicated were mated to males carrying the different bluetail chromosomes. Embryos from each cross were collected and stained in parallel to assay for β-galactosidase expression. Genotype of the mother is indicated on each embryo as either +/+ or Trl/+. Position of the anterior border of PS12 is indicated by the vertical arrow.

Susan Schweinsberg, et al. Genetics. 2004 November;168(3):1371-1384.
5.
F <span style="font-variant: small-caps" class="small-caps">igure</span>  5.—

F igure 5.—. From: The Enhancer-Blocking Activity of the Fab-7 Boundary From the Drosophila Bithorax Complex Requires GAGA-Factor-Binding Sites.

GAGA sites are required but not sufficient for enhancer-blocking activity in the ftz:hsp70-LacZ assay. A β-galactosidase assay was performed on all embryos in parallel to detect LacZ expression. Therefore, expression levels can be directly compared among embryos in all panels. (Left) A lateral view of stage 10 embryos, in which LacZ expression is driven by the UPS enhancer and can be detected in even-numbered parasegments. (Right) A ventral view of stage 14 embryos, in which LacZ expression is directed by the NE enhancer and can be detected in the central nervous system. All embryos are oriented such that anterior is to the left and posterior is to the right. Embryos from representative transgenic lines are shown in each case and are as follows: random DNA, five binding sites for Su(Hw), the wild-type 1.2-kb Fab-7 fragment, a mutant 1.2-kb Fab-7 fragment with mutations in GAGAG sites 1–5, a mutant 1.2-kb fragment with mutations in GAGAG sites 3–4, and a mutant 1.2-kb fragment with mutations in GAGAG sites 1–2.

Susan Schweinsberg, et al. Genetics. 2004 November;168(3):1371-1384.
6.
F <span style="font-variant: small-caps" class="small-caps">igure</span>  2.—

F igure 2.—. From: The Enhancer-Blocking Activity of the Fab-7 Boundary From the Drosophila Bithorax Complex Requires GAGA-Factor-Binding Sites.

GAGA factor binds to GAGAG sequences of the Fab-7 boundary. Binding of GAGA factor to its binding sites in this region was tested by incubating the nuclear extract with a DNA affinity matrix and then probing the bound protein in a Western blot with anti-GAGA antibody (A) and gel-mobility-shift experiments (B and C). (A) Nuclear extracts were incubated with affinity matrix beads containing different DNA sequences. As described in materials and methods, proteins associating with the affinity matrix beads after washing were eluted with 0.4 m salt and analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting. The nitrocellulose filters were probed with GAGA-519 (top, I) and GAGA-581 (bottom, II) isoform specific antibodies. The corresponding isoforms recognized by these antibodies are indicated (by numbers) next to the bands. (B and C) Gel-shift experiments. (B) An HS1 subfragment probe spanning GAGAG sites 3 and 4 was incubated with NE in the absence of any competitor, lane NE, or in the presence of increasing amount (10- to 50-fold excess over the labeled probe) of cold competitor DNA. WT and mu indicate the wild-type and mutated versions, respectively, of the same subfragment. Lane “—,” probe incubated without NE. (C) Super shift with GAGA antibody: a gel-mobility-shift experiment with an HS1 subfragment probe spanning GAGAG sites 5 and 6. Lane “—,” probe without nuclear extract. Lane NE, nuclear extract alone. In the next two lanes increasing amounts (0.5 and 1.0 μl) of GAGA antibody (from Carl Wu) were included in the incubation mix. NE, nuclear extract.

Susan Schweinsberg, et al. Genetics. 2004 November;168(3):1371-1384.

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