Article Figures & Data
Figures
- Fig. 1.
Alignment of eh1/GEH and octapeptide motifs of different homeodomain proteins from Drosophila melanogaster (D. mel.), Xenopus laevis (X. lae.), mouse (Mus musculus [M. mus.]), andCaenorhabditis elegans (C. ele.). S59 is a protein expressed in muscle precursor cells (20), and Msh is involved in patterning the neuroectoderm and embryonic muscles (14, 32, 40). Note the presence of a strongly conserved seven-amino-acid core which always starts with a Phe residue (position 3 in the alignment). This Phe residue is not conserved in a related motif known as the octapeptide (reviewed in reference43; see Discussion). Two octapeptide sequences from the Sparkling (Spa [25]) and Pax-5 (2) proteins are shown for comparison. See references 43and 57 for more detailed sequence alignments.
- Fig. 2.
GscR directs Gro-dependent repression via the eh1/GEH motif. (A) Diagram of Hairy derivatives expressed under the control of the hb promoter. The Gsc domains are represented by grey boxes; the position of the eh1/GEH (GEH) motif is indicated by a black box. (B to D) Effects on Sxl expression ofhb-hgsc (B and C) andhb-hgscΔGEH (D) in wild-type (B and D) andgro mutant (C) embryos. Repression by HairyGscrequires the eh1/GEH motif and endogenous Gro activity.
- Fig. 3.
Binding of Hairy derivatives to Gro in vitro. (A) GST-Hairy fusions were immobilized on glutathione-Sepharose beads and incubated with 35S-labeled Gro protein. After the beads were washed, the bound Gro protein was detected by SDS-PAGE and autoradiography. Hairy and HairyGsc bind Gro with high affinity. In contrast, little or no binding is detected with Hairy1-286 (which lacks the C-terminal 51 amino acids of the protein) and the HairyGscΔGEH chimera. (B) Coomassie staining of the gel shown in panel A, demonstrating the integrity of the different GST fusions after the binding reaction.
- Fig. 4.
The WRPW and eh1/GEH motifs are sufficient for repression in vivo and binding to Gro in vitro. (A) Diagram of the HairyWRPW and HairyGEH derivatives. (B and C) Expression of HairyWRPW (B) and HairyGEH-17 (C) under the control of the hb promoter causes efficient repression of Sxl. (D) Binding of GST-Hairy derivatives to Gro in vitro. HairyWRPW, HairyGEH-17, and HairyGEH-9 bind to Gro with similar efficiencies; in contrast, HairyGEH-9m, which carries a mutation in a conserved Phe residue within the eh1/GEH element, does not interact with Gro.
- Fig. 5.
Gro does not form oligomeric complexes. Embryo nuclear extracts were centrifuged through a glycerol gradient, and the different fractions collected were examined for the presence of Gro and TAF-80 proteins. The migration of markers (BSA and aldolase) and known endogenous complexes (TFIID and the Brahma complex [Brm-C]) is depicted by arrows. Gro migrates with an apparent size similar to its predicted molecular mass (80 kDa). In contrast, TAF-80 (which also has a molecular mass of 80 kDa) migrates with an apparent mass of ∼600 to 700 kDa because it forms part of the TFIID complex. (B) Gel filtration analysis of endogenous and purified recombinant Gro by Sephacryl S-300 chromatography. Fractions collected were examined for the presence of Gro by SDS-PAGE and Western blot analysis with an antibody directed against Gro. The elution volume (Ve) is indicated. Only the gels that contained Gro are shown. Positions of the protein standards are indicated as follows: T, thyroglobulin; F, ferritin; C, catalase; A, aldolase, B, BSA. (C) The position of the approximate Gro peak is indicated on the calibration curve.
Tables
- Table 1.
Effects of Hairy derivatives on female viability
Construct No. of progeny from the cross +/+ females × hb-/Y males Females Males hb-hg s c 0 59 hb-hgscΔGEH 57 62
