XB-IMG-150173
Xenbase Image ID: 150173
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Fig. 1. Summary of the predicted structure of the missense and loss-of-function alleles in GLI2. (a) The WT architecture of GLI2 is depicted in the top bar (WT construct 1; ZF = Zn finger). Shown below are the apparently unique missense variants seen in human HPE patients (red diamonds), putative polymorphisms (blue arrow), and HUT102 sequence variants (yellow triangles). Three of these variations predict missense changes (red diamonds). These three changes [V104M, 310G>A (construct 3), D88N, 262G>A (construct 7), and N273S, 818 A>G (construct 10)] behaved identically with the normal gene and are likely rare polymorphisms (Fig. 3c and data not shown). Similarly, apparent polymorphisms (found in normal controls), such as K410R, 1229 A>G (blue arrow, construct 6), behaved identically to the WT cDNA (construct 1) in all assays performed, as did the putative HUT102 reference cDNA variants (yellow triangles, constructs 12-14). The predicted architecture of the disease-related variants is shown below. Construct 2 represents the predicted outcome of a hypothetical RNA splicing event removing exon 5. Confirmation of this form was not attempted, and the related construct 8 represents the more likely form. Failure to execute alternative splicing predicts premature termination within intron 5 (hatched bar; construct 8). Constructs 5 (2274del1), 4 and 15 (W113X, 339G>A), and 16 (R168X, 502C>T) represent the predicted protein structures that are prematurely truncated. Note that constructs 4 and 15 are identical truncation mutations except that construct 4 includes also the V104M missense mutation (as does construct 3). (b) Western analysis of COS-7 cells transfected with N-myc-tagged GLI2 alleles and probed with anti-myc antibody to verify expression. Untransfected cells show no bands (data not shown). Cotransfected lacZ mRNA encoded the β-galactosidase (β-Gal) control protein. The arrow marks the predicted full-length protein, and the arrowheads identify two consistently seen smaller proteins that could represent processed forms or stable degradation products. Although these smaller bands resemble those seen with frog Gli2 (6), their significance is unknown and will require further study. (c) The deletions caused by variants 2 and 5 produce the expected truncated proteins that are smaller than the WT full-length product encoded by construct 1. Note that the smaller processed bands are still formed in both, but in allele 2 they migrate faster, indicating that an apparently specific cleavage occurs between the zinc fingers and the site of truncation of construct 5. (d) The predicted truncations of the rest of the alleles cause smaller proteins of the expected sizes (constructs 4, 8, 15, and 16). Image published in: Roessler E et al. (2003) Copyright © 2003. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View |