Halka kitap okutmak bahsi etrafında

Taha Toros Arşivi, Dosya No: 111-Kütüphanelerİstanbul Kalkınma Ajansı (TR10/14/YEN/0033) İstanbul Development Agency (TR10/14/YEN/0033

Novel ENU-Induced Mutation in Tbx6 Causes Dominant Spondylocostal Dysostosis-Like Vertebral Malformations in the Rat

Congenital vertebral malformations caused by embryonic segmentation defects are relatively common in humans and domestic animals. Although reverse genetics approaches in mice have provided information on the molecular mechanisms of embryonic somite segmentation, hypothesis-driven approaches cannot adequately reflect human dysmorphology within the population. In a N-ethyl-N-nitrosourea (ENU) mutagenesis project in Kyoto, the Oune mutant rat strain was isolated due to a short and kinked caudal vertebra phenotype. Skeletal staining of heterozygous rats showed partial loss of the cervical vertebrae as well as hemivertebrae and fused vertebral blocks in lumbar and sacral vertebrae. In homozygous embryos, severe displacement of the whole vertebrae was observed. The Oune locus was genetically mapped to rat chromosome 1 using 202 backcross animals and 50 genome-wide microsatellite markers. Subsequently, a miss-sense mutation in the Tbx6 gene was identified in the critical region. Although the mutation is located within the T-box domain near a predicted dimmer-interface, in vitro experiments revealed that the Tbx6 variant retains normal DNA binding ability and translational efficiency. However, the variant has decreased transcriptional activation potential in response to Notch-mediated signaling. Recently, it was reported that a dominant type of familial spondylocostal dysostosis is caused by a stoploss mutation in TBX6. Thus, we propose that partial dysfunction of Tbx6 leads to similar congenital vertebral malformations in both humans and rats. The Oune strain could be a unique animal model for dominant spondylocostal dysostosis and is useful for molecular dissection of the pathology of congenital vertebral malformations in humans

Translational efficiency and transcription activation ability of <i>Tbx6</i> proteins.

<p>(A) Western blotting analysis of S protein-tagged mTbx6 proteins. Cell lysate from transfectant of the <i>mTbx6</i> and <i>mTbx6</i>^<i>Oune</i> expression constructs (Tbx6 and Tbx6 mut, respectively), in which the coding region of wild type and <i>Oune</i> mutant <i>mTbx6</i> are tagged with partial S protein sequences in N-terminus, was used with anti-S protein (the upper panel) and anti-USF2 (the lower panel) antibodies. Signals of S protein tagged mTbx6 are indicated by the arrow. (B) <i>In vitro</i> translation assays for mTbx6 and mTbx6 mut. Difference of translational efficiency between wild type and mutant mTbx6 was not observed. The S-tag mTbx6 constructs showed multiple translational initiations (tagged protein: asterisk). (C) Transcriptional activation properties of <i>Tbx6</i> and <i>Tbx6</i>^<i>Oune</i> using a <i>Mesp2</i> promoter-luciferase reporter construct. <i>rTbx6</i>^<i>Oune</i> activates transcription less effectively than <i>rTbx6</i> when a Notch intracellular domain (NICD) expression construct was cotransfected into C2C12 cultured cells. (D) Transcriptional activation properties of a mixture of <i>Tbx6</i> and <i>Tbx6</i>^<i>Oune</i> constructs. Half-and-half of rTbx6 and rTbx6 mut constructs with NICD showed intermediate levels of luciferase activities. Assays were performed in triplicate. One-way analysis of variance was performed on data from all experiments, and significance was determined using Turkey's post hoc test. ns, not significant. mTbx6 and rTbx6 represents mouse and rat Tbx6, respectively.</p

Electrophoretic mobility shift assay (EMSA) of <i>Tbx6</i> and <i>Tbx6</i>^<i>Oune</i>.

<p>(A) The <i>Tbx6</i>^<i>Oune</i> allele did not influence the DNA binding ability of the T-box binding consensus sequences. <i>Tbx6</i> and <i>Tbx6</i>^<i>Oune</i> (Tbx6 mut) showed no difference in binding ability to the Tbind probe, two T-box gene binding sites, and to the Tbind-half probe, a single binding site, judged from intensity of shifted bands. Free binding probes were shown at the bottom. (B) DNA binding ability of mutant Tbx6 was not changed. The Tbind probe concentration was diluted to one sixteenth, but no difference was detected between Tbx6-wt and mut. mTbx6 and rTbx6 represents mouse and rat Tbx6, respectively.</p