Characterization of a novel Gli5 gene during embryonic development in Xenopus laevis

Mai, Ming (1999) Characterization of a novel Gli5 gene during embryonic development in Xenopus laevis. Masters thesis, Memorial University of Newfoundland.

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Abstract

Human Gli oncogene gene was first isolated from its amplification in a glioblastoma cell line, DM-259 MG. Because Gli gene is the vertebrate homolog of the Drosophila segment polarity gene, Ci (cubitus interruptus), isolation of the Xenopus Gli gene and analysis of its function in embryonic development were performed. Using RT-PCR and 5' RACE-PCR, 2.1 Kb of the 5' end of Xenopus Gli5 gene was cloned and sequenced. Sequence comparison found that region one and the zinc finger motif of Xenopus Gli5 were very similar to other Glis. In region one and the zinc finger region, the amino acid identities between XGli5 and Chicken Gli2/4, XGli4, XGli3, XGli1, Human Gli3, HGli1 are 98.6%, 100%, 78.5%, 78.5%, 81,5%, 65.7% and 92%, 97%, 95%, 91.3%, 94%, 87.3% respectively. In addition to these two conserved regions, the GF (gain of function) region and the region located between GF region and region one are also conserved among Gli family. But, outside of these conserved regions the homology is less conserved. -- From Northern Hybridization and RT-PCR analysis, it was found that although Xenopus Gli5 was expressed at all stages of embryonic development, the peak of expression was at stage 8. This expression pattern is different from that of known Xenopus Gli genes in embryonic development. Using affinity purified XGli5 antibodies, through competitive Western blotting analysis, the XGli5 protein expression was detected in stage 28 embryos as a 190 KDa band. -- A XGli5 fusion protein was made in the pGEX.KT vector. GST+Gli5 protein was purified by GST affinity chromatography. The fusion protein was used to test the specificity of XGli5 polyclonal antibodies and whether Xenopus Gli5 can bind the same specific DNA sequence as those in other Gli protein. According to the results of electrophoretic mobility shift assays (EMSA), XGli5 can specifically bind the same Gli binding sequence, 5'-GACCACCCA- 3' as other Glis. Therefore, it is likely that XGli5 is a transcription factor as other Glis which can regulate transcription by binding specific promoter. To explore the relation between XGli5 expression and FGF induction, animal pole explants from Xenopus embryos were incubated with FGF (100ng/ml) for different lengths of time and then total RNA was extracted. The level of Xenopus Gli5 gene expression was examined by RT-PCR. Preliminary results showed that FGF induced Gli5 expression in explants. -- Overall, a new number of XGli gene family, XGli5 was isolated. The full length of XGli5 cDNA is 8.5 Kb and encodes a 190 Kda protein. Because XGli5 can also bind the core Gli binding sequence, XGli5 may function as a transcription factor. In the early Xenopus embryonic development, FGF, the mesoderm induction signal, can also induce XGli5 expression.

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