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Gene
2005 Oct 10;359:73-80. doi: 10.1016/j.gene.2005.04.042.
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Cloning and functional characterization of the Xenopus orthologue of the Treacher Collins syndrome (TCOF1) gene product.
Gonzales B
,
Yang H
,
Henning D
,
Valdez BC
.
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Treacher Collins syndrome (TCS) is an autosomal dominant disorder of craniofacial development caused by mutations in the TCOF1 gene, which encodes the nucleolar phosphoprotein treacle. We previously reported a function for mammalian treacle in ribosomal DNA gene transcription by its interaction with upstream binding factor. As an initial step in the development of a TCS model for frog the cDNA that encodes the Xenopus laevis treacle was cloned. Although the derived amino acid sequence shows a poor homology with its mammalian orthologues, Xenopus treacle has 11 highly homologous direct repeats near the center of the protein molecule similar to those present in its human, dog and mouse orthologues. Comparison of their amino acid compositions indicates conservation of predominant specific amino acid residues. Antisense-mediated down-regulation of treacle expression in X. laevis oocytes resulted in inhibition of rDNA gene transcription. The results suggest evolutionary conservation of the function of treacle in ribosomal RNA biogenesis in higher eukaryotes.
Fig. 1.
Alignment of the cDNA-derived amino acid sequences from human (H, GenBank accession no. NM_000356), dog (D, GenBank accession no. NM_001003057), mouse (M, GenBank accession no. NM_011552), and frog (F, GenBank accession no. AY731504) using ClustalW and BOXSHADE programs. Red letters show identical amino acids, blue letters indicate similarity and black letters show absence of homology. The dashed line shows absence of corresponding sequence.
Fig. 2.
Alignment of the 11 repeats in the cDNA-derived amino acid sequence of Xenopus laevis treacle. Color codes are similar to those described in Fig. 1. The repeats are arranged in decreasing homology.
Fig. 3.
Characterization of X. laevis treacle (xtreacle). (A) Indirect immunofluoresence staining shows nucleolar localization of the antigen which co-localizes with nucleolin/C23 (xnucleolin) in X. laevis kidney A6 cells. (B) Western blot analysis of A6 cell nuclear extract using 1 : 10,000 anti-xtreacle antibody recognizes a ∼160 kDa antigen.
Fig. 4.
Down-regulation of xtreacle expression using antisense oligodeoxynucleotides. (A) X. laevis oocytes were microinjected with antisense oligodeoxynucleotides (32 ng per oocyte) and total RNA was isolated after 24 h. RT-PCR was used to determine the levels of expressions of mRNA for Xenopus nucleolar protein B23 (xB23) and Tcof1 (xTcof1) in the same reaction mix containing 50 ng total RNA. One oligo in each pair of primers was 32P-end labeled. The phosphorous image signals were analyzed using the ImageQuant software and the level of xTcof1 expression, after normalizing against xB23, was calculated relative to the negative control (see numbers below the image). The antisense target sequences in the xTcof1 mRNA are shown below the panel. BV579 is an unrelated oligo used as negative control. (B) Western blot analysis using anti-xtreacle antibody and protein extracts from oocytes incubated for 5 days after microinjection with BV579 control oligo or BV1222 xTcof1 antisense. The membrane was stripped and re-probed with anti-xnucleolin antibody to check protein loading. Numbers on the right refer to molecular weight markers in kDa.
Fig. 5.
Xenopus treacle functions in rDNA gene transcription. Frog oocytes were microinjected with the indicated oligodeoxynucleotides (32 ng/oocyte). After 72 h at 18 °C, oocytes were microinjected with [α-32P]-GTP. Total RNA was isolated after 1 h. (A) The level of xTcof1 mRNA was determined by RT-PCR using 50 ng total RNA and measured relative to xB23. (B) Equal amount of RNA (2 ug) was loaded onto 1.2% agarose-formaldehyde gel, blotted onto nylon membrane and stained with methylene blue. The membrane was exposed overnight on a phosphorous imager cassette and analyzed. BV1222-treated samples are shown in duplicate.
