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Biochem Biophys Res Commun
2007 Sep 14;3611:74-8. doi: 10.1016/j.bbrc.2007.06.158.
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Identification and preliminary function study of Xenopus laevis DRR1 gene.
Zhao XY, Liang SF, Yao SH, Ma FX, Hu ZG, Yan F, Yuan Z, Ruan XZ, Yang HS, Zhou Q, Wei YQ.
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Xenopus laevis has recently been determined as a novel study platform of gene function. In this study, we cloned Xenopus DRR1 (xDRR1), which is homologous to human down-regulated in renal carcinoma (DRR1) gene. Bioinformatics analysis for DRR1 indicated that xDRR1 shared 74% identity with human DRR1 and 66% with mouse DRR1, and the phlogenetic tree of DRR1 protein was summarized. The xDRR1 gene locates in nuclei determined by transfecting A549 cells with the recombinant plasmid pEGFP-N1/xDRR1. RT-PCR analysis revealed that xDRR1 gene was expressed in all stages of early embryo development and all kinds of detected tissues, and whole-mount in situ hybridization showed xDRR1 was mainly present along ectoderm and mesoderm. Furthermore, xDRR1 expression could suppress A549 cell growth by transfecting with plasmid pcDNA3.1(+)/xDRR1. xDRR1 probably plays important roles involving in cell growth regulation and Xenopus embryo development.
Fig. 1.
Comparison of DRR1 proteins from X. laevis (GenBank protein_id: AAH82866) with those of other species. (A) xDRR1 and hDRR1 proteins (GenBank protein_id: NP_009108) are homologous proteins. There is a coiled domain (underlined) and a signal peptide (bold) in hDRR1 protein and xDRR1. Asterisks (∗) indicate amino acid residues that are conserved across species. Colons (:) indicate strong similarity between protein xDRR1 and hDRR1. Dots (.) indicate weak similarity. (B) Phlogenetic tree of the DRR1 protein.
Fig. 2.
Expression pattern of xDRR1 gene in developing embryos and tissues from an adult frog. (A) Temporal expression profile of the xDRR1 mRNA detected by RT-PCR. (B) Tissue expression profile of xDRR1. The relative expression ratio of xDRR1 was shown in Xenopus different developing stages (C) and several different organs (D). The ODC expression was taken as control. (E) Spatial expression of xDRR1 using whole-mount in situ hybridization analysis. Numbers indicate developmental stage. At stage 21, expression is mainly detectable in neuroectoderm. At stages 25/26, the transcripts are present in brain, heart, notochord, and somites.
Fig. 3.
Cellular localization of the xDRR 1 protein. The A549 cells were transfected with expression vector pEGFP-N1/xDRR1, which expresses a fusion protein consisting of DRR 1 and EGFP epitope, and then detected under fluorescence microscope (×200). Included in the picture are cells which show the fusion protein (strong green) in nuclei. (For interpretation of the references to colors in this figure legend, the reader is referred to the web version of this article.
Fig. 4.
Suppression of cell growth by re-expression of xDRR1 gene. The xDRR1 plasmids were transfected into A549 cells. At the indicated time points, the cells were subjected to proliferation analysis using MTS and the absorbance detected at 490 nm. The cells transfected with the xDRR 1 plasmids were suppressed. Each bar represents the mean ± standard deviation (n = 6). ∗P < 0.05.
fam107a (family with sequence similarity 107 member A ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 21, lateral view, anteriorleft, dorsal up.
fam107a (family with sequence similarity 107 member A ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 26, lateral view, anteriorleft, dorsal up.
