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Gene
2003 Dec 11;322:105-12. doi: 10.1016/j.gene.2003.08.016.
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Cloning and characterization of Xenopus laevis drg2, a member of the developmentally regulated GTP-binding protein subfamily.
Ishikawa K
,
Azuma S
,
Ikawa S
,
Morishita Y
,
Gohda J
,
Akiyama T
,
Semba K
,
Inoue Ji
.
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The developmentally regulated GTP-binding protein (DRG) subfamily is an uncharacterized member of the Obg family, an evolutional branch of GTPase superfamily proteins. GTPases act as molecular switches regulating diverse cellular processes. DRG2 and DRG1 comprise the DRG subfamily in eucaryotes. Although drg1 was first identified as a gene predominantly expressed during early development of the mouse central nervous system, comparative analysis of drg2 and drg1 expression during embryogenesis has never been reported, and the biochemical properties of the DRG family proteins remain to be elucidated. Thus, we first cloned Xenopus drg2 (Xdrg2) and examined the temporal and spatial expression patterns of Xdrg2 mRNA in comparison to those of Xdrg1. Both Xdrg2 and Xdrg1 are induced at late gastrula and subsequently increased during later stages of embryos (stage 13-41). Whole-mount in situ hybridization showed that Xdrg2 and Xdrg1 expression patterns are almost identical except that only Xdrg2 expression is detected in the stage 22pronephric anlage. Strong transcripts of both genes are also observed at this stage in neural crest cells, blood islands, and developing eyes, and in brain, eyes, otic vesicle, branchial arches, pronephroses, spinal cord, notochord, head mesenchyme, and somites at stages 27 and 32. Northern blot analysis of adult tissues revealed that both genes are expressed highly in ovary and testis and rather moderately in other organs, except that Xdrg1 transcripts are scarcely detected in heart, lung, and liver. Accordingly, transcription or stability of Xdrg2 and Xdrg1 mRNAs may be regulated by different mechanisms. In addition, by generating recombinant XDRG2 and XDRG1 proteins, we found the RNA binding activity of these proteins in vitro. Our results suggest that the DRG proteins may play their physiological roles via RNA binding.
Fig. 1. Comparison of amino acid sequences of the DRG subfamily proteins. Amino acid sequences of the DRG family proteins from X. laevis (x), mouse (m), and human (h) were aligned by means of the ClustalX software program. Residues identical to Xenopus DRG2 are marked in black. G1, G2, G3, and G4, which comprise the core of GTPase, are indicated by straight lines. The TGS domain is marked with an arrow. The Swiss-Prot accession numbers are: Q9QXB9 (mouse DRG2), P55039 (human DRG2), P43690 (Xenopus DRG1), P32233 (mouse DRG1), Q9Y295 (human DRG1). The nucleotide sequence of Xenopus drg2 has been submitted to DDBJ/EMBL/GenBank (accession number AB104728).
Fig. 2. RNA binding activity of XDRG2 and XDRG1. GST-fusion XDRG2, XDRG1 or GST proteins (270 ng) were preincubated with indicated concentrations of free poly(U) RNA in 150 Al of binding buffer, and pulled down by poly(U) agarose beads. Captured protein was analyzed by Western blotting using polyclonal anti-GST antibody. In each ‘input’ lane, 7.5% of the total protein in the reaction vial was loaded as a detection standard. Arrowheads denote the full-length GST-DRG2, GST-DRG1 or GST.
Fig. 3. Temporal expression of Xdrg2 (A) and Xdrg1 (B) transcripts during
X. laevis development. Total RNAs isolated from embryos at indicated
stages were separated for Northern blotting. The same membranes were
reprobed with Xenopus ODC probe for RNA quality control. Ethidium
bromide (EtBr) stained rRNAs are shown as loading control.
Fig. 4. Spatial expression of Xdrg2 and Xdrg1 transcripts during Xenopus development. (A and B) Ventral views, anteriorleft; (C and D) dorsal views, anteriorleft; (E –J) lateral views, anteriorleft; (GV–JV) higher magnification image of the anterior part of the embryo depicted in (G), (H), (I), and (J), respectively; (K and L) clearing of embryo by benzyl alcohol/benzyl benzoate (2:1); (M and N) transverse sections of embryos. Positions of the sections are indicated as dotted lines in (IV) and (JV); (O–Q) enlarged lateral view of boxed area in (S), anteriorleft; (R) deduced region of Xdrg2 and Xdrg1 expressions in a somite depicted in purple, anteriorleft; (S) schematic diagram at stage 29; abbreviations: ba, branchial arch; bcs, branchial crest segment; bi, blood islands; cnc, cranial neural crest cells; de, developing eyes; e, eyes; fb, forebrain; hb, hindbrain; hcs, hyoid crest segment; hm, headmesenchyme; le, lens; mb, midbrain; mcs, mandibular crest segment; nc, notochord; ov, otic vesicle; pr, pronephros; pra, pronephric anlage; sc, spinal cord; tnc, trunk neural crest; asterisks indicate intersomite boundaries.
Fig. 5. Specificity of probes in whole-mount in situ hybridization. Lateral views of embryos at stage 32. Hybridization reaction was performed using Xdrg2
(A–C) or Xdrg1 (D–F) digoxigenin-labeled antisense RNA probe in the absence (A and D) or presence of 50-fold excess unlabeled antisense Xdrg2 (B and E)
or Xdrg1 (C and F) RNA probe. The chromogenic reaction was performed for 2 h (A–C) or 3.5 h (D– F).
Fig. 6. Tissue-specific expression of Xdrg2 (A) and Xdrg1 (B) mRNA in adult Xenopus. Total RNAs isolated from the indicated adult tissues were separated for Northern blotting. The same membranes were reprobed with Xenopus EF-1aS probe for RNA quality control. The band of EF-1aS was scarcely detected in the lane of ovary, because the second EF-1a variant (EF-1aO) rather than EF-1aS is expressed mainly in oocytes (Dje et al.,1990). Ethidium bromide (EtBr) stained rRNAs are shown as loadingcontrol.
drg2 (developmentally regulated GTP binding protein 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, lateral view, anteriorleft, dorsal up.
drg2 (developmentally regulated GTP binding protein 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 27, lateral view, anteriorleft, dorsal up.
drg2 (developmentally regulated GTP binding protein 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anteriorleft, dorsal up.
