Nagano M et al. (1997), Cloning of Xenopus Dr1 (TBP-binding repressor) ...

XB-ART-16866

Biochem Biophys Res Commun 1997 Feb 24;2313:561-5. doi: 10.1006/bbrc.1997.6120.

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Cloning of Xenopus Dr1 (TBP-binding repressor) and its expression in oocytes and early embryos.

Nagano M , Koga C , Tashiro K , Kugawa F , Shiokawa K .

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With a final goal to study how TBP-binding repressor Dr1 regulates transcription in Xenopus early embryos, we cloned its cDNA from Xenopus liver cDNA library. The cDNA was 1,986 bp long, with the open reading frame coding for 175 amino acids, whose sequence was highly homologous to its human counterpart. Xenopus Dr1 mRNA was expressed from the earliest stage of oogenesis, inherited as maternal mRNA at a high level, but its level became low at and after the neurula stage where RNA synthetic activity is strongly activated. Dr1 mRNA occurred in larger amounts in the animal half than in the vegetal half in 8-cell stage embryos, and in neurula and tailbud stage embryos its distribution was slightly larger in the anterior part than in the posterior part. These data show that Dr1 mRNA is expressed in a temporally and spatially regulated manner, and its occurrence at higher levels in earlier stage embryos may be responsible for their low transcriptional activity.

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Species referenced: Xenopus laevis
Genes referenced: dr1 tbp

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	FIG. I. eDNA a nd deduced amino acid sequences of Xenopus laev/s Dr I. The amino acid sequence Is s hown by s ingle letter codes, the stop codon by the asterisk. the three potential amphlpathlc ahelical regions underlined by thin Unes (a-c). the In-frame stop codon by a double underline. and the polyadenylation signal by a thick underline. The region b Is required for the binding to TBP. the region c. rich In glutamine and alanine residues. for mediating Dr! repression acttvlty.The hiStone fold region required for tnteracllon with DRAPI (Drl -assoctated polypeptide) Is shown between bent arrows.
	FIG. 2. Alignment of total amino acid sequences of Xenopus and human Ori s. Asteris ks signify Identity. The bar s hows the gap or the amino acid missing. For boxes a, b, and c and bent arrows, see the legend to Ftg. I.
	FIG. 3. Expression of Drl mRNA during oogenesis and oocyte maturation. RNAs equivalent to 2 oocytes were loaded. The major band (6.5 kb) appeared to represent Dr I mRNA. Results of double hybridization carried out wtth rR.\IA eDNA as a probe are given to assess the loading eqtvalency. Lanes I to 6 are for oocytes at stages I. II. Ill. IV. V. and VI. respectively. Lanes 7 to 10 are for stage VI oocytes treated with progesterone for 2.5. 5.0. 7.5. and 8.5 hrs, respectively.
	FIG. 4. Expression of Drl mRNA during early embryogenesis of Xenopus laevls. (A) Ftve pg of total RNAs were loaded on the get throught stages. The major band (6.5 kb) as Drl mRNA. and ISS and 28S rRNAs obtained by double hybridization are gtven. UnfertilIzed egg (lane 1), and embryos at stages 1 (lane 2). 4 (lane 3). 6.5 (lane 4). 8 (lane 5). 10 {lane 6). 12 (lane 7). 15 (lane 8). 18 {lane 9). 23 (lane 1 0). 25 (lane 11). and 27 (lane 12) were used. (B) The relative Intensity of the radioactivity of Drl mRNA In A was measured In a BAS 2000 densitometer. and normalized taking the level oflhe signal In lane 7 as 1.0. Altogether. results of three Independent expert ments (circle. rectangle. triangle) are given, together with the mean value (lozenge).
	FIG. 5. Spatial distribution of Dr1 mRNA at 8-Cell stage. (A) Embryos were dissected into four portions as indicated. (B) Five mg of total RNAs were loaded (W, whole embryo; lane 1, animal-dorsal region; lane 2, vegetal-dorsal region; lane 3, animal-ventral region; lane 4, vegetal-ventral region).
	FIG. 6. Spatial distribution of Dr1 mRNA at neurula (stage 23) and tailbud (stage 27) stages. (A) Embryos were dissected along the lines. (B) Five mg of total RNAswere loaded.Wn,whole neurula embryo; lane 1, anterior; lane 2, posterior; lane 3, dorsal; and lane 4, ventral region of neurula embryo. Wt, whole tailbud embryo; lane 5, anterior; lane 6, trunk; and lane 7; tail region of the tailbud embryo.