Stickland JE et al. (1992), A Xenopus borealis homeobox gene expressed pref...

XB-ART-23561

Gene 1992 Jul 15;1162:269-73.

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A Xenopus borealis homeobox gene expressed preferentially in posterior ectoderm.

Stickland JE , Sharpe CR , Turner PC , Hames BD .

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We have isolated a new homeobox-encoding gene (XhoxB.1) from Xenopus borealis which has a homeobox exon identical to that of the murine Hox1.7 gene, except for one amino acid. XhoxB.1 transcripts of 2.1 and 7.0 kb are first detected at the late gastrula stage, accumulate until the tailbud stage and are most abundant in the posterior third of the dorsal part of the embryo.

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Species referenced: Xenopus
Genes referenced: hoxa9 hoxb1 mt-tr ncoa6 trna

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	Fig. 1. Restriction map and sequence of the XhoxB. ! genomic DNA clone. (A) The upper horizontal line represents the X. boreafis DNA insert in phage AEMBL3. E. EcoRI; B, Barn HI', S, SolI. Part of the subcloned BamHI-EcoRI fragment was sequenced on both strands and is shown in (B). The homeobox is shown as a filled box and the Sau3A-EcoRI fragment used as the RNase protection assay probe is indicated. (B) Nucleotide sequence of the homeobox exon of XhoxB. I and the deduced aa sequence. The m 241-420 encode the homeodomain (boxed). The arrowhead indicates a potential accepter splice site. Methods, Construction and screening of the genomic library, isolation and identification of positive plaques and cloning procedures were done by standard methods (Maniatis et al., 1982) using a random primed (Feinberg and Vogelstein, 1983) mixed homeobox probe consisting of an ! 18-bp XIHboxl probe (Carrasco et al., 1984), a 200-bp A,tp probe (Gather et al., 1983) and a 248.bp BglII-Ddel fragment of the XIHboxl gene containing the Y-terminal two thirds of the homeobox together with 85 bp of 3'-flanking sequence, Sequencing was performed by the method of Sanger et al. (1977). GenBank accession No. X61916.
	Fig. 2. Homeodomain and flanking sequence comparison. XhoxB.l is compared with mouse Hox 1.7 (Rubin et al., 1987), X. laevis XIHbox6 (Sharpe et al., 1987) and Drosophila Antp and Abd-B (Regulski et ai., 1985) homeodomain regions. The start of the homeobox is numbered + 1 and asterisks indicate stop codons. Identical aa are boxed.
	Fig. 3. Developmental expression of XhoxB'~ 1. (Panel A) The RNase protection assay was performed according to Melton et al. (1985). The 257-nt long probe was transcribed from the subclone which contained the 223- bp Sau3A-EcoRl fragment. Size (nt) markers (SM) were 32P-labelled pBR322 Hinfl fragments. The RNA from two embryos of the successive developmental stages shown was analysed in each case. U-E, unfertilized egg; B, blastula (stages 8-9); MG, mid-gastrula (12-13); N, neurula (14- 17); T, tailbud (26); H, hatched tadpole (42); t, yeast tRNA control. (Panel B) Northern blot analysis of total RNA from dissected embryos. During dissection of dejellied stage 26 embryos, tissue explants were maintained in modified Barth's solution prior to RNA extraction (Sharpe et al., 1987). The RNA from four whole embryos or embryo fragments was fractionated on 1.2% agarose/2.2 M formaldehyde gels and blotted to Gene- Screen membranes (Maniatis et al., 1982) The same Sau3A-EcoRl probe as in (panel A) was used. Arrowheads indicate the 2.1-kb and 7.0-kb XhoxB. 1 transcripts and the positions of 28S and 18S rRNA are shown.
	Fig. 4. XhoxB. 1 expression along the anterior/posterior axis. (a) Stage 26 embryo dissection scheme used. (Panel b) RNase protection analysis was carried out as described in Fig. 3A using RNA from four embryo fragments or two whole embryos. W, whole embryo; V, ventral', P, posterior; C, central; A, anterior; Pr, undigested probe DNA; S, Hinfl-cut pBR322 size (nt) markers (see Fig. 3A, lane SM).