Marklew S et al. (1994), Isolation of a novel RXR from Xenopus that most...

XB-ART-20941

Biochim Biophys Acta 1994 Aug 02;12183:267-72.

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Isolation of a novel RXR from Xenopus that most closely resembles mammalian RXR beta and is expressed throughout early development.

Marklew S , Smith DP , Mason CS , Old RW .

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In a search for nuclear receptors that may mediate the teratogenic effects of the potential morphogen, retinoic acid, on the early development of Xenopus we have isolated a novel Xenopus RXR that most closely resembles the mammalian beta-type RXR. Xenopus RXR beta mRNA is expressed throughout early embryogenesis, and functions as an accessory protein to enhance the DNA-binding of other members of the nuclear receptor superfamily.

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Species referenced: Xenopus laevis
Genes referenced: rxra rxrb

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	Fig. 1. Nucleotide sequence, and putative amino acid sequence of xRXRfl, xRXR~ cDNA was isolated as a single EcoRI fragment, the nucleotide sequence of which is shown. The flanking EcoRI sites have been omitted from the sequence since these were added during cDNA cloning. Translation of the longest open reading frame from the first in-~ame ATG codon is shown.
	Fig. 2. Amino acid sequence comparison of xRXR/3 to other RXRs. (A) Sequence alignment of xRXR/3 and hRXR/31 putative amino acid sequences. Sequences were aligned with the Clustal V program [45]. Vertical lines indicate identical amino acids, and dots conservative amino acid differences. Regions of sequence enclosed by lines are the putative C (DNA binding) and E (ligand binding) domains, respectively. (B) Percentage identity of the domains of xRXR/3 to some other RXRs. Location of the domains of xRXR/3 is illustrated in (A). Amino acid sequences were aligned with the Clustal V program to calculate percentage identities. For the A/B domain the percentage identity is in the region of overlap. The prefixes h, m and x refer to human, mouse and Xenopus respectively, hRXR/31 and hRXR/32 are N-terminal variants, xRXR/3 extends only 4 amino acids into the region of variation. Sources of sequences are: hRXR/31, 46; hRXR/32, 47; mRXRa/¢3/7, 24; xRXRa/y, 13.
	Fig. 3. Band shift assays to show that xRXRI3 enhances the binding of hVDR to a DR-3 DNA element, mRNA-injected embryos, and control (uninjected) embryos were used to make extract for band shift assays. Band shift assays contained a total of 6 /zl of embryo extract (equivalent to 1.2 embryos) and 0.25 ng of end-labelled duplex DR-3 oligonucleotide (see Materials and methods). Embryo extracts were mixed in the assays as indicated. Protein-DNA complexes were resolved on a 4% polyacrylamide gel, and the gel dried and autoradiographed.
	Fig. 4. Temporal expression of xRXR/3 transcripts in early embryogenesis. A portion of the xRXR/3 cDNA from the E domain (see methods) was transcribed in vitro in the presence of radiolabelled nucleotides and the resulting antisense transcript used in RNase protection assays with nucleic acid extracted from Xenopus laevis embryos of the developmental stages shown (embryos were staged according to 40). E = egg; NONE = control assay with no RNA added, Arrows indicate the position of undigested probe (PROBE); partially digested probe (PD); xRXR/3 transcript protected probe (xRXR/3).