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Gene
2002 May 15;2901-2:53-61. doi: 10.1016/s0378-1119(02)00587-5.
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Molecular cloning and expression study of Xenopus latent TGF-beta binding protein-1 (LTBP-1).
Quarto N
.
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Latent transforming growth factor beta binding protein-1 (LTBP-1) is important in regulating the localization and activation of transforming growth factor beta. In this paper is reported the isolation of the full-length Xenopus LTBP-1 cDNA from screening a neurulaembryo cDNA library. Sequence analysis of XLTBP-1 cDNA revealed an open reading frame of 4518 bp encoding a 1398 amino acid protein with a molecular mass of 154.1 kDa and an isoelectric point of 4.65. The Xenopus XLTBP-1 shares 61 and 65% amino acid identity with the mouse and human LTBP-1, respectively. It contains 17 epidermal growth factor-like motifs and four eight-cysteine repeats (8-Cys). RNase protection assay revealed that XLTBP-1 is a maternal and zygotic gene, while whole-mount in situ hybridization analysis performed on embryos at different stages showed that during early Xenopus development, XLTBP-1 mRNA is expressed in the Spemann organizer, prechordal and chordal mesoderm, and later on in the organizer derived tissues. These findings suggest an important role for XLTBP-1 in embryo axis formation.
Fig. 1.
Nucleotide and predicted amino acid sequences derived from Xenopus LTBP-1 cDNA. (A) The putative signal sequence at the NH2-terminus is in lower case letters. Potential N-linked glycosylation sites are in bold. The eight-cysteine repeats are underlined. The XLTBP-1 EMBL Accession number is AJ421626. The stop codon is indicated by an asterisk. (B) Schematic representation of the XLTBP-1 protein structure.
Fig. 2.
Comparison between the Xenopus, mouse and human LTBP-1 protein. The protein sequences were aligned using the pileup GCG 9.0 program. Identical residues between Xenopus, mouse and human XLTBP-1 are in yellow, non-conserved residues are in green and conserved residues between mouse and human are in blue. The human LTBP-1 Accession number is NM000627 (Kanzaki et al., 1990); the mouse LTBP-1 Accession number is AF022889 (Noguera et al., GenBank).
Fig. 3.
Temporal expression of XLTBP-1 during embryo development. Total RNA (20 μg) from embryos at different stages was analyzed by RNase protection assay for the presence of XLTBP-1 mRNA. The probe alone without RNase is a positive control, while the probe plus yeast RNA digested with RNase is a negative control. ODC, a constantly expressed message, was used as a loading control.
Fig. 4.
Spatial expression pattern of XLTBP-1. Whole-mount in situ hybridization on albino embryos at different stages: (A) gastrula (stage 10.5), the arrow indicates the dorsal blastopore lip; (B) mid-neurula (stage 18); (C) late neurula (stage 21); (D) early tailbud; (E) late tailbud; (F) dorsal view; (L) enlargement of (E); (B–E) lateral view with anterior end to the left; (G) swimming tadpole stage, lateral view with anterior end to the left; (H) dorsal view; (I) enlargement of (G); (M) embryo stage 55. Embryos were cleared in 2:1 benzyl-benzoate/benzyl alcohol to visualize deep staining.
Fig. 5.
XLTBP-1 transcript localizes in the DMZ of gastrulaembryo. DMZ and VMZ were dissected from gastrula embryos at stage 10.5 and analyzed by RT-PCR for the expression of XLTBP-1. Chordin and XWnt-8 were used as dorsal and ventralmesoderm markers to ensure the accuracy of dissections, Xbra is a pan-mesodermal marker and EF1-α 1 is a loading control.
