Herzberg F et al. (1990), Identification of Ca2(+)-dependent cell adhesio...

XB-ART-25794

Differentiation 1990 Jul 01;441:1-7. doi: 10.1111/j.1432-0436.1990.tb00530.x.

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Identification of Ca2(+)-dependent cell adhesion molecules in Xenopus by the use of interspecies homology.

Herzberg F , Pöting A , Wedlich D .

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Ca2(+)-dependent cell adhesion molecules (CAMs) are transmembrane glycoproteins structurally and functionally related in mammalian and avian species. This suggests that Ca2(+)-dependent CAMs consist of an evolutionary conserved gene family. Antibodies or cDNA probes specific either to the extracellular part or the cytoplasmic domain of uvomorulin were compared for their ability to detect corresponding molecules in Xenopus. Only antibodies directed against the evolutionary highly conserved cytoplasmic domain afforded a clear membrane staining on sections of Xenopus embryos or on cultured Xenopus epithelial cells. However, these antibodies recognized different polypeptides of 156, 140 and 128 kDa in immunoblots prepared from cell lysates of epithelial, neural, muscle and embryonic tissues. In concordance with the antibody analysis, signals in Northern hybridizations were only obtained when the cDNA probe encoding the cytoplasmic domain of uvomorulin was used. Here again, this cDNA probe revealed different mRNA species of 4.3, 4.1, 3.8 and 3.2 kb in the studied cell types. These results provide further direct evidence that the Ca2(+)-dependent CAMs are evolutionary conserved. The variety of polypeptides and transcripts observed in Xenopus indicates that several members of this gene family were detected by the use of probes specific to conserved sequences. More important, with this approach we also identified members of this gene family in the early stages of Xenopus development. Since these proteins were present in mature eggs but not in oocytes, we assume a maternal store of Ca2(+)-dependent CAM RNAs whose translation might be initiated during egg maturation.

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Species referenced: Xenopus laevis
Genes referenced: cdh1

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	Fig. 1. Scheme of the used probes in relation to the uvomorulin protein. TM: transmembrane domain. Probes for extracellular domains: anti-gp 84 and F20A, probes for cytoplasmatic domains: anti-UMc and H3
	Fig. 2. Immunoblots of total SDS extracts of different cell lines and organs: EAX 1875 cells ( A ) , A6 cells incubated with anti-gp84 (B), A6 cells treated with tunicamycin (C), A6 cells digested with trypsin in the presence of Ca" (TC-cells) (D), brain ( E ) . kidney (F),li ver (G). Except lane B (control), all blots werc incubated with anti-UMc
	Fig. 3. Northern hybridization of poly(A)+ RNA from EAX 1875 cells probed with mouse cDNA clones H3 (lane A ) and F20A ( h e B) and from liver (lane C) and brain (lane D) probed with mouse cDNA clone (H3). The same filters probed with Xenopus EF-1 a (lunc A-D, line below). Autoradiographs were exposed for 14 days (H3 and F20A) or overnight (EF-la). Lanes A , B and C represent 2,s pg poly(A)+ RNA
	Fig. 4a-c. Immunofluorescence staining of different cell lines and sections of embryos and tadpoles: A6 cells (a). A6 cells incubated with anti-gp 84 (b), fibroblasts (c), frozen sections of a stage 7 blastula (d, e), stage 10 1/2 gastrula (9 and a stage 18 neurula (g), sections of stage 45 tadpole showing parts of the pancreas and intestinc (h) and a part of the brain (i). Except b (control), all stainings were performed using anti-UMc. BC: blastocoel, rn: mesoderm, c : ectoderm, E: epidermis, NT: neural tube, F: fibrous cortex. Y : ventricle, P: pancreas, I: intestine. Burs in a-c: 20 pm, in d-i: 50 pm
	Fig. 5. Immunoblots of Con A sepharose precipitates from oocytes and embryonic stages: oocytes (A), mature eggs (E), 32 cell stage (C), stage 7 blastula ( D ) , stage 10 1/2 gastrula ( E ) and stage 18 neurula (F)