Molecular cloning of the mouse cell adhesion molecule uvomorulin: cDNA contains a B1-related sequence.

Abstract

A clone (F20) containing coding sequences for the cell adhesion molecule uvomorulin was isolated by immunological techniques from a cDNA library in the expression vector Agtll.' The ,B-galactosidase-uvomorulin fusion protein was used to affiity purify anti-uvomorulin antibodies. Affinity-purified antibodies recognized uvomorulin from cell lysates of embryonal carcinoma cells and reacted with the cell surface of embryonal carcinoma cells. The 1.8-kilobase cDNA insert hybridized to a single 4.3-kilobase poly(A)+ RNA species found only in cells expressing uvomorulin. Part of the nontranslated 3' sequences of the cloned uvomorulin cDNA is homologous to the interspersed B1 repeat of the mouse genome.Cell-cell interactions during embryonic development and in tissue organization involve specific cell-adhesion molecules (CAM) that are functionally defined by antibodies that interfere with cell-cell adhesion (1-5).Uvomorulin is involved in the compaction process of mouse morulae (6, 7). Later in development uvomorulin is expressed on epithelial cells independent of their germ-layer origin (8, 9)'. In adult tissue, the localization of uvomorulin is restricted to the intermediate junctions of intestinal epithelial cells (10). Anti-uvomorulin Fab fragments block the aggregation of primary mouse embryonic liver cells (11), and the rat anti-uvomorulin monoclonal antibody DECMA-1 disrupts the monolayer of canine kidney epithelial (MDCK) cells (34) indicating that uvomorulin also serves as an adhesive factor on adult cells.Uvomorulin has been characterized as a 120-kDa cell surface glycoprotein (gpl20) from which an 84-kDa fragment (gp84) can be released by trypsin digestion in the presence of Ca2l (7). Antibodies prepared against each of these two proteins recognize two additional proteins with apparent molecular sizes of 102 (plO2) and 92 (gp92) kDa (8, 9), respectively. 'The relation of these proteins to gpl20 is beginning to be understood (11), and it is clear that at least p102 is a true cellular component also expressed on the cell surface (D.V. and R.K., unpublished results). Molecular studies on uvomorulin require molecular probes to establish the relation of the different proteins that share common antigenic sites and a possible homology of uvomorulin with other CAM.The present paper describes the molecular cloning of uvomorulin cDNA and the uvomorulin mRNA and gives an initial characterization of part of the uvomorulin gene.MATERIALS AND METHODS Cells. The following cells were used: embryonal carcinoma (EC) cells [PCC4azal, F9 (12,13), and 3/A/1-Dl-an EC cell-derived embryonic fibroblast cell line (14)] and NIH/3T3 cells.Antibodies. The following rabbit anti-uvomorulin sera were used: anti-gpl20 and anti-gp84 (9) and anti-p102 (D.V. and R.K., unpublished data). A rat monoclonal antibody (DECMA-1) has been selected against gp84 (34). This monoclonal antibody blocks cell adhesion in a functional cell aggregation assay and recognizes the known set of uvomorulin proteins in immunoprecipitation and immunoblot exper...

Sequence analysis of a cDNA clone encoding the liver cell adhesion molecule, L-CAM.

“…The mRNA encoding uvomorulin has been reported to contain sequences homologous to the rat B1 repetitive sequence (34). We find no evidence for any repetitive elements in the cDNA clone for L-CAM; all regions of the cDNA clone hybridize to a single size species on RNA transfer blots.…”

Chromosomal mapping of the structural gene coding for the mouse cell adhesion molecule uvomorulin.

“…Uvomorulin cDNA clones have been isolated that hybridize to a single 4.3-kilobase (kb) mRNA (11,12). Southern blot analyses of murine genomic DNA revealed a single structural gene coding for uvomorulin.…”

cDNA Cloning and Sequence Analysis of a Novel Calcium Binding Protein with Oligoproline Motif.

“…Abbreviations: G buffer; 50 mMTris-HCl buffer, pH 9.6 containing 5 mMglutathione, GST; glutathione S-transferase, MBP; maltose binding protein, PBS(-); Dulbecco's phosphate buffered saline without Ca2+ and Mg2+; PWA; pokeweed agglutinin, SSC; 0.15 M NaCl, 0.015 Msodium citrate. et al (14). The library, constructed from mRNAsof F9 embryonal carcinoma cells, has been described (8).…”