Abstract
The neural cell adhesion molecule N-CAM has been identified in a number of species and comprises at least three major cell surface polypeptides of different molecular structures and tissue distributions. We report here the isolation and characterization of cDNA clones encoding two of the three major forms of N-CAM from a human neuroblastoma cDNA libary. One of the clones, NII-6, provides the first complete sequence of a small cytoplasmic domain (140 kDa) form of the molecule in humans and differs in a number of respects from cDNA clones derived from human muscle. These differences include the presence of a 30-bp insert in the fourth immunoglobulin-like domain of N-CAM, a 3-bp insert in the extracellular portion of the molecule, and an additional 6 pb in the middle of the membrane-spanning segment. Based on the analysis of a genomic DNA clone spanning these regions of N-CAM, the first two differences arise by alternate splicing of RNA and occur in some, but not all clones; the additional 6 bp may reflect a genetic polymorphism. A second cDNA clone, NI-10, encodes the complete sequence of a segment that is specific to the large cytoplasmic domain (180 kDa) polypeptide of human N-CAM and is very similar to corresponding segments of mouse, chicken, and rat N-CAM. This sequence also arises by alternative splicing of RNA. In addition, we have identified a genomic DNA segment encoding sequences specific to the third, small surface domain (120 kDa) polypeptide of N-CAM. The data presented here and previously define the DNA sequences of the membrane-bound forms and known variants of human N-CAM. From these sequences, a wide variety of probes can be generated for investigating the expression of particular N-CAM polypeptides in normal and pathological tissues.
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Barbas, J.A., Chaix, J.-C., Steinmetz, M., Goridis, C. (1988). Differential splicing and alternative polyadenylation generates distinct NCAM transcripts and proteins in the mouse. EMBO J. 7: 625–632
Barthels, D., Vopper, G., Wille, W. (1988). NCAM-180, the large isoform of the neural cell adhesion molecule of the mouse, is encoded by an alternatively spliced transcript. Nucleic Acids Res. 16: 4217–4225
Barton, C.H., Dickson, G., Gower, H.J., Rowett, L.H., Putt, W., Elson, V., Moore, S.E., Goridis, C., Walsh, F.S. (1988). Complete sequence and in vitro expression of a tissiue-specific phosphatidylinositol-linked N-CAM isoform from skeletal muscle. Development 104: 165–173
Bhat, S., Silberberg, D.H. (1988). Adult human brain expresses four different molecular forms of neural cell adhesion molecules. Neurochem, Int. 13: 487–491
Cunningham, B.A., Hemperly, J.J., Murray, B.A., Prediger, E.A., Brackenbury, R., Edelman, G.M. (1987). Neural cell adhesion molecule: Structure, immunoglobulin-like domains, cell surface modulation, and alternative RNA splicing. Science 236: 799–806
Dickson, G., Gower, H.J., Barton, C.H., Prentice, H.M., Elsom, V.L., Moore, S.E., Cox, R.D., Quinn, C., Putt, W., Walsh, F.S. (1987). Human muscle neural cell adhesion molecule (N-CAM): Identification of a muscle-specific sequence in the extracellular domain. Cell 50: 1119–1130
Edelman, G.M. (1988). Morphoregulatory molecules. Biochemistry 27: 3533–3543
Gennarini, G., Hirsch, M.-R., He, H.-T., Hirn, M., Finne, J., Goridis, C. (1986). Differential expression of mouse neural cell-adhesion molecule (N-CAM) mRNA species during brain development and in neural cell lines. J. Neurosci. 6: 1983–1990
Goblet, C., Prost, E., Whalen, R.G. (1989). One-step amplification of transcripts in total RNA using the polymerase chain reaction. Nucleic Acids Res. 17: 2144
Goridis, C., Wille, W. (1988). The three size classes of mouse NCAM proteins arise from a single gene by a combination of alternative splicing and use of different polyadenylation sites. Neurochem. Int. 12: 269–272
Harrelson, A.L., Goodman, C.S. (1988). Growth cone guidance in insects: Fasciclin II is a member of the immunoglobulin superfamily. Science 242: 700–708
He, H.-T., Barbet, J., Chaix, J.-C., Goridis, C. (1986). Phosphatidylinositol is involved in the membrane attachment of NCAM-120, the smallest component of the neural cell adhesion molecule. EMBO J. 5: 2489–2494
Hemperly, J.J., Edelman, G.M., Cunnigham, B.A. (1986a). cDNA clones of the neural cell adhesion molecule (N-CAM) lacking a membrane-spanning region consistent with evidence for membrane attachment via a phosphatidylinositol intermediate. Proc. Natl. Acad. Sci. U.S.A. 83: 9822–9826
Hemperly, J.J., Murray, B.A., Edelman, G.M., Cunningham, B.A. (1986b). Sequence of a cDNA clone encoding the neural cell adhesion molecule N-CAM. Proc. Natl. Acad. Sci. U.S.A. 83: 3037–3041
Maniatis, T., Frisch, E.F., Sambrook, J., (eds). (1982). Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
McClain, D.A., Edelman, G.M. (1982). A neural cell adhesion molecule from human brain. Proc. Natl. Acad. Sci. U.S.A. 79: 6380–6384
Mihovilovic, M., Lee, J.E. (1989). An efficient method for sequencing PCR amplified DNA. Biotechniques 7: 14–15
Moore, S.E., Thompson, J., Kirkness, V., Dickson, J.G., Walsh, F.S. (1987). Skeletal muscle neural cell adhesion molecule (N-CAM): Changes in protein and mRNA species during myogenesis of muscle cell lines. J. Cell Biol. 105: 1377–1386
Murray, B.A., Hemperly, J.J., Prediger, E.A., Edelman, G.M., Cunnigham, B.A. (1986a). Alternatively spliced mRNAs code for different polypeptide chains of the chicken neural cell adhesion molecule (N-CAM). J. Cell Biol. 102: 189–193
Murray, B.A., Owens, G.C., Prediger, E.A., Crossin, K.L., Cunnigham, B.A., Edelman, G.M. (1986b). Cell surface modulation of the neural cell adhesion molecule resulting from alternative mRNA splicing in a tissue-specific developmental sequence. J. Cell Biol. 103: 1431–1439
Owens, G.C., Edelman, G.M., Cunningham, B.A. (1987). Organization of the neural cell adhesion molecule (N-CAM) gene: Alternative exon usage as the basis for different membrane-associated domains. Proc. Natl. Acad. Sci. U.S.A. 84: 294–298
Pollerberg, G.E., Burridge, K., Krebs, K.E., Goodman, S.R., Schachner, M. (1987). The 180-kD component of the neural cell adhesion molecule N-CAM is involved in cell-cell contacts and cytoskeleton-membrane interactions. Cell Tissue Res. 250: 227–236
Prediger, E.A., Hoffman, S., Edelman, G.M., Cunningham, B.A. (1988). Four exons encode a 93-base-pair insert in three neural cell adhesion molecule mRNAs specific for chicken heart and skeletal muscle. Proc. Natl. Acad. Sci. U.S.A. 85: 9616–9620
Ramos, P., Safaei, R., Kayalar, C., Ellis, L. (1989). Isolation and sequence of λgt11 cDNA clones encoding the 5B4 antigen expressed on sprouting neurons. Mol. Brain Res. 5: 297–303
Rougon, G., Marshak, D.R (1986). Structural and immunological characterization of the amino-terminal domain of mammalian neural cell adhesion molecules. J. Biol. Chem. 261: 3396–3401
Sadoul, K., Meyer, A., Low, M.G., Schachner, M. (1986). Release of the 120 kDa component of the mouse neural cell adhesion molecule N-CAM from cell surfaces by phosphatidylinositol-specific phospholipase C. Neurosci. Lett. 72: 341–346
Santoni, M.-J., Barthels, D., Barbas, J.A., Hirsch, M.-R., Steinmetz, M., Goridis, C., Wille, W. (1987). Analysis of cDNA clones that code for the transmembrane forms of the mouse neural cell adhesion molecule (NCAM) and are generated by alternative RNA splicing. Nucleic Acids Res. 15: 8621–8641
Santoni, M.-J., Barthels, D., Vopper, G., Boned, A., Goridis, C., Wille, W. (1989). Differential exon usage involving an unusual splicing mechanism generates at least eight types of NCAM cDNA in mouse brain. EMBO J. 8: 385–392
Seeger, M.A., Haffley, L., Kaufman, T.C. (1988). Characterization of amalgam: A member of the immunoglobulin superfamily fromDrosophila. Cell 55: 589–600
Small, S.J., Shull, G.E., Santoni, M.-J., Akeson, R. (1987). Identification of a cDNA clone that contains the complete coding sequence for a 140-kD rat NCAM polypeptide. J. Cell Biol. 105: 2335–2345
Small, S.J., Haines, S.L., Akeson, R.A. (1988). Polypeptide variation in an N-CAM extracellular immunoglobulin-like fold is developmentally regulated through alternative splicing. Neuron 1: 1007–1017
Thompson, J., Dickson, G., Moore, S.E., Gower, H.J., Putt, W., Kenimer, J.G., Barton, C.H., Walsh, F.S. (1989). Alternative splicing of the neural cell adhesion molecule gene generates variant extracellular domain structure in skeletal muscle and brain. Genes Dev. 3: 348–357
von Heijne, G. (1986). A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 14: 4683–4690
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Hemperly, J.J., DeGuglielmo, J.K. & Reid, R.A. Characterization of cDNA clones defining variant forms of human neural cell adhesion molecule N-CAM. J Mol Neurosci 2, 71–78 (1990). https://doi.org/10.1007/BF02876913
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DOI: https://doi.org/10.1007/BF02876913