The NCAMS (neural cell adhesion molecules) are a group of cell surface transmembrane polypeptides derived from a single gene by alternative splicing. NCAMs have been proposed to mediate a number of processes in neural development through cell-cell adhesion and interaction. Our previous work has identified structural variation in NCAM polypeptides near the region of the molecule believed to be directly involved in adhesion and has shown that some but not all neuronal cells express these alternative NCAM forms. There is also structural variation elsewhere in NCAM. We propose to determine more fully the sequence identify and relative abundances of these multiple NCAM polypeptide forms by using the polymerase chain reaction to amplify the NCAM mRNAs found in brain and other tissues. This technique will both identify preciously undiscovered exons and also determine which of the alternative exons are actually combined to form polypeptides. Changes in NCAM forms during development and preferential combinations of alternative external and cytoplasmic domains will be determined. Complete cDNAs corresponding to major NCAM populations will be constructed and used to transfect neuronal and fibroblast lines. To determine the biologic potentials of these structurally distinct NCAMs, the transfected cell lines will be tested with 4 assays which model developmental events: a short term aggregation assay, longer term """"""""sorting-out"""""""" assay, an axonal growth and """"""""stop"""""""" assay, and as axonal choice assay. By isolating NCAM isoform as the single variable between cell lines, we will thus determine which NCAM forms are capable of mediating short and longer term preferential cell association and preferential axonal elongation or directional choice. Antibodies for these distinct NCAM forms will be produced to determine which individual cell types express them. The DNA sequence elements which restrict NCAM expression to neurons, glial, and muscle cells in adult mammals will be identified. We will determine whether distinct or common DNA promoter and enhancer elements control expression in these cell types. Enhancer elements will be identified by DNAse hypersensitivity assays and """"""""enhancer trap"""""""" techniques. These studies will give insight into neural specific gene expression. Finally, using our previous identification of a heparin binding domain on NCAM, we will determine which amino acid regions are directly involved in cell adhesion. NCAMs altered by in vitro mutagenesis will be transfected into cell lines for testing in the biologic assays listed. These combined experiments will establish the degree of NCAM expression and how at the molecular level NCAM causes cell adhesion. This detailed analysis of the roles of NCAM in neural development will further our understanding of development defects that might be caused by changes in NCAM expression.
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