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Isolation of mouse N-CAM-related cDNA: detection and cloning using monoclonal antibodies
Abstract:
Clones coding for the mouse neural cell adhesion molecule (N-CAM) were isolated from a cDNA library prepared in the expression vector lambda gt 11 from mRNA extracted from a mouse neuroblastoma cell line. This library was screened with two anti-N-CAM monoclonal antibodies directed against different sites on the molecule and with rabbit anti-N-CAM serum. Two clones were identified with the first monoclonal antibody, three with the second one, none reacted with both. The relevance of these cDNA clones to N-CAM was confirmed by several observations. First, cDNA sequences detected with one monoclonal antibody cross-hybridized with those identified by the other antibody. Second, the different fusion proteins all bound the rabbit serum in addition to one monoclonal antibody. Finally, the probes hybridized to discrete mRNA species of sufficient lengths to code for the very large N-CAM polypeptides in RNA preparations from N-CAM-expressing, but not from N-CAM-negative cells. An additional mRNA species not seen in embryonic brain was expressed in adult mouse brain. Genomic blot experiments indicated that sequences corresponding to one of our probes are present only a few times in the mouse genome.
Insights
Researchers isolated mouse neural cell adhesion molecule (N-CAM) cDNA clones using monoclonal antibodies and rabbit serum. These clones confirm N-CAM gene expression in specific cells and tissues, revealing novel mRNA patterns in adult mouse brain.
Area of Science:
- Neuroscience
- Molecular Biology
- Genetics
Background:
- Neural cell adhesion molecule (N-CAM) plays a crucial role in neuronal development and function.
- Understanding N-CAM gene expression is vital for deciphering neural circuit formation and plasticity.
Purpose of the Study:
- To isolate and characterize cDNA clones encoding mouse N-CAM.
- To confirm the relevance of these clones to N-CAM expression.
- To investigate N-CAM mRNA expression patterns in different developmental stages and cell types.
Main Methods:
- Construction of a lambda gt 11 cDNA expression library from mouse neuroblastoma mRNA.
- Screening the library using monoclonal antibodies against N-CAM and rabbit anti-N-CAM serum.
- Cross-hybridization, fusion protein binding assays, and Northern blot analysis to validate cDNA clones and analyze mRNA expression.
- Genomic Southern blot analysis to assess gene copy number.
Main Results:
- Isolation of multiple cDNA clones encoding parts of mouse N-CAM.
- Confirmation of clone relevance through antibody binding and cross-hybridization.
- Detection of N-CAM mRNA in N-CAM-expressing cells but not in N-CAM-negative cells.
- Identification of an additional N-CAM mRNA species in adult mouse brain not present in embryonic brain.
- Genomic analysis indicated low copy number for N-CAM sequences.
Conclusions:
- The isolated cDNA clones are relevant to mouse N-CAM.
- N-CAM expression is cell-type specific and developmentally regulated.
- Novel N-CAM mRNA transcripts are present in the adult mouse brain, suggesting complex regulation or isoforms.