Abstract
The combined factors that regulate the expression of cell adhesion molecules (CAMs) during development of the nervous system are largely unknown. To identify such factors for Ng-CAM, the neuron-glia CAM, constructs containing portions of the 5' end of the Ng-CAM gene were examined for activity after transfection into N2A neuroblastoma and NIH3T3 cells. Positive regulatory elements active in both cell types included an Ng-CAM proximal promoter with SP1 and cAMP response element motifs extending 447 base pairs upstream of a single RNA start site and a region within the first exon corresponding to 5'-untranslated sequences. Negative regulatory elements included five neuron-restrictive silencer elements (NRSEs) and a binding site for Pax gene products in a 305-base pair segment of the first intron. Constructs containing the promoter together with the entire first intron were active in N2A cells but were silenced in NIH3T3 cells. This silencer activity was mapped to the NRSEs. In contrast, the Pax motif inhibited activity of Ng-CAM constructs in both cell types. The DNA elements defined in these transfection experiments were examined for their ability to bind nuclear factors. The region within the first exon formed a DNA-protein complex after exposure to nuclear extracts prepared from both NIH3T3 and N2A cells. The NRSE region formed a more prominent complex with proteins prepared from NIH3T3 cells than it did with extracts from N2A cells. A member of the Pax protein family, Pax-3 bound to the Pax motif. Mutations introduced within the Pax motif in its ATTA sequence eliminated this binding whereas mutations in its GTTCC sequence did not, suggesting that paired homeodomain interactions are important for the recognition of Pax-3 by this DNA target sequence. The combined data suggest that negative regulation by NRSEs and Pax proteins may play a key role in the place-dependent expression patterns of Ng-CAM during development.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U31086
Structure of the 5Ј End of the Ng-cell adhesion molecules (CAMs) Gene: Identification of the Proximal Promoter and Regulatory Sequences in the First Intron—A cosmid clone was isolated after screening with a 32P-labeled 400-base pair EcoRI-BstXI fragment derived from the 5Ј end of the Ng-CAM cDNA sequence [3]
The borders for the first seven exons and introns were located by restriction mapping and Southern blotting and their sequences were determined using oligonucleotide primers derived from the Ng-CAM cDNA sequence (Fig. 1)
Summary
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U31086. Ng-CAM, L1, NrCAM, neurofascin/ABGP from vertebrates and neuroglian from Drosophila [14] comprise a subfamily of neural CAMs containing six immunoglobulin domains and five fibronectin type III repeats Molecules of this Ng-CAM subfamily are expressed prominently in axonal pathways in both the central nervous system and peripheral nervous system and are involved in neurite fasciculation and outgrowth. One region of the first intron was found to contain five neuron-restrictive silencer elements (NRSEs) which extinguished expression of the Ng-CAM gene in a fibroblast cell line, but not a neuroepithelial cell line Another region contained GTTCC and ATTA sequences characteristic of binding sites for Pax proteins. Our studies suggest that the NRSEs and Pax motif may play critical roles in the place-dependent expression of Ng-CAM in the nervous system
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