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The expression of the N-type voltage-gated calcium channel α1B gene is restricted to neurons by a 5′- upstream region (-3992 to -1788) that contains negative regulatory element(s) that are active in nonneuronal cells. A 39 bp DNA element, which is repeated nine times in a head-to-tail fashion, was found within the same region. To examine whether this direct repeat (DR) may function as a negatively acting cisregulatory element, several fusion plasmids, DR- 110α1BLUC (1X), DR-SV40LUC (1X, 2X), in which one or two copies of the DR fragment were subcloned upstream of the homologous and heterologous promoters, were transiently transfected into HeLa and NS20Y cells. The promoter activity of DR-110α1BLUC (1X) decreased to approximately 17% of the 110α1BLUC construct in HeLa cells. The expression of the DRSV40LUC (1X) and DR-SV40LUC (2X) plasmids was also reduced to 50 to 23% of the levels that were observed in the pGL2-Promoter in the same cells. However, no repression of the DR constructs was observed in NS20Y cells. An electrophoretic mobility shift assay showed that two DR-specific complexes were detected in HeLa cells, but not in NS20Y cells. In addition, Southwestern blotting revealed the presence of approximately 33 and 43 kDa proteins in HeLa cells. Overall, these results suggest that a 39 bp DNA element might act as repressor in non-neuron cells through the specific interactions of the DNA-proteins.