A homopurine:homopyrimidine sequence derived from the rat neuronal cell adhesion molecule-encoding gene alters expression in transient transfections.
A 178-bp homopurine-homopyrimidine (R:Y) sequence is located upstream from the transcription start point (tsp) of the rat neuronal cell adhesion molecule-encoding gene (NCAM). This R:Y sequence contains several mirror repeats. Such sequences have been proposed to regulate gene expression. To determine its effect on gene transcription, a DNA fragment containing the R:Y sequence was cloned into a luciferase-encoding (luc) expression vector. Transient transfection assays with the R:Y-luc constructs were performed in cell lines which constitutively express (B104 rat neuronal cells and C6 rat glial cells) or lack (H411E rat liver cells and L mouse fibroblast cells) NCAMs. In its natural orientation, the R:Y sequence caused a 2.5-fold reduction in luc expression in B104 and H411E cells, but had a statistically insignificant effect in C6 and L cells. The magnitude of the R:Y sequence reduction in luc expression was position and orientation dependent (varying from 2- to 5.5-fold). To determine if nuclear protein(s) specifically bind the sequence, gel retardation assays of a DNA fragment containing the R:Y sequence were carried out with nuclear extracts from these four cell lines. Specific DNA-protein interaction was found with B104 and H411E nuclear extracts, but not with C6 and L cell nuclear extracts. Competition experiments indicate that the (AGG):(TCC) repeat segment within the rat R:Y sequence may constitute the protein-binding domain. These results indicate that the R:Y sequence may have a negative effect on gene transcription in certain cell lines. In correlation with this negative effect, these same cell lines also contain nuclear proteins which bind the sequence.
Division of Basic Research, Children's Hospital Research Foundation, Cincinnati, OH 45229.
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Chen
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Reyes
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Akeson
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D015816:Cell Adhesion Molecules, Neuronal / Q000235:genetics*; D002460:Cell Line; D001517:Coleoptera / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid; D014158:Transcription, Genetic; D014162:Transfection
D015816:Cell Adhesion Molecules, Neuronal; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D008156:Luciferases