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.
Chen
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A
Division of Basic Research, Children's Hospital Research Foundation, Cincinnati, OH 45229.
Reyes
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A
Akeson
R
R
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