Elsevier

Gene

Volume 128, Issue 2, 30 June 1993, Pages 211-218
Gene

A homopurine:homopyrimidine sequence derived from the rat neuronal cell adhesion molecule-encoding gene alters expression in transient transfections

https://doi.org/10.1016/0378-1119(93)90565-KGet rights and content

Abstract

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 fibro blast 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 H41 IE 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.

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    Dedicated to the memory of Dr. R. Akeson.

    ∗∗

    Present address: CoCensys Inc., 213 Technology Drive, Irvine, CA 92718, USA,

    Deceased.

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