Insulators are a new class of genetic elements that attenuate enhancer function directionally. Previously, we characterized in sea urchin a 265-bp-long insulator, termed sns. To test insulator activity following stable integration in human cells, we placed sns between the CMV enhancer and a tk promoter up-stream of a GFP transgene of plasmid or retroviral vectors. In contrast to controls, cells transfected or transduced with insulated constructs displayed a barely detectable fluorescence. Southern blot and PCR ruled out vector rearrangement following integration into host DNA; RNase protection confirmed the enhancer blocking activity. Finally, we demonstrate that two cis-acting sequences, previously characterized in sea urchin, are also specific binding sites for human proteins. We conclude that sns interferes with enhancer promoter interaction also in a human chromatin context. The relatively small size, evolutionary conservation and apparent lack of enhancer specificity might result useful in gene transfer experiments in human cells. © 2001 Academic Press.

Di Simone, P., Di Leonardo, A., Costanzo, G., Melfi, R., Spinelli, G. (2001). The sea urchin sns insulator blocks CMV enhancer following integration in human cells. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 284(4), 987-992 [10.1006/bbrc.2001.5082].

The sea urchin sns insulator blocks CMV enhancer following integration in human cells

DI LEONARDO, Aldo;MELFI, Raffaella;SPINELLI, Giovanni
2001-01-01

Abstract

Insulators are a new class of genetic elements that attenuate enhancer function directionally. Previously, we characterized in sea urchin a 265-bp-long insulator, termed sns. To test insulator activity following stable integration in human cells, we placed sns between the CMV enhancer and a tk promoter up-stream of a GFP transgene of plasmid or retroviral vectors. In contrast to controls, cells transfected or transduced with insulated constructs displayed a barely detectable fluorescence. Southern blot and PCR ruled out vector rearrangement following integration into host DNA; RNase protection confirmed the enhancer blocking activity. Finally, we demonstrate that two cis-acting sequences, previously characterized in sea urchin, are also specific binding sites for human proteins. We conclude that sns interferes with enhancer promoter interaction also in a human chromatin context. The relatively small size, evolutionary conservation and apparent lack of enhancer specificity might result useful in gene transfer experiments in human cells. © 2001 Academic Press.
Settore BIO/11 - Biologia Molecolare
Settore BIO/18 - Genetica
Di Simone, P., Di Leonardo, A., Costanzo, G., Melfi, R., Spinelli, G. (2001). The sea urchin sns insulator blocks CMV enhancer following integration in human cells. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 284(4), 987-992 [10.1006/bbrc.2001.5082].
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10447/198559
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