An insulator element from the sea urchin Hemicentrotus pulcherrimus suppresses variation in transgene expression in cult
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O R I GI N A L P A P E R
S. Nagaya á K. Yoshida á K. Kato á K. Akasaka A. Shinmyo
An insulator element from the sea urchin Hemicentrotus pulcherrimus suppresses variation in transgene expression in cultured tobacco cells Received: 31 July 2000 / Accepted: 8 December 2000 / Published online: 3 March 2001 Ó Springer-Verlag 2001
Abstract Specialized DNA sequences known as insulators protect genes from both the positive and negative in¯uences of nearby chromatin. Many insulators have been identi®ed in various species; however, few function in multiple species. We have shown that an insulator from the Ars (arylsulfatase) gene of the sea urchin Hemicentrotus pulcherrimus functions in plant cells. Normally, expression of an introduced chimeric GUS gene is inactivated in approximately 30% of transformed tobacco BY2 clones. Transgenes containing the Ars insulator, however, were expressed in all transformed tobacco BY2 cells. The insulator did not aect the copy number, the chromosomal position of transgene integration or maximum expression levels. These results suggest that the insulator functions to suppress the variation normally associated with transgene expression in tobacco BY2 cells. Key words Insulator element á Position eects á Tobacco á Transgene
Introduction The technology for introducing foreign genes into plants provides a powerful tool for investigating the function of speci®c genes, and generating useful, genetically modi®ed, plants having the potential to yield products with
Communicated by W. HoÈrz S. Nagaya á K. Yoshida (&) á K. Kato á A. Shinmyo Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan E-mail: [email protected] Tel.: +81-743-725461 Fax: +81-743-725469 K. Akasaka Graduate School of Science, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8526, Japan
industrial or pharmaceutical applications. There are a number of technical limitations that must be overcome before transgenic techniques realize their maximum potential. A major problem is that transgene expression is notoriously variable between dierent, independently generated, transgenic plants. The eukaryotic genome, organized in a complex, heterogeneous structure termed chromatin, is not homogeneous with respect to transcriptional activity. The variation in transgene expression is due to random integration into the chromosome. It is theoretically possible to suppress variation in transgene expression by ensuring that the transgene is protected from the in¯uence of the chromosomal environment nearby. Matrix attachment regions (MARs) are de®ned as DNA elements that bind speci®cally to the nuclear matrix in vitro. According to the loop model (Mirkovitch et al. 1984) of chromatin structure, MARs may organize chromatin loops, which are attached at their bases to the nuclear matrix, thus de®ning domains of independent gene regulation. Variation in the expression level of transgenes inserted between a pair of MARs has been studied previously (reviewed by Holmes-Davis and Coma
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