| Transgenes are common tools for studying gene expression and regulation in plants. However, little is known about the role chromatin organization plays in transgene expression. To address the question of how chromatin-organizing elements affect transgene expression, I studied the Matrix Attachment Regions (MARs) and introns from the developmentally regulated tomato Heat Shock Cognate 80 gene (HSC80). HSC80 is highly expressed in shoot and root apices (Koning, et al., 1992, Plant Physiol. 100: 801--811) and expression of HSC80 transgenes in Arabidopsis requires the 5' MAR and promoter, the introns, and the 3' MAR and downstream region (Chinn et al., 1996, Plant Mol. Biol. 32: 959--968). Several pieces of evidence suggest that chromatin organization is important in the regulation of HSC80: the promoter is sufficient to drive expression in a transient assay; the introns and downstream elements are necessary only for stable transgene expression; and expression from stably integrated transgenes is often variegated (Chinn et al., 1996, Plant Mol. Biol. 32: 959--968).;Two hypotheses about HSC80 regulation were tested here. First, do the HSC80 MARs and introns function in a generalized way and affect heterologous transgene expression? Second, is HSC80 transgene expression dependent upon DNA replication?;To test these hypotheses, a novel reporter gene system intended to generate visible gene expression was developed for Arabidopsis. The maize Lc gene has been shown to increase anthocyanin levels and trichome numbers in Arabidopsis thaliana (Lloyd et al., 1992, Science 258: 1773--1775). However, the high levels of Lc conferred by the CaMV35S promoter were toxic to the plants and subsequently the Arabidopsis adenine phosphoribosyltransferase (APT) promoter (Moffatt et al., 1994, Gene 143: 211--216) was used to confer lower levels of Lc expression. Unexpectedly, in most transgenic plants, Lc expression levels were too low for visible expression and subsequently Lc mRNA levels were analyzed by RT-PCR.;Analysis of the transgenic Arabidopsis showed that the HSC80 MARs and introns had no effect on heterologous transgene expression, while the yeast 'ARS1' MAR repressed transgene expression. In addition, two DNA replication modifiers, kinetin and hydroxyurea, affected HSC80 transgene expression, suggesting a role for DNA replication in HSC80 expression. |
