Mol Gen Genet (2992) 235:389-396 © Springer-Verlag 1992 Effect of T-DNA configuration on transgene expression Peter Breyne, Godelieve Gheysen, Anni Jacobs, Marc Van Montagu, and Ann Depicker Laboratorium voor Genetica, UniversiteitGent, K.L. Ledeganckstraat35, B-9000 Gent, Belgium Received May 13, 1992 / AcceptedJune 12, 1992 Summary. T-DNA vectors were constructed which carry a /~-glucuronidase (gusA) gene fused to the promoter of the nopaline synthase (nos) gene and the 3' end of the octopine synthase (ocs) gene. This reporter gene was cloned at different locations and orientations towards the right T-DNA border. For each construct, between 30 and 60 stably transformed calli were analysed for /?-glucuronidase activity. Depending on the T-DNA con- figuration, distinct populations of gusA-expressing calli were obtained. Placing the reporter gene in the middle of the T-DNA results in relatively low expression levels and a limited inter-transformant variability. Placing the gene with its promoter next to the right border led to an increase in both the mean activity and the variability level. With this construct, some of the calli expressed the gusA gene at levels four to five times higher than the mean. In all these series, at least 30% of the calli contained reporter gene activities that were less than half of the mean expression level. Separating the gusA gene from the right T-DNA border by an additional 3'-untranslated region, derived from the nos gene, re- sulted in an increase in the mean expression to a level almost four times higher than that of constructions car- rying the reporter gene in the middle of the T-DNA. Moreover, the number of transformants with extremely low activities decreased by at least 50% and this resulted in significantly lower inter-transformant variability inde- pendently of the orientation of the reporter gene on the T-DNA. Key words: Agrobacterium tumefaciens - Inter-transfor- mant variability - Nicotiana tabacum - Position effect - Transcription interference Introduction Agrobacterium-mediated plant cell transformation usu- ally results in the stable integration of one or a few Correspondence to : M. Van Montagu copies of a well-defined T-DNA region (reviewed in Gheysen et al. 1989). Most often, the expression pattern of the transgenes that are in the plant genome is qualita- tively correct (e.g. Okamuro et al. 1986; Bogusz et al. 1990). However, the quantitative expression levels can vary widely between independent transformants (Dean et al. 1988; Gendloff et al. 1990; Peach and Velten 1991) and there is usually no positive correlation between the T-DNA copy number and the expression level of the transgenes, when limited numbers of individual transfor- mants are compared (e.g. Odell et al. 1987; Shirsat et al. 1989; Gendloff et al. 1990; Hobbs et al. 1990). Inter-transformant variability may be caused by sev- eral molecular and genetic phenomena: the T-DNA that enters the plant nucleus upon transformation lacks a predetermined chromatin structure and DNA modifica- tion pattern; random methylation (reviewed by Selker 1990), mutation, rearrangements (Gheysen et al. 1990) or interactions with host cell factors, occurring prior to, during or after integration might greatly influence the expression of the transgene, independently of the site of insertion (discussed in Peach and Velten 1991). On the other hand, however, inter-transformant vari- ability is explained in many cases by position effects: transgene expression will be variably affected by the structural and functional properties of the chromatin region flanking the T-DNA integration site. Firstly, the local chromatin structure and modification pattern of the flanking DNA can determine the state of the inte- grated T-DNA (e.g. Coates et al. 1987; Peerbolte et al. 1986). Secondly, the higher-order chromatin structure of the region surrounding the integration site might in- fluence gene expression. Since eukaryotic DNA seems to be organized in looped domains, which function as independent genomic units (Eissenberg and Elgin 1991), the specific loop in which the T-DNA has inserted may partially determine the DNA structure and the overall expression level of the present genes. Thirdly, transgene expression can be influenced directly by neighbouring plant regulatory sequences; it is thought that the T- DNA preferentially integrates in transcribed DNA re-