Studies on position effects and gene tagging in transgenic plants.

Transgenic plant technology has greatly facilitated the study of plant gene structure and function. It has, however, become apparent that the position of integration into the genome can influence transgene expression both quantitatively and qualitatively. One aim of the work presented in this thesis was to investigate the effects of a chicken B-globin gene matrix attachment region to insulate against these position effects in transgenic tobacco (Nicotiana tabacum). It was found that, while this sequence mediates position-independent transgene expression in mice, it does not do so in transgenic tobacco. Concurrently, a second aim of this thesis was to identify plant genes that are expressed either constitutively or in the majority of cell types. Such gene loci may contain native plant DNA elements that could confer position-independent transgene expression and would, furthermore, provide additional promoters for genetic engineering. The approach was to introduce a promoterless gusA transgene, situated adjacent to the left border repeat of a T-DNA, to generate plant gene-reporter gene fusions in vivo following transformation of potato (Solanum tuberosum). Expression of the transgene is only expected if the gene has become integrated downstream from an active endogenous plant promoter, thereby tagging it and facilitating its subsequent isolation. A transgenic line was identified exhibiting transgene expression in leaf, stem, root and tuber tissue. Isolation of genomic flanking DNA by inverse PCR identified the site of T-DNA integration as being within a locus containing patatin genes, and analysis of transgene expression reconciled the expression level and pattern to that expected from a class I patatin gene promoter.