On the stability of sequences inserted into viral genomes

Willemsen, Anouk; Zwart, Mark P

Several studies in the 1980s already reported the possibility of inserting foreign DNA into specific sites of the cauliflower mosaic virus (CaMV) genome without greatly affecting viral infectivity or function (Gronenborn et al. 1981; Howell, Walker, and Walden 1981; Dixon, Koenig, and Hohn 1983; Brisson et al. 1984; Lefebvre, Miki, and Laliberté 1987) . In two of these studies, functional bacterial genes were introduced into the CaMV genome, where a fragment of the lac operator (Gronenborn et al. 1981 ) and the DHFR gene (Brisson et al. 1984) were successfully expressed. In these studies, issues regarding the stability of the insert were raised, where the lac operator was lost after five successive transfers and extended growth of the plants, and deletions in the DHFR gene started appearing after the second and third transfers. On the contrary, an inserted mammalian metallothionein gene appeared to be stable and functional in the CaMV genome (Lefebvre, Miki, and Laliberté 1987) . These studies suggest that the differences in stability of inserts in the CaMV genome depend on at least two factors. First, the site of the insert seems to be important as many inserts are lethal for the virus (Gronenborn et al. 1981; Howell, Walker, and Walden 1981; Dixon, Koenig, and Hohn 1983) . Second, the size of the insert is important, as CaMV can accept only small foreign genes due to viral encapsidation limits (Gronenborn et al. 1981; Lefebvre, Miki, and Laliberté 1987) .