A link between DNA methylation and epigenetic silencing in transgenic Volvox carteri

Epigenetic silencing of foreign genes introduced into plants poses an unsol ved problem for transgenic technology. Here we have used the simple multice llular green alga Volvox carteri as a model to analyse the relation of DNA methylation to transgenic silencing. Volvox DNA contains on average 1.1% Ei -methylcytosine and 0.3% N6-methyladenine, as revealed by electrospray mass spectrometry and phosphoimaging of chromatographically separated P-32-labe lled nucleotides. In two nuclear transformants of V.carteri, produced in 19 93 by biolistic bombardment with a foreign arylsulphatase gene (C-ars), the transgene is still expressed in one (Hill 181), but not in the other (Hill 183), after an estimated 500-1000 generations. Each transformant clone con tains multiple intact copies of C-ars, most of them integrated into the gen ome as tandem repeats. When the bisulphite genomic sequencing protocol was applied to examine two select regions of transgenic C-ars, we found that th e inactivated copies (Hill 183) exhibited a high-level methylation (40%) of CpG dinucleotides, whereas the active copies (Hill 181) displayed low-leve l (7%) CpG methylation. These are average values from 40 PCR clones sequenc ed from each DNA strand in the two portions of C-ars. The observed correlat ion of CpG methylation and transgene inactivation in a green alga will be d iscussed in the light of transcriptional silencing.