EPIGENETIC TRANSCRIPTIONAL SILENCING AND 5-AZACYTIDINE-MEDIATED REACTIVATION OF A COMPLEX TRANSGENE IN RICE

Despite a growing number of reports indicating non-Mendelian inheritan ce of transgene expression in monocots, no detailed description of the structure and stability of the transgene exists for transformants gen erated by direct DNA-transfer techniques, making the cause for these o bservations difficult to determine. In this paper we describe the comp lex organization of Btt cryIIIA and bar transgenes in rice (Oryza sati va L.) that displayed aberrant segregation in R-1 progeny. Silencing r ather than rearrangement of the bar gene was implicated because the he rbicide-sensitive R-1 plants had a DNA hybridization profile identical to that of the resistant R-0 parent and R-1 siblings. Genomic DNA ana lysis revealed substantial methylation of the Ubi1/bar sequences in si lenced plants and, to a lesser degree, in herbicide-resistant plants, suggesting that the transgene locus was potentiated for silencing. Nuc lease protection and nuclear run-on assays confirmed that silencing wa s due to transcriptional inactivation. Treatment of R-2 progeny of sil enced plants with 5-azacytidine resulted in demethylation of the Ubi1 promoter and reactivation of bar gene expression, demonstrating a func tional relationship for methylation in gene silencing. These findings indicate that methylation-based silencing may be frequent in cereals t ransformed by direct DNA protocols that insert multiple, often rearran ged sequences.