Rice Genetics IV - IRRI books - International Rice Research Institute

eflect different causes for silencing. For example, the presence of a multicopy locusmay trigger silencing at relatively distant loci such as the super-silencer loci describedby Matzke et al (1994) and Vaucheret (1993), whereas the apparently potently silencedlocus (both uidA and bar are inactive) in JDV95 may have only local (cis)effects. It is noteworthy that the int-uidA gene was preferentially silenced in theseexperiments and it is tempting to attribute this to its being driven by the 35S promoter,which has been implicated in many cases of transgene silencing (Iyer et al 2000).Interestingly, a reactivation of 35S/int-uidA was observed in JDV95 × JDV88.The reactivation of the silenced 35S/int-uidA in JDV88 in the presence of (or by) thetransgene locus in JDV95 is unique in that interaction between two silenced loci resultedin reactivation of the silenced gene. This observation implies that the interactionamong the silenced loci is dependent on distance because the crosses betweenJDV95 and other functional loci (JDV85, JDV86) and silenced loci (JDV83, JDV90)did not show any effect on the reporter gene expression. The remote possibility existsthat the activation observed represents complementation of mutations caused bytransgene insertion. Transgene inactivation resulting from interacting loci has previouslybeen shown to be chromosomal-location-dependent (Schmulling and Rohrig1995). Addressing the physical distance between transgene loci is critical in understandinginactivation and reactivation, but remains a challenge because current FISHtechnology does not allow estimation of the native physical distance between twotransgene loci on different chromosomes. We are examining segregating hybrid linesto determine whether the modification of the functional locus by the multicopytransgene locus is reversible.That transgene silencing is caused by various mechanisms involving modificationsof DNA, RNA, and chromatin structure has now been rigorously established.However, the transgene-silencing phenomena we have observed in rice are complicated,for example, AzaC and TSA treatment reactivated GUS expression in linesJDV90 and JDV92 but not in JDV95. Although multicopy transgene events are mostfrequently silenced (Flavell 1994, Garrick et al 1998, Henikoff 1998, Kumpatla andHall 1999), silencing can occur for a single intact transgene locus (Table 1) and evenfor a single gene flanked by active transgenes at a single locus (JDV88); thus, theremay or may not be epistatic interactions between transgene loci.Alleviating transgene silencingAs silencing of desirable transgenes is a substantial complication in biotechnologicaldevelopment of novel crops, there is considerable interest in avoiding gene silencingor in overcoming its effects. Insight into the mechanisms of both TGS and PTGS israpidly increasing (Cogoni and Macino 1999b, Fagard and Vaucheret 2000, Iyer et al2000) and several mutations (mostly in Arabidopsis) have now been shown to affectgene silencing. Several groups, including our own (Kumpatla et al 1998), are exploringstrategies for evading silencing by introducing sequence heterogeneity into theRCg2 promoter. The suppression of methylation systems and the reduction of opportunitiesfor heterochromatinization, for example, by the insertion of enhancer ele-Gene silencing and its reactivation in transgenic rice 475