Plants have selleck chem Sorafenib a more complex and sophisti cated gene silencing apparatus than animals do and make use of cytosine methylation at multiple sites in combination with histone modifications and harbor a vast variety of small RNAs. Plants even have a signal transmission pathway for small RNAs, which can act as mobile signals to direct RdDM systemically. The very active systemic spreading of the silencing signal through the phloem was first observed in the solan aceous plants, tobacco and tomato and later demonstrated also for Arabidopsis. From our initial 113 independent sense expression N. attenuata lines we omitted 43% after three generations due to indications of gene silencing. N.
attenuata has a highly sophisticated suite of defenses against herbivores and it might be, that this plant also has an active methylation apparatus to protect its genome against genetic manipulations, which Inhibitors,Modulators,Libraries Michael Wassenegger once aptly called a gene silencing based resistance against transgene overexpression. Factors influencing transgene silencing the gene dosage effect Factors which have often been shown to increase the probability of transgene silencing are the transgene copy number and the strength of expression. In addition, T DNA rearrangements, read through tran scripts or improperly terminated or non polyadenylated mRNA are also associated with transgene silencing. Certainly position effects and integration into heterochromatin have been frequently reported in asso Inhibitors,Modulators,Libraries ciation with local gene silencing, but integrations into euchromatin can be similarly silenced and more re cent studies suggest that overall, the insertion position plays only a minor role.
Strong viral promoters, such as the 35S cauliflower mosaic virus promoter, were thought to produce aberrant RNA after exceeding a certain threshold of expression. The progressive methylation of the Inhibitors,Modulators,Libraries 35S promoter and the observed downregulation of the transgene in the lines ICE 4. 4, PNA 1. 2 and PNA 10. 1 might be mainly induced by the presence of two T DNA copies in close proximity to each other. These complex insertions at a sin gle locus can trigger transgene silencing as shown in earlier studies. Any form of repeated T DNA ar rangement appears to increase the overall silencing probability. But despite the intensity, the methy Inhibitors,Modulators,Libraries lation increase occurred relatively late in these lines and the loss of the resistance marker was not revealed until the T3 generation.
We hypothesize that Inhibitors,Modulators,Libraries the expression of the two T DNA copies remains below a threshold level when plants are hemizygous. Once Ganetespib OSA homozygous in the T2, these thresholds are exceeded and the sum of the four T DNA copies likely initiate the silencing process. This scenario would explain why also all hemizygous T2 sibling plants of these lines were in conspicuous and showed no abnormal segregation.