Gene silencing
Frequently, expression of transgenes becomes suppressed in transgenic plants after they have been down for one or
more generations, this is called gene silencing. Gene silencing is often accompanied by DNA methylation of
transgenes; usually methylation of C occurs in the symmetrical CG and CNG (N=any one of the four DNA bases) sequences, but sometimes asymmetrical sequences are also methylated. In general, higher the number of copies of a transgene, more frequent is their hypermethylation and transgene inactivation, Gene silencing mechanisms are not well understood, but they can be of two types: (1) transcriptional silencing and (i) post-transcriptional silencing.
Transcriptional silencing is generally due to promoter methylation, while post-transcriptional silencing can involve
methylation of the coding sequence. The latter does not directly affect transcription. Gene silencing occurs most likely due to RNA transcript degradation.
Transcriptional Silencing - It denotes a suppression of transcription of the transgenes, and is the result of methylation of the promoter region of transgenes This is the most frequent when multiple copies of a transgene are integrated either as a concatamer or joined head-to-tail, at the same locus or at different sites in the same genome. It is postulated that some mechanism detects homology between different chromosomal regions, and when multiple homologous sequences are detected these sequences are subjected to hypermethylation and gene silencing.
Another mechanism of transgene silencing is due to integration of the transgene into a hypermethylated chromosomal region. The hypermethylation pattern of the chromosomal region may spread into the transgene either reducing its transcription or producing variable expression patterns similar to the variegation position effects so well documented in Drosophila. Thus the site of transgene integration may markedly affect its expression level mainly by
hypermethylation and chromatin condensation (due to heterochromatin proximity), which influences the general
availability of transgenes general for transcription.
In case of certain transgenes primary transformants (plants obtained directly from transformed cells) show a
satisfactory and stable expression of the transgene. However, the transgene becomes methylated and thereby inactivated in the subsequent generations. This effect is more pronounced when the transgenic plants are exposed to environmental stress. In tissue cultures, environmental stress is mimicked by the addition of propionic or butyric
acid; this induces hypermethylation in potentially unable transgenes. This type of de nove methylation is usually
confined to transgenes. For example a maize (monocot) gene encoding dihydroflavonol-4-reductase became
methylated when transferred into petunia (dicot),, while the same gene from gerbera (dicot) was not affected. This problem can be overcome by suitably changing the base sequences of transgenes so as to enhance codon usage in eliminate the mRNA function/stability affecting sequences, and to obtain isochore compositions similer to those of the host plant species without influencing amino acid sequences of the encoded proteins. Post-Transcriptional Silencing- The proposed mechanisms of post-transcriptional silencing are the same as those described for the phenomenon of co suppression.
Gene silencing is mediated by double-stranded RNA, this phenomenon is a multistep process. The dsRNA is
processed by Dicer (an RNase III enzyme) into short molecules; in case of plans, these molecules are either short
(21-22 nucleotides, nt) or long (24-26 nt). The short siRNAs most likely lead to mRNA degradation In contrast, the
long RNAs seem to be involved in RNA directed methylation of homologous DNA that spreads to the promoter
DNA as well Both these effects are highly sequence specific and affect only homologous RNA/DNA sequences.
Amelioration of Gene Silencing, Stable transgene expression can be obtained by screening a large number of
transformants and by a selection for stable expression in a breeding programme based on transgenic plants.