Characterising transgene silencing in single-copy Arabidopsis thaliana lines: DNA methylation in view of genomic and transcriptional context
Year: 2004
Type: dissertation
Abstract: A total of nine single-copy transgenic Arabidopsis thaliana lines displaying varying degrees of silencing of a nos promoter-nptII marker gene (encoding kanamycin-resistance) have been studied, with emphasis on two of these, line P4 and P10. Both of these lines show a very high frequency of nptII-silencing in one subline, but little or no silencing in other sublines, indicating epigenetic mechanisms. \nOne candidate is DNA methylation, which has been associated both with silencing on a transcriptional level (TGS) and with post-transcriptional gene silencing (PTGS). Using the bisulphite-mediated genomic DNA sequencing method, the methylation status of all cytosine positions in a sequence consisting of the nos promoter and the 5’-end of the nptII gene were investigated for both lines. The results show a very high degree of methylation for the silenced sublines, also in asymmetric cytosines, and a significantly lower degree of methylation in the sublines with little or no silencing. When comparing the promoter and transcribed regions, a preference for methylation of the former is observed, indicating that the nptII gene is subject to TGS.\nMany models of silencing have evoked the presence of aberrant transcripts. Using RT-PCR, sense transcripts covering the promoter were detected for both of the investigated lines. Counter to what was expected, however, these RNAs were detected in sublines showing little or no silencing, whereas none or very low levels were detected in silenced plants. Promoter analyses and characterisation of the sequences flanking the nptII construct indicate that these transcripts may originate in cryptic promoter elements or as an extension of a recently reported endogenous transcript. \nBased on these results and recent models of RNA-directed DNA methylation, a scenario is presented where the detected promoter transcripts are targeted and processed in the silenced sublines by components involved in silencing, resulting in sequence-specific methylation of the corresponding DNA.
Author: Morten Christoph Eike
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2253486976