Retrotransposons are mobile genetic elements that transpose by reverse transcription of element RNA, followed by insertion of the cDNA into new positions of the host genome. Although they are major constituents of eukaryotic genomes, many facets of their biology remain to be understood. Transposition is generally rare, suggesting that it is subject to tight regulation. However, only the first regulatory step (transcriptional induction) is currently amenable to investigation in higher eukaryotes. To investigate the complete life cycle of a long terminal repeat (LTR) retrotransposon in plants, we established a synthetic biology program on tobacco retrotransposon Tto1, and achieved transposition in whole plants triggered by an inducible promoter. The engineered element, iTto (inducible Tto1), is a novel tool for analysis of retrotransposition in plants. In addition, it allows to explore the potential of an inducible retrotransposon for insertional mutagenesis.Electronic supplementary materialThe online version of this article (doi:10.1007/s11693-010-9053-4) contains supplementary material, which is available to authorized users.
Ty1/copia group retrotransposon Tto1 from tobacco was put under control of an inducible promoter for expression in Arabidopsis thaliana. The system was used to analyze intermediates of the transposition process. The Tto1 RNA 5' region has a complex structure and contains several AUG codons. We therefore sought to experimentally define the translation initiation site. Constructs starting at various positions within the structural gag region were expressed in planta and functionally characterized. We found that gag proteins starting at the first ATG of the gag-pol ORF (ATG1), but also those starting at the third ATG of the gag-pol ORF (ATG3), can form virus-like particles (VLPs). However, gag protein expressed by the inducible Tto1 element had a size similar to gag starting at ATG1, and mutation of ATG1 in the inducible element abolished reverse transcription. This suggested that translation initiation at ATG1 is essential for the Tto1 life cycle. To support this conjecture, gag protein starting at ATG1, or gag protein shortened amino-terminally by nine amino acids (starting at the second ATG of the gag region, ATG2), was co-expressed with Tto1 carrying mutations at ATG1 and ATG2. Trans-complementation of the defective Tto element by gag starting at ATG1, but not by gag starting at ATG2, defines ATG1 as the functional translation initiation site.
Improvements to massively parallel sequencing have allowed the routine recovery of natural and induced sequence variants. A broad range of biological disciplines have benefited from this, ranging from plant breeding to cancer research. The need for high sequence coverage to accurately recover single nucleotide variants and small insertions and deletions limits the applicability of whole genome approaches. This is especially true in organisms with a large genome size or for applications requiring the screening of thousands of individuals, such as the reverse-genetic technique known as TILLING. Using PCR to target and sequence chosen genomic regions provides an attractive alternative as the vast reduction in interrogated bases means that sample size can be dramatically increased through amplicon multiplexing and multi-dimensional sample pooling while maintaining suitable coverage for recovery of small mutations. Direct sequencing of PCR products is limited, however, due to limitations in read lengths of many next generation sequencers. In the present study we show the optimization and use of ultrasonication for the simultaneous fragmentation of multiplexed PCR amplicons for TILLING highly pooled samples. Sequencing performance was evaluated in a total of 32 pooled PCR products produced from 4096 chemically mutagenized Hordeum vulgare DNAs pooled in three dimensions. Evaluation of read coverage and base quality across amplicons suggests this approach is suitable for high-throughput TILLING and other applications employing highly pooled complex sampling schemes. Induced mutations previously identified in a traditional TILLING screen were recovered in this dataset further supporting the efficacy of the approach.
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