Universal platform for quantitative analysis of DNA transposition

<p>Abstract</p> <p>Background</p> <p>Completed genome projects have revealed an astonishing diversity of transposable genetic elements, implying the existence of novel element families yet to be discovered from diverse life forms. Concurrently, several better understood transposon systems have been exploited as efficient tools in molecular biology and genomics applications. Characterization of new mobile elements and improvement of the existing transposition technology platforms warrant easy-to-use assays for the quantitative analysis of DNA transposition.</p> <p>Results</p> <p>Here we developed a universal <it>in vivo </it>platform for the analysis of transposition frequency with class II mobile elements, i.e., DNA transposons. For each particular transposon system, cloning of the transposon ends and the cognate transposase gene, in three consecutive steps, generates a multifunctional plasmid, which drives inducible expression of the transposase gene and includes a mobilisable <it>lacZ</it>-containing reporter transposon. The assay scores transposition events as blue microcolonies, papillae, growing within otherwise whitish <it>Escherichia coli </it>colonies on indicator plates. We developed the assay using phage Mu transposition as a test model and validated the platform using various MuA transposase mutants. For further validation and to illustrate universality, we introduced IS<it>903 </it>transposition system components into the assay. The developed assay is adjustable to a desired level of initial transposition via the control of a plasmid-borne <it>E. coli </it>arabinose promoter. In practice, the transposition frequency is modulated by varying the concentration of arabinose or glucose in the growth medium. We show that variable levels of transpositional activity can be analysed, thus enabling straightforward screens for hyper- or hypoactive transposase mutants, regardless of the original wild-type activity level.</p> <p>Conclusions</p> <p>The established universal papillation assay platform should be widely applicable to a variety of mobile elements. It can be used for mechanistic studies to dissect transposition and provides a means to screen or scrutinise transposase mutants and genes encoding host factors. In succession, improved versions of transposition systems should yield better tools for molecular biology and offer versatile genome modification vehicles for many types of studies, including gene therapy and stem cell research.</p>

Abstract licence: CC BY-NC-ND

A person counts once a day per paper. We keep no cookie and no address: a reader is a hash that changes every day. Any month under five reads shows no number.