Tn5 in Vitro Transposition

Abstract

This communication reports the development of an efficient in vitro transposition system for Tn5. A key component of this system was the use of hyperactive mutant transposase. The inactivity of wild type transposase is likely to be related to the low frequency of in vivo transposition. The in vitro experiments demonstrate the following: the only required macromolecules for most of the steps in Tn5 transposition are the transposase, the specific 19-bp Tn5 end sequences, and target DNA; transposase may not be able to self-dissociate from product DNAs; Tn5 transposes by a conservative "cut and paste" mechanism; and Tn5 release from the donor backbone involves precise cleavage of both 3 and 5 strands at the ends of the specific end sequences.Transposition is a process that gives rise to a variety of genomic rearrangements such as insertions, deletions, inversions, and chromosome fusions (1). Because transposition can play a profound role in genome evolution and in a variety of genetic diseases, how this process occurs and how its frequency is determined is of considerable interest. Bacterial transposons, such as Tn5, are convenient tools for studying these two questions.The transposition process can be understood in the context of the following model (2, 3). The first step in transposition involves the sequence-specific binding of the transposon-encoded transposase (Tnp) 1 to the specific end sequences that define the ends of the transposable element. The second step involves formation of a synaptic complex in which the ends of the transposable element are brought together through Tnp oligomerization. In some well studied cases such as Tn10 (4, 5), synaptic complex formation is facilitated by one or more host proteins. The third step involves a Tnp-mediated nucleolytic attack on the phosphodiester bonds adjacent to the ends of the transposable element. The precise nature of these cleavages (whether just the 3Ј ends of the transposon are liberated or both the 3Ј and the 5Ј ends and the exact location of the cleavage in the case of 5Ј end release) is a special property of the system being studied. The fourth step involves Tnp binding to the target DNA sequence (target capture). Different transposons demonstrate various degrees of sequence specificity at this step. The fifth step involves a concerted nucleophilic attack by the transposon 3Ј-OH ends on phosphodiester bonds in both strands of

The BMAL1 C terminus regulates the circadian transcription feedback loop

“…To this end, we generated an expression library of random mutant (Flag-tagged) BMAL1 proteins by using a commercially available Tn5 transposon-based insertion system that introduces 19 aa in-frame (18). In this way, we obtained 30 in-frame protein mutants, named Flag-BMAL1-Bm1 to Flag-BMAL1-Bm30, that were sequenced to determine the position of the 19-aa insertion.…”

Active integration: new strategies for transgenesis

“…There is currently work being done with PB transposase to increase the transpositional efficiencies to that of retroviral vectors. There are methods to accomplish this goal such as PCR random mutagenesis or alanine substitutions utilizing mutagenic PCR primers (Goryshin and Reznikoff 1998;Yant et al 2004;Pledger and Coates 2005;Keravala et al 2006). One of us, Thomas Ryan, is pursuing PB active transgenesis in embryonic stem cells for the production of transgenic animals.…”

Chromosomal Deletion Formation System Based on Tn5 Double Transposition: Use For Making Minimal Genomes and Essential Gene Analysis

“…They have long been used in the generation of knockout mutations. With the advent of in vitro transposition systems (Devine and Boeke 1994;Gwinn et al 1997;Akerley et al 1998;Goryshin and Reznikoff 1998;Griffin IV et al 1999;Haapa et al 1999), the use of transposons in genome analysis has been greatly expanded to include, for instance, their being applied as mobile primer binding sites in high-throughput sequencing efforts Shevchenko et al 2002). In this communication, we will describe another powerful application of DNA transposition that combines in vitro and in vivo Tn5-based technologies to generate random deletions in the Escherichia coli K12 genome.…”