ABSTRACT Tn5 was one of the first transposons to be identified ( 10 ). As a result of Tn5's early discovery and its simple macromolecular requirements for transposition, the Tn5 system has been a very productive tool for studying the molecular mechanism of DNA transposition. These studies are of broad value because they offer insights into DNA transposition in general, because DNA transposition is a useful model with which to understand other types of protein-DNA interactions such as retroviral DNA integration and the DNA cleavage events involved in immunoglobulin gene formation, and because Tn5-derived tools are useful adjuncts in genetic experimentation.
- SourceAvailable from: Francisco J. López de Saro
[Show abstract] [Hide abstract]
- "Although asymmetries derived from the first two have been analyzed extensively, only a few transpososomes have been studied in structural detail (reviewed in Dyda et al. 2012). Although transpososomes consist of homomultimeric transposases, major conformational and functional asymmetries (e.g., sequential cleaving of DNA ends) have been found, for example, in the transposition pathways of Tn5 (Reznikoff 2008), Mu (Montaño et al. 2012), IS91 (Garcillán-Barcia et al. 2001), IS3 (Sekine et al. 1999), or IS200 (Ronning et al. 2005). In all cases, if β binds preferentially to one of the transposases, then an orientation bias during insertion on DNA could be the result (fig. "
ABSTRACT: Insertion Sequences (ISs) are small transposable elements widespread in bacterial genomes, where they play an essential role in chromosome evolution by stimulating recombination and genetic flow. Despite their ubiquity, it is unclear how ISs interact with the host. Here we report a survey of the orientation patterns of ISs in bacterial chromosomes with the objective of gaining insight into the interplay between ISs and host chromosomal functions. We find that a significant fraction of IS families present a consistent and family-specific orientation bias with respect to chromosomal DNA replication, especially in Firmicutes. Additionally, we find that the transposases of up to 9 different IS families with different transposition pathways interact with the β sliding clamp, an essential replication factor, suggesting that this is a widespread mechanism of interaction with the host. While we find evidence that the interaction with the β sliding clamp is common to all bacterial phyla, it also could explain the observed strong orientation bias found in Firmicutes, since in this group β is asymmetrically distributed during synthesis of the leading or lagging strands. Besides the interaction with the β sliding clamp, other asymmetries also play a role in the biased orientation of some IS families. The utilization of the highly conserved replication sliding clamps suggests a mechanism for host regulation of IS proliferation and also a universal platform for IS dispersal and transmission within bacterial populations and among phyllogenetically distant species.Genome Biology and Evolution 03/2014; 6(3). DOI:10.1093/gbe/evu052 · 4.23 Impact Factor
[Show abstract] [Hide abstract]
- "It is an effective in vivo mutagen and has numerous post-genomic applications such as the delivery of sequence bar codes and deep-sequencing primers. Hyperactivity depends on three point mutations, which inactivate the transposon’s natural autoregulatory mechanisms (11,12). Although two of the mutations were selected from random libraries, M56A was informed by prior knowledge of autoregulation. "
ABSTRACT: New applications for transposons in vertebrate genetics have spurred efforts to develop hyperactive variants. Typically, a genetic screen is used to identify several hyperactive point mutations, which are then incorporated in a single transposase gene. However, the mechanisms responsible for the increased activity are unknown. Here we show that several point mutations in the mariner transposase increase their activities by disrupting the allostery that normally serves to downregulate transposition by slowing synapsis of the transposon ends. We focused on the conserved WVPHEL amino acid motif, which forms part of the mariner transposase dimer interface. We generated almost all possible single substitutions of the W, V, E and L residues and found that the majority are hyperactive. Biochemical analysis of the mutations revealed that they disrupt signals that pass between opposite sides of the developing transpososome in response to transposon end binding. In addition to their role in allostery, the signals control the initiation of catalysis, thereby preventing non-productive double-strand breaks. Finally, we note that such breaks may explain the puzzling 'self-inflicted wounds' at the ends of the Mos1 transposon in Drosophila.Nucleic Acids Research 12/2013; 42(4). DOI:10.1093/nar/gkt1218 · 9.11 Impact Factor
[Show abstract] [Hide abstract]
- "As negative regulation was not required, only the tetA gene was amplified. The kanR gene, amplified from pZErO-2, encodes a aminophosphotransferase, APH(3′)-II isolated from transposon tn5, which confers resistance to kanamycin via enzymatic inactivation [2,16]. "
ABSTRACT: Background The cloning of gene sequences forms the basis for many molecular biological studies. One important step in the cloning process is the isolation of bacterial transformants carrying vector DNA. This involves a vector-encoded selectable marker gene, which in most cases, confers resistance to an antibiotic. However, there are a number of circumstances in which a different selectable marker is required or may be preferable. Such situations can include restrictions to host strain choice, two phase cloning experiments and mutagenesis experiments, issues that result in additional unnecessary cloning steps, in which the DNA needs to be subcloned into a vector with a suitable selectable marker. Results We have used restriction enzyme mediated gene disruption to modify the selectable marker gene of a given vector by cloning a different selectable marker gene into the original marker present in that vector. Cloning a new selectable marker into a pre-existing marker was found to change the selection phenotype conferred by that vector, which we were able to demonstrate using multiple commonly used vectors and multiple resistance markers. This methodology was also successfully applied not only to cloning vectors, but also to expression vectors while keeping the expression characteristics of the vector unaltered. Conclusions Changing the selectable marker of a given vector has a number of advantages and applications. This rapid and efficient method could be used for co-expression of recombinant proteins, optimisation of two phase cloning procedures, as well as multiple genetic manipulations within the same host strain without the need to remove a pre-existing selectable marker in a previously genetically modified strain.BMC Research Notes 03/2013; 6(1):85. DOI:10.1186/1756-0500-6-85