Flexibility in MuA Transposase Family Protein Structures: Functional Mapping with Scanning Mutagenesis and Sequence Alignment of Protein Homologues

Peer reviewe

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

Large-scale data integration framework provides a comprehensive view on glioblastoma multiforme

Background: Coordinated efforts to collect large-scale data sets provide a basis for systems level understanding of complex diseases. In order to translate these fragmented and heterogeneous data sets into knowledge and medical benefits, advanced computational methods for data analysis, integration and visualization are needed.Methods: We introduce a novel data integration framework, Anduril, for translating fragmented large-scale data into testable predictions. The Anduril framework allows rapid integration of heterogeneous data with state-of-the-art computational methods and existing knowledge in bio-databases. Anduril automatically generates thorough summary reports and a website that shows the most relevant features of each gene at a glance, allows sorting of data based on different parameters, and provides direct links to more detailed data on genes, transcripts or genomic regions. Anduril is open-source; all methods and documentation are freely available.Results: We have integrated multidimensional molecular and clinical data from 338 subjects having glioblastoma multiforme, one of the deadliest and most poorly understood cancers, using Anduril. The central objective of our approach is to identify genetic loci and genes that have significant survival effect. Our results suggest several novel genetic alterations linked to glioblastoma multiforme progression and, more specifically, reveal Moesin as a novel glioblastoma multiforme-associated gene that has a strong survival effect and whose depletion in vitro significantly inhibited cell proliferation. All analysis results are available as a comprehensive website.Conclusions: Our results demonstrate that integrated analysis and visualization of multidimensional and heterogeneous data by Anduril enables drawing conclusions on functional consequences of large-scale molecular data. Many of the identified genetic loci and genes having significant survival effect have not been reported earlier in the context of glioblastoma multiforme. Thus, in addition to generally applicable novel methodology, our results provide several glioblastoma multiforme candidate genes for further studies. Anduril is available at http://csbi.ltdk.helsinki.fi/anduril/ The glioblastoma multiforme analysis results are available at http://csbi.ltdk.helsinki.fi/anduril/tcga-gbm

Transposition-based method for the rapid generation of gene-targeting vectors to produce Cre/Flp-modifiable conditional knock-out mice

Peer reviewe

Application of the bacteriophage Mu-driven system for the integration/amplification of target genes in the chromosomes of engineered Gram-negative bacteria—mini review

The advantages of phage Mu transposition-based systems for the chromosomal editing of plasmid-less strains are reviewed. The cis and trans requirements for Mu phage-mediated transposition, which include the L/R ends of the Mu DNA, the transposition factors MuA and MuB, and the cis/trans functioning of the E element as an enhancer, are presented. Mini-Mu(LR)/(LER) units are Mu derivatives that lack most of the Mu genes but contain the L/R ends or a properly arranged E element in cis to the L/R ends. The dual-component system, which consists of an integrative plasmid with a mini-Mu and an easily eliminated helper plasmid encoding inducible transposition factors, is described in detail as a tool for the integration/amplification of recombinant DNAs. This chromosomal editing method is based on replicative transposition through the formation of a cointegrate that can be resolved in a recombination-dependent manner. (E-plus)- or (E-minus)-helpers that differ in the presence of the trans-acting E element are used to achieve the proper mini-Mu transposition intensity. The systems that have been developed for the construction of stably maintained mini-Mu multi-integrant strains of Escherichia coli and Methylophilus methylotrophus are described. A novel integration/amplification/fixation strategy is proposed for consecutive independent replicative transpositions of different mini-Mu(LER) units with “excisable” E elements in methylotrophic cells

Mu transpososome activity-profiling yields hyperactive MuA variants for highly efficient genetic and genome engineering

The phage Mu DNA transposition system provides a versatile species non-specific tool for molecular biology, genetic engineering and genome modification applications. Mu transposition is catalyzed by MuA transposase, with DNA cleavage and integration reactions ultimately attaching the transposon DNA to target DNA. To improve the activity of the Mu DNA transposition machinery, we mutagenized MuA protein and screened for hyperactivity-causing substitutions using an in vivo assay. The individual activity-enhancing substitutions were mapped onto the MuA–DNA complex structure, containing a tetramer of MuA transposase, two Mu end segments and a target DNA. This analysis, combined with the varying effect of the mutations in different assays, implied that the mutations exert their effects in several ways, including optimizing protein–protein and protein–DNA contacts. Based on these insights, we engineered highly hyperactive versions of MuA, by combining several synergistically acting substitutions located in different subdomains of the protein. Purified hyperactive MuA variants are now ready for use as second-generation tools in a variety of Mu-based DNA transposition applications. These variants will also widen the scope of Mu-based gene transfer technologies toward medical applications such as human gene therapy. Moreover, the work provides a platform for further design of custom transposases.Peer reviewe