The Sleeping Beauty (SB) transposon system is a promising new method for establishing persistent transgene expression in vivo. We applied the SB system for enhancing transgenesis in Saanen dairy goat fibroblast cells. We constructed a pKT2/CMV-EGFP-IRES-PURO vector and investigated the influence of transposon and transposase vector ratios on transfection efficiency in the Saanen goat fibr...

The ciliate Paramecium tetraurelia must eliminate approximately 60,000 short sequences from its genome to generate uninterrupted coding sequences in its somatic macronucleus. In this issue of Genes & Development, Baudry and colleagues (pp. 2478-2483) identify the protein that excises these noncoding sequences: a domesticated piggyBac transposase that has been adapted to remove what are likely t...

Transposition (the movement of discrete segments of DNA, resulting in rearrangement of genomic DNA) initiates when transposase forms a dimeric DNA-protein synaptic complex with transposon DNA end sequences. The synaptic complex is a prerequisite for catalytic reactions that occur during the transposition process. The transposase-DNA interactions involved in the synaptic complex have been of gre...

Mu DNA transposition proceeds through a series of higher-order nucleoprotein complexes called transpososomes. The structural core of the transpososome is a tetramer of the transposase, Mu A, bound to the two transposon ends. High-resolution structural analysis of the intact transposase and the transpososome has not been successful to date. Here we report the structure of Mu A at 16-angstroms an...

Complete and partial sequences of mariner-like elements (MLEs) have been reported for hundreds of species of animals, but only two have been identified in plants. On the basis of these two plant MLEs and several related sequences identified by database searches, plant-specific degenerate primers were derived and used to amplify a conserved region of MLE transposase genes from a variety of plant...

A key step in cut-and-paste DNA transposition is the pairing of transposon ends before the element is excised and inserted at a new site in its host genome. Crystallographic analyses of the paired-end complex (PEC) formed from precleaved transposon ends and the transposase of the eukaryotic element Mos1 reveals two parallel ends bound to a dimeric enzyme. The complex has a trans arrangement, wi...

Nonreplicative transposition by Tn10/IS10 involves three chemical steps at each transposon end: cleavage of the two strands plus joining of one strand to target DNA. These steps occur within a synaptic complex comprising two transposon ends and monomers of IS10 transposase. We report four transposase mutations that individually abolish each of the three chemical steps without affecting the syna...

We describe discovery in Beta vulgaris L. of Coe1, a DNA transposase gene within putative long terminal repeats (LTRs), and other retrotransposon-like features including both a retroviral-like hypothetical gene and an Rvt2-domain reverse transcriptase pseudogene. The central DNA transposase gene encodes, in eight exons, a predicted 160-KDa protein producing BLAST alignments with En/Spm-type tra...

The transposase family of proteins mediate DNA transposition or retroviral DNA integration via multistep phosphoryl transfer reactions. For Tn10 and phage Mu, a single active site of one transposase protomer catalyzes the successive transposition reaction steps. We examined phosphorothioate stereoselectivity at the scissile position for all four reaction steps catalyzed by the Tn10 transposase....

A soluble single-point mutant of full-length Mos1 mariner transposase (MW = 40.7 kDa) has been overexpressed in Escherichia coli, purified to 95% homogeneity and crystallized. This provides the first example of the crystallization of a eukaryotic transposase. The native crystals diffract to 2.5 A resolution and show tetragonal symmetry, with unit-cell parameters a = b = 44.5, c = 205.6 A. Multi...