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Alterations in tissue-specific gene expression greatly affect cell function. Transcription factors (TFs) interact with cis-acting binding sites in noncoding enhancer promoter regions. Transposable elements (TEs) are abundant and similarly represented among mammalian genomes. TEs are important in gene regulation, but their function is not well understood. We have characterized a TE containing functional TF-binding sites for the carcinogen-activated dioxin receptor xenobiotic responsive element (XRE) and the epithelial-mesenchymal transition regulator Slug (Slug site). A Mus promoter database was scanned for XREs to predict coregulation with other TFs. We identified an overrepresented (1,398 genes) B1 retrotransposon containing XRE and Slug sites within 35 bp of each other (designated as B1-X35S). This B1-X35S retrotransposon differed from classic Bls by the presence of the Slug site and by its differential nucleotide conservation outside the X35S region. Phylogenetically, B1-X35S appeared recently in evolution, close to the B1-B subfamily. Comparative gene expression in 61 mouse tissues revealed that B1-X35S-containing genes had lower median expression levels than those with canonical B1 TEs, suggesting a repressive role for X35S. Indeed, X35S was functional and able to bind aryl hydrocarbon (dioxin) receptor (AhR) and Slug and, importantly, to repress cis-reporter genes. Moreover, AhR and Slug were recruited to X35S in vivo and repressed the endogenous expression of X35S-containing genes. Our results demonstrate the existence of a widely present B1 subfamily in the mouse. Because AhR and Slug are relevant in tumor development and differentiation, X35S may represent a genome-wide regulatory mechanism and a tool to modulate gene expression.

The multiple steps of DNA transposition take place within a large complex called the transpososome, in which a pair of transposon DNA ends are synapsed by a multimer of the transposase protein. The final step, a DNA strand transfer reaction that joins the transposon ends to the target DNA strands, entails no net change in the number of high-energy chemical bonds. Physiology demands that, despite remaining stably associated with the transpososome, the strand transfer products undergo neither the reverse reaction nor any further cleavage reactions. Accordingly, when the Mu or Tn10 strand transfer complex was produced in vitro through transposase-catalyzed reaction steps, reverse reactions were undetectable. In contrast, when the Mu or Tn10 strand transfer complexes were assembled from DNA already having the structure of the strand transfer product, we detected a reaction that resembled reversal of target DNA strand transfer. The stereoselectivity of phosphorothioate-containing substrates indicated that this reaction proceeds as the pseudoreversai of the normal target DNA strand transfer step. Comparison of the reactivity of closely related Mu substrate DNA structures indicated that the configuration of the flanking DNA outside of the transposon sequence plays a key role in preventing the transposon end cleavage reaction after the strand transfer step. DNA transposition | phage Mu | recombination

An active miniature inverted repeat transposable element (MITE), mPing, was discovered by computer-assisted analysis of rice genome sequence. The mPing element is mobile in rice cell culture and in a few rice strains where it has been amplified to > 1,000 copies during recent domestication. However, determination of the transposase source and characterization of the mechanism of transposition have been hampered by the high copy number of mPing and the presence of several candidate autonomous elements in the rice genome. Here, we report that mPing is active in Arabidopsis thaliana, where its transposition is catalyzed by three sources of transposase from rice: the autonomous Ping and Pong elements and by a cDNA derived from a Ping transcript. In addition to transposase, the product of a second element-encoded ORF of unknown function is also required for mPing transposition. Excision of mPing in A. thaliana is usually precise, and transposed copies usually insert into unlinked sites in the genome that are preferentially in or near genes. As such, this will be a valuable assay system for the dissection of MITE transposition and a potentially powerful tagging system for gene discovery in eukaryotes. autonomous element | PIF/Harbinger | transgenic

The use of novel nonviral vector system, the Sleeping Beauty (SB) transposon (*Tn) is investigated to insert a wild-type beta-globin expression cassette into the human genone for sustained expression of beta-globin. It is found that SB-Tn system is a promising nonviral vector for efficient genomic insertion conferring stable, persistent erythroid-specific expression of [beta]-globin.

Exported proteins of the animal pathogen Corynebacterium pseudotuberculosis is identified using the reporter transposon-based system TnFuZ. Results revealed that thirty-four out of 1,500 mutants had detectable alkaline phosphatase (PhoZ) activity.

A cold-sensitive Listeria monocytogenes mutant intended cld-14 was obtained by transposon Tn917 mutagenesis and the gene interrupted by Tn917 in cld-14 was the Listeria monocytogenes LMOf2365_1485 homolog that exhibits 45.7% homology to the Bacillus subtilis yqfF locus. PgpH designated as LMOf2365_1485 might play a critical role due to its predicted membrane location in sensing the environmental temperature and altering cellular (p)ppGpp levels to allow the organism to adapt to low temperatures.

A simple and high-throughput transposon-mediated mutagenesis system employing two different types of transposons in combination with direct genomic DNA amplification and thermal asymmetric interlaced PCR (TAIL-PCR) is developed. This method is useful in furthering genetic understanding of cellular life and facilitating the functional analysis of microorganisms.

A dimerization mutant F460K is characterized to demonstrate that the dimeric form of the enzyme Metnase or SETMAR with unusual active site plasticity is required for its DNA cleavage, DNA-binding, and nonhomologous end joining activities. The observed clustering of substitutions located in alpha helices 4 and 5 within the putative DNA-binding site is found to be consistent with loss of transposition specific DNA cleavage activity and acquisition of DNA repair specific cleavage activity.