Development of mPing-based Activation Tags for Crop Insertional Mutagenesis

Modern plant breeding increasingly relies on genomic information to guide crop improvement. Although some genes are characterized, additional tools are needed to effectively identify and characterize genes associated with crop traits. To address this need, the mPing element from rice was modified to serve as an activation tag to induce expression of nearby genes. Embedding promoter sequences in mPing resulted in a decrease in overall transposition rate; however, this effect was negated by using a hyperactive version of mPing called mmPing20. Transgenic soybean events carrying mPing-based activation tags and the appropriate transposase expression cassettes showed evidence of transposition. Expression analysis of a line that contained a heritable insertion of the mmPing20F activation tag indicated that the activation tag induced overexpression of the nearby soybean genes. This represents a significant advance in gene discovery technology as activation tags have the potential to induce more phenotypes than the original mPing element, improving the overall effectiveness of the mutagenesis system.

Compensating for over-production inhibition of the Hsmar1 transposon in Escherichia coli using a series of constitutive promoters

Background Transposable elements (TEs) are a diverse group of self-mobilizing DNA elements. Transposition has been exploited as a powerful tool for molecular biology and genomics. However, transposition is sometimes limited because of auto-regulatory mechanisms that presumably allow them to cohabit within their hosts without causing excessive genomic damage. The papillation assay provides a powerful visual screen for hyperactive transposases. Transposition is revealed by the activation of a promoter-less lacZ gene when the transposon integrates into a non-essential gene on the host chromosome. Transposition events are detected as small blue speckles, or papillae, on the white background of the main Escherichia coli colony. Results We analysed the parameters of the papillation assay including the strength of the transposase transcriptional and translational signals. To overcome certain limitations of inducible promoters, we constructed a set of vectors based on constitutive promoters of different strengths to widen the range of transposase expression. We characterized and validated our expression vectors with Hsmar1, a member of the mariner transposon family. The highest rate of transposition was observed with the weakest promoters. We then took advantage of our approach to investigate how the level of transposition responds to selected point mutations and the effect of joining the transposase monomers into a single-chain dimer. Conclusions We generated a set of vectors to provide a wide range of transposase expression which will be useful for screening libraries of transposase mutants. The use of weak promoters should allow screening for truly hyperactive transposases rather than those that are simply resistant to auto-regulatory mechanisms, such as overproduction inhibition (OPI). We also found that mutations in the Hsmar1 dimer interface provide resistance to OPI in bacteria, which could be valuable for improving bacterial transposon mutagenesis techniques.

A series of constitutive expression vectors to accurately measure the rate of DNA transposition and correct for auto-inhibition

BackgroundTransposable elements (TEs) form a diverse group of DNA sequences encoding functions for their own mobility. This ability has been exploited as a powerful tool for molecular biology and genomics techniques. However, their use is sometimes limited because their activity is auto-regulated to allow them to cohabit within their hosts without causing excessive genomic damage. To overcome these limitations, it is important to develop efficient and simple screening assays for hyperactive transposases.ResultsTo widen the range of transposase expression normally accessible with inducible promoters, we have constructed a set of vectors based on constitutive promoters of different strengths. We characterized and validated our expression vectors with Hsmar1, a member of the mariner transposon family. We observed the highest rate of transposition with the weakest promoters. We went on to investigate the effects of mutations in the Hsmar1 transposase dimer interface and of covalently linking two transposase monomers in a single-chain dimer. We also tested the severity of mutations in the lineage leading to the human SETMAR gene, in which one copy of the Hsmar1 transposase has contributed a domain.ConclusionsWe generated a set of vectors to provide a wide range of transposase expression which will be useful for screening libraries of transposase mutants. We also found that mutations in the Hsmar1 dimer interface provides resistance to overproduction inhibition in bacteria, which could be valuable for improving bacterial transposon mutagenesis techniques.

Metaproteomics Reveals Abundant Transposase Expression in Mutualistic Endosymbionts

ABSTRACTTransposases, enzymes that catalyze the movement of mobile genetic elements, are the most abundant genes in nature. While many bacteria encode an abundance of transposases in their genomes, the current paradigm is that the expression of transposase genes is tightly regulated and generally low due to its severe mutagenic effects. In the current study, we detected the highest number of transposase proteins ever reported in bacteria, in symbionts of the gutless marine wormOlavius algarvensiswith metaproteomics. At least 26 different transposases from 12 different families were detected, and genomic and proteomic analyses suggest that many of these are active. This high expression of transposases indicates that the mechanisms for their tight regulation have been disabled or no longer exist.IMPORTANCEThe expansion of transposable elements (TE) within the genomes of host-restricted symbionts and pathogens plays an important role in their emergence and evolution and might be a key mechanism for adaptation to the host environment. However, little is known so far about the underlying causes and evolutionary mechanisms of this TE expansion. The current model of genome evolution in host-restricted bacteria explains TE expansion within the confines of the paradigm that transposase expression is always low. However, recent work failed to verify this model. Based on our data, we hypothesize that increased transposase expression, which has not previously been described, may play a role in TE expansion, and could be one explanation for the sometimes very rapid emergence and evolution of new obligate symbionts and pathogens from facultative ones.

The autoregulation of a eukaryotic DNA transposon

How do DNA transposons live in harmony with their hosts? Bacteria provide the only documented mechanisms for autoregulation, but these are incompatible with eukaryotic cell biology. Here we show that autoregulation of Hsmar1 operates during assembly of the transpososome and arises from the multimeric state of the transposase, mediated by a competition for binding sites. We explore the dynamics of a genomic invasion using a computer model, supported by in vitro and in vivo experiments, and show that amplification accelerates at first but then achieves a constant rate. The rate is proportional to the genome size and inversely proportional to transposase expression and its affinity for the transposon ends. Mariner transposons may therefore resist post-transcriptional silencing. Because regulation is an emergent property of the reaction it is resistant to selfish exploitation. The behavior of distantly related eukaryotic transposons is consistent with the same mechanism, which may therefore be widely applicable.

332 INDUCIBLE RED FLUORESCENT PROTEIN (RFP) EXPRESSION IN PORCINE FIBROBLASTS AND TRANSGENIC CLONED EMBRYOS USING piggyBac TRANSPOSITION

Transgenic pigs are promising animal resources for human disease models and organ donors for xenotransplantation, because they resemble humans anatomically and physiologically. Transgenic pigs have been produced from transfected donor cells using several gene delivery systems including retrovirus infection. Recently, it has been reported that piggyBac (PB) transposition is a highly efficient tool in producing transgenic mice. This study investigated the use of PB transposition to establish transgenic cells and produce transgenic cloned embryos in pigs. We constructed plasmid DNA with red fluorescence protein (RFP) expressed by tetracycline-dependent cassette (from Addgene) with PB site using gateway cloning. We co-transfected porcine fibroblasts with the structured plasmid vector (pB-TET-DsRed), pB-rtTA (from Addgene), and a transposase expression vector pCy43 (Sanger Insitute, Hinxton, UK) using Fugene HD. After 24 h, 2 μg mL–1 doxycycline was added to the culture medium to turn on RFP expression. After 48 h of culture, 1 mg mL–1 neomycin was added to select stable RFP transfectants. Selected fibroblasts were cultured for 9 days without doxycycline, thus reducing RFP expression. After establishment of inducible RFP-expressing cells, the cells were used for somatic cell nuclear transfer. Embryos were cultured in porcine zygote medium-3, and 2 μg mL–1 doxycycline was added 5 days later. As a result, RFP expression was detected in the blastocysts. In conclusion, this study demonstrated that the inducible RFP gene in porcine fibroblasts and embryos was controlled by PB transposition system. Furthermore, this system could be a means of delivering an exogenous gene into porcine somatic cells and embryos for transgenic research. This study was supported by grants from MKE (#2009-67-10033839, #2009-67-10033805), IPET (#109023-05-1-CG000), NRF (#M10625030005-10N250300510), and BK21 program.

A hobo Transgene That Encodes the P-Element Transposase in Drosophila melanogaster: Autoregulation and Cytotype Control of Transposase Activity

Drosophila were genetically transformed with a hobo transgene that contains a terminally truncated but otherwise complete P element fused to the promoter from the Drosophila hsp70 gene. Insertions of this H(hsp/CP) transgene on either of the major autosomes produced the P transposase in both the male and female germlines, but not in the soma. Heat-shock treatments significantly increased transposase activity in the female germline; in the male germline, these treatments had little effect. The transposase activity of two insertions of the H(hsp/CP) transgene was not significantly greater than their separate activities, and one insertion of this transgene reduced the transposase activity of P(ry+, Δ2-3)99B, a stable P transgene, in the germline as well as in the soma. These observations suggest that, through alternate splicing, the H(hsp/CP) transgene produces a repressor that feeds back negatively to regulate transposase expression or function in both the somatic and germline tissues. The H(hsp/CP) transgenes are able to induce gonadal dysgenesis when the transposase they encode has P-element targets to attack. However, this ability and the ability to induce P-element excisions are repressed by the P cytotype, a chromosomal/cytoplasmic state that regulates P elements in the germline.