Extrachromosomal transposition of the transposable element Minos in embryos of the cricket Gryllus bimaculatus

Effective germline transformation of insects has been shown to depend on the right choice of transposon system and selection marker. In this study the promoter region of a Gryllus cytoplasmic actin (GbA3/4) gene was isolated and characterized, and was used to drive the expression of Minos transposase in embryos of the cricket Gryllus bimaculatus. Active Minos transposase was produced in these embryos as monitored through established transposon excision and interplasmid transposition assays. In contrast, Drosophila melanogaster hsp70 promoter, previously used to express Minos transposase in a number of insect species and insect cell lines, failed to produce any detectable Minos transposase activity, as recorded by using the very sensitive transposon excision assay. In addition, the GbA3/4 promoter was found to drive expression of enhanced green fluorescent protein (eGFP) predominantly in vitellophages of the developing Gryllus eggs when a plasmid carrying a GbA3/4 promoter-eGFP fusion gene was transiently injected into embryos. These results strongly support the use of Minos transposons marked with the GbA3/4 promoter-eGFP for the genetic transformation of this emerging model insect species.     

Germline Transformation of Drosophila Virilis Mediated by the Transposable Element Hobo

A laboratory strain of Drosophila virilis was genetically transformed with a hobo vector carrying the miniwhite cassette using a helper plasmid with an hsp70-driven hobo transposase-coding sequence. The rate of transformation was 0.5% per fertile G0 animal. Three transgenic insertions were cloned and characterized and found to be authentic hobo insertions. These results, together with the known wide-spread distribution of hobo in diverse insect species, suggest that hobo and related transposable elements may be of considerable utility in the germline transformation of insects other than D. melanogaster.     

Use of the piggyBac transposon for germ-line transformation of insects

Germ-line transformation of insects is now possible with four independent transposable element vector systems. Among these, the TTAA-insertion site specific transposon, piggyBac, discovered in Trichoplusia ni, is one of the most widely used. Transformations have been achieved in a wide variety of dipterans, lepidopterans, and a coleopteran, and for many species, piggyBac transposition was first tested by plasmid-based mobility assays in cell lines and embryos. All plasmid and genomic insertions are consistent with the duplication of a TTAA insertion site, and most germ-line integrations appear to be stable, though this is largely based on stable marker phenotypes. Of the vector systems presently in use for non-drosophilids, piggyBac is the only one not currently associated with a superfamily of transposable elements, though other elements exist that share its TTAA insertion site specificity. While functional piggyBac elements have only been isolated from T. ni, nearly identical elements have been discovered in a dipteran species, Bactrocera dorsalis, and closely related elements exist in another moth species, Spodoptera frugiperda. It appears that piggyBac has recently traversed insect orders by horizontal transmission, possibly mediated by a baculovirus or other viral system. This interspecies movement has important implications for the practical use of piggyBac to create transgenic insect strains for field release.     

Hermes, a Functional Non-Drosophilid Insect Gene Vector from Musca Domestica

Hermes is a short inverted repeat-type transposable element from the house fly, Musca domestica. Using an extra-chromosomal transpositional recombination assay, we show that Hermes elements can accurately transpose in M. domestica embryos. To test the ability of Hermes to function in species distantly related to M. domestica we used a nonautonomous Hermes element containing the Drosophila melanogaster white (w(+)) gene and created D. melanogaster germline transformants. Transgenic G(1) insects were recovered from 34.6% of the fertile G(0) adults developing from microinjected w(-) embryos. This transformation rate is comparable with that observed using P or hobo vectors in D. melanogaster, however, many instances of multiple-element insertions and large clusters were observed. Genetic mapping, Southern blotting, polytene chromosome in situ hybridization and DNA sequence analyses confirmed that Hermes elements were chromosomally integrated in transgenic insects. Our data demonstrate that Hermes elements transpose at high rates in D. melanogaster and may be an effective gene vector and gene-tagging agent in this species and distantly related species of medical and agricultural importance.     

In vitro transposition of ISY100, a bacterial insertion sequence belonging to the Tc1/mariner family

The Synechocystis sp. PCC6803 insertion sequence ISY100 (ISTcSa) belongs to the Tc1/mariner/IS630 family of transposable elements. ISY100 transposase was purified and shown to promote transposition in vitro. Transposase binds specifically to ISY100 terminal inverted repeat sequences via an N-terminal DNA-binding domain containing two helix-turn-helix motifs. Transposase is the only protein required for excision and integration of ISY100. Transposase made double-strand breaks on a supercoiled DNA molecule containing a mini-ISY100 transposon, cleaving exactly at the transposon 3' ends and two nucleotides inside the 5' ends. Cleavage of short linear substrates containing a single transposon end was less precise. Transposase also catalysed strand transfer, covalently joining the transposon 3' end to the target DNA. When a donor plasmid carrying a mini-ISY100 was incubated with a target plasmid and transposase, the most common products were insertions of one transposon end into the target DNA, but insertions of both ends at a single target site could be recovered after transformation into Escherichia coli. Insertions were almost exclusively into TA dinucleotides, and the target TA was duplicated on insertion. Our results demonstrate that there are no fundamental differences between the transposition mechanisms of IS630 family elements in bacteria and Tc1/mariner elements in higher eukaryotes.     

What's buzzing? Mosquito genomics and transgenic mosquitoes

Genome projects and associated technologies are now being established for mosquito species that are vectors of human disease. The recent announcement of an award by the National Institute of Allergy and Infectious Diseases (NIAID) to Celera Genomics to sequence the Anopheles gambiae genome will further accelerate the completion of the sequencing of this genome. Completion of the An. gambiae sequence will mean that the genomes of all three organisms involved in the transmission of falciparum malaria--the mosquito, the parasite, and the human--will have been sequenced. This will greatly facilitate the identification of genes and pathways involved in the transmission of malaria. The recent genetic transformation of An. gambiae with the piggyBac transposable element and the transformation of another important malarial vector, Anopheles stephensi using the Minos element, now provide researchers with powerful tools with which to genetically manipulate these medically important vector species. Here we review the recent progress made in the extension of contemporary tools of modern genetics and genomics into these medically important insects.     

Transposable elements as tools for genomics and genetics in Drosophila.

The P-element has been the workhorse of Drosophila genetics since it was developed as a tool for transgenesis in 1982; the subsequent development of a variety of systems based on the transposon have provided a range of powerful and flexible tools for genetics and genomics applications. P-element insertions are frequently used as starting-points for generating chromosomal deletions to remove flanking genes, either by screening for imprecise excision events or by selecting for male recombination events. Elements that utilise the yeast FLP/FLP recombination target (FRT) site-specific recombination system have been widely used to generate molecularly marked mitotic clones for mosaic analysis, extending the reach of this powerful genetic tool to virtually all areas of developmental biology. P-elements are still widely used as traditional mutagenesis reagents and form the backbone of projects aimed at generating insertions in every predicted gene in the fly genome. In addition, vectors based on the FLP/FRT system are being used for genome-wide applications, including the development of molecularly-mapped deletion and duplication kits. In addition to these 'traditional' genetic approaches, a variety of engineered elements have been developed for a wide range of transgenic applications, including enhancer trapping, gene-tagging, targeted misexpression, RNA interference (RNAi) delivery and homologous recombination/gene replacement. To complement the use of P-elements, alternative transposon vectors have been developed. The most widely used of these are the lepidopteran element piggyBac and a Drosophila hydei transposon, Minos. In total, a range of transposon vectors offers the Drosophila biologist considerable flexibility and sophistication in manipulating the genome of the fly and has allowed rapid advances in all areas of developmental biology and genome science.     

基于PIPE现象的质粒克隆位点序列设计与功能评价

分子克隆是现代生物学研究的核心技术之一,是基因工程、蛋白质工程中的重要手段。为提高分子克隆实验的操作效率,本研究设计并合成基于聚合酶引物不完全延伸（polymerase incomplete primer extension,PIPE）现象的质粒克隆位点序列。并以此为基础统一相关引物的设计方案,避免传统酶切--连接法中需针对不同载体MCS序列设计不同引物的缺点。该方案利用13 bp定长接头序列,在同一体系中使用2对引物、2种线性化模板同时扩增载体和插入片段,通过20个循环,在1次PCR过程中即合成可供转化使用的带缺口质粒产物。在NEB Q5酶系统中,利用此法将3种荧光素酶序列插入pET-15b及pET-21b(+)载体,均获得成功。且利用商品化感受态细胞（转化效率 > 5×108 cfu/μg）转化后所获得转化子数量均在300个以上,其中含插入片段的阳性克隆比例可达85%以上。基于本方案的设计及作用原理,可将其应用于10 kb以内载体和插入片段的快速重组。且具有通用性强、耗时少、阳性克隆得率高和成本低等优点,是传统DNA重组方法的有益补充,可作为各实验室的常规分子克隆手段之一。     

Transposon-free insertions for insect genetic engineering

Methods involving the release of transgenic insects in the field hold great promise for controlling vector-borne diseases and agricultural pests. Insect transformation depends on nonautonomous transposable elements as gene vectors. The resulting insertions are stable in the absence of suitable transposase, however, such absence cannot always be guaranteed. We describe a method for post-integration elimination of all transposon sequences in the pest insect Medfly, Ceratitis capitata. The resulting insertions lack transposon sequences and are therefore impervious to transposase activity.     

Transformation and transposon mutagenesis of Leifsonia xyli subsp. xyli, causal organism of ratoon stunting disease of sugarcane

Conditions have been developed for genetic transformation and insertional mutagenesis in Leifsonia xyli subsp. xyli (Lxx), the causal organism of ratoon stunting disease (RSD), one of the most damaging and intractable diseases of sugarcane internationally. Transformation frequencies ranged from 1 to 10 colony forming units (CFU)/microg of plasmid DNA using Clavibacter/Escherichia coli shuttle vectors pCG188, pDM302, and pDM306 and ranged from 50 to 500 CFU/microg using cosmid cloning vectors pLAFR3 and pLAFR5-km. The transformation/transposition frequency was 0 to 70 CFU/microg of DNA, using suicide vectors pUCD623 and pSUP2021 containing transposable elements Tn4431 and Tn5, respectively. It was necessary to grow Lxx in media containing 0.1% glycine for electroporation and to amplify large plasmids in a dam-/dcm- E. coli strain and purify the DNA by anion exchange. To keep selection pressure at an optimum, the transformants were grown on nitrocellulose filters (0.2-microm pore size) on media containing the appropriate antibiotics. Transposon Tn4431 containing a promoterless lux operon from Vibrio fischeri and a tetracycline-resistance gene was introduced on the suicide vector pUCD623. All but 1% of the putative transposon mutants produce light, indicating transposition into functional Lxx genes. Southern blot analysis of these transformants indicates predominantly single transposon insertions at unique sites. The cosmid cloning vector pLAFR5-km was stably maintained in Lxx. The development of a transformation and transposon mutagenesis system opens the way for molecular analysis of pathogenicity determinants in Lxx.     

Complete sequence of the binary vector Bin 19

Despite the widespread use of Bin 19 as a vector for plant transformation, detailed sequence information on its T-DNA region has only recently become available. We now show that the non-T-DNA region, like the T-DNA region, contains several superfluous insertions and find that some functional elements may not contain optimal sequences. Knowledge of the complete 11 777 bp sequence will aid in the construction of exceptionally efficient derivative vectors of approximately half this size. Precise knowledge of restriction sites and removal of unnecessary sequences will facilitate plasmid manipulations and plant transformation.     

The Drosophila gene disruption project: progress using transposons with distinctive site specificities

The Drosophila Gene Disruption Project (GDP) has created a public collection of mutant strains containing single transposon insertions associated with different genes. These strains often disrupt gene function directly, allow production of new alleles, and have many other applications for analyzing gene function. Here we describe the addition of ～7600 new strains, which were selected from >140,000 additional P or piggyBac element integrations and 12,500 newly generated insertions of the Minos transposon. These additions nearly double the size of the collection and increase the number of tagged genes to at least 9440, approximately two-thirds of all annotated protein-coding genes. We also compare the site specificity of the three major transposons used in the project. All three elements insert only rarely within many Polycomb-regulated regions, a property that may contribute to the origin of "transposon-free regions" (TFRs) in metazoan genomes. Within other genomic regions, Minos transposes essentially at random, whereas P or piggyBac elements display distinctive hotspots and coldspots. P elements, as previously shown, have a strong preference for promoters. In contrast, piggyBac site selectivity suggests that it has evolved to reduce deleterious and increase adaptive changes in host gene expression. The propensity of Minos to integrate broadly makes possible a hybrid finishing strategy for the project that will bring >95% of Drosophila genes under experimental control within their native genomic contexts.     

Frequency,composition and mobility of Escherichia coli-derived transposable elements in holdings of plasmid repositories

By providing the scientific community with uniform and standardized resources of consistent quality, plasmid repositories play an important role in enabling scientific reproducibility. Plasmids containing insertion sequence elements (IS elements) represent a challenge from this perspective, as they can change the plasmid structure and function. In this study, we conducted a systematic analysis of a subset of plasmid stocks distributed by plasmid repositories (The Arabidopsis Biological Resource Center and Addgene) which carry unintended integrations of bacterial mobile genetic elements. The integration of insertion sequences was most often found in, but not limited to, pBR322-derived vectors, and did not affect the function of the specific plasmids. In certain cases, the entire stock was affected, but the majority of the stocks tested contained a mixture of the wild-type and the mutated plasmids, suggesting that the acquisition of IS elements likely occurred after the plasmids were acquired by the repositories. However, comparison of the sequencing results of the original samples revealed that some plasmids already carried insertion mutations at the time of donation. While an extensive BLAST analysis of 47 877 plasmids sequenced from the Addgene repository uncovered IS elements in only 1.12%, suggesting that IS contamination is not widespread, further tests showed that plasmid integration of IS elements can propagate in conventional Escherichia coli hosts over a few tens of generations. Use of IS-free E. coli hosts prevented the emergence of IS insertions as well as that of small indels, suggesting that the use of IS-free hosts by donors and repositories could help limit unexpected and unwanted IS integrations into plasmids.     

AT-rich sequences from the arbuscular mycorrhizal fungus Gigaspora rosea exhibit ARS function in the yeast Saccharomyces cerevisiae

Autonomous replicating sequences are DNA elements that trigger DNA replication and are widely used in the development of episomal transformation vectors for fungi. In this paper, a genomic library from the mycorrhizal fungus Gigaspora rosea was constructed in the integrative plasmid YIp5 and screened in the budding yeast Saccharomyces cerevisiae for sequences that act as ARS and trigger plasmid replication. Two genetic elements (GrARS2, GrARS6) promoted high-rates of yeast transformation. Sequence analysis of these elements shows them to be AT-rich (72-80%) and to contain multiple near-matches to the yeast autonomous consensus sequences ACS and EACS. GrARS2 contained a putative miniature inverted-repeat transposable element (MITE) delimited by 28-bp terminal inverted repeats (TIRs). Disruption of this element and removal of one TIR increased plasmid stability several fold. The potential for palindromes to affect DNA replication is discussed.     

Post-integration behavior of a Mos1 mariner gene vector in Aedes aegypti

The post-integration behavior of insect gene vectors will determine the types of applications for which they can be used. Transposon mutagenesis, enhancer trapping, and the use of transposable elements as genetic drive systems in insects requires transposable elements with high rates of remobilization in the presence of transposase. We investigated the post-integration behavior of the Mos1 mariner element in transgenic Aedes aegypti by examining both germ-line and somatic transpositions of a non-autonomous element in the presence of Mos1 transposase. Somatic transpositions were occasionally detected while germ-line transposition was only rarely observed. Only a single germ-line transposition event was recovered after screening 14,000 progeny. The observed patterns of transposition suggest that Mos1 movement takes place between the S phase and anaphase. The data reported here indicate that Mos1 will be a useful vector in Ae. aegypti for applications requiring a very high degree of vector stability but will have limited use in the construction of genetic drive, enhancer trap, or transposon tagging systems in this species.     

Multiple events of horizontal transfer of the Minos transposable element between Drosophila species

In this study the Minos element was analyzed in 26 species of the repleta group and seven species of the saltans group of the genus Drosophila. The PCR and Southern blot analysis showed a wide occurrence of the Minos transposable element among species of the repleta and the saltans groups and also a low number of insertions in both genomes. Three different analyses, nucleotide divergence, historical associations, and comparisons between substitution rates (d(N) and d(S)) of Minos and Adh host gene sequences, suggest the occurrence of horizontal transfer between repleta and saltans species. These data reinforce and extend the Arca and Savakis [Genetica 108 (2000) 263] results and suggest five events of horizontal transfer to explain the present Minos distribution: between D. saltans and the ancestor of the mulleri and the mojavensis clusters; between D. hydei and the ancestor of the mulleri and the mojavensis clusters; between D. mojavensis and D. aldrichi; between D. buzzatii and D. serido; and between D. spenceri and D. emarginata. An alternative explanation would be that repeated events of horizontal transfer involving D. hydei, which is a cosmopolitan species that diverged from the others repleta species as long as 14Mya, could have spread Minos within the repleta group and to D. saltans. The data presented in this article support a model in which distribution of Minos transposon among Drosophila species is determined by horizontal transmission balanced by vertical inactivation and extinction.     

Instability in tyrR Strains of Plasmids Carrying the Tyrosine Operon: Isolation and Characterization of Plasmid Derivatives with Insertions or Deletions

The transformation of tyrR strains of Escherichia coli with multicopy plasmids which carry the tyrosine operon gave rise to modified plasmids with either insertions or deletions. The effect of each of these insertions or deletions was to decrease the level of expression of this operon. It is proposed that plasmid instability arose as a direct consequence of the metabolic effects of an overproduction of the enzymes coded for by the tyrosine operon. The results have significant implications for the cloning of genes that are repressed by the product of a regulatory gene. Since the predominant plasmid modification observed was the insertion of an IS1 element near the regulatory region of the tyrosine operon, the results also suggest a role for IS1 elements in the regulation of gene expression.     

Instability of Tn5 inserts in cyanobacterial cloning vectors.

Transposon Tn5 was used to produce random insertions in two hybrid cloning vectors for the unicellular cyanobacterium Anacystis nidulans. The transposon-containing plasmids were used to localize essential replication functions and to characterize the stability of large inserts in these vectors. The effect of the insertions on plasmid function was tested by transformation into a derivative of A. nidulans that had been cured of the endogenous plasmid used to construct the vectors. A region of approximately 4 kilobases was essential for successful plasmid transformation and replication. This region has also been shown to be involved in plasmid replication by deletion analysis. High rates of excision of Tn5 inserts within this region and restoration of normal replication function were observed when transformants were selected by using a resistance marker outside the replication region in the absence of selection for the transposon-coded kanamycin resistance. Transposon inserts outside this region were not deleted.     

Minos transposon causes germline transgenesis of the ascidian Ciona savignyi

An ascidian, Ciona savignyi, is regarded as a good experimental animal for genetics because of its small and compact genome for which a draft sequence is available, its short generation time and its interesting phylogenic position. ENU-based mutagenesis has been carried out using this animal. However, insertional mutagenesis using transposable elements (transposons) has not yet been introduced. Recently, one of the Tc1/mariner superfamily transposons, Minos, was demonstrated to cause germline transgenesis in the related species Ciona intestinalis. In this report, we show that Minos has the ability to transpose from DNA to DNA in Ciona savignyi in transposition assays. Although the activity was slightly weaker than in Ciona intestinalis, Minos still caused germline transgenesis in Ciona savignyi. In addition, one insertion seemed to have caused an enhancer trapping. These results indicate that Minos provides a potential tool for transgenic techniques such as insertional mutagenesis in Ciona savignyi.