Cloning and characterization of piggyBac- like elements in lepidopteran insects

PiggyBac-like elements (PLE) are widespread in variety of organisms, however, few of them are active or have an intact transposon structure. To further define the distribution PLEs in Lepidoptera, where the original active piggyBac IFP2 was discovered, and potentially isolate new functional elements, a survey for PLEs by PCR amplification and Southern dot blots was performed. Two new PLEs, AyPLE and AaPLE, were successfully isolated from the noctuid species, Agrotis ypsilon and Argyrogramma agnate, respectively. These elements were found to be closely related to each other by sequence similarity, and by sharing the same 16 bp inverted terminal repeat sequences. The AyPLE1.1 and AaPLE1.1 elements are structurally intact having characteristic TTAA target site duplications, inverted terminal repeats and intact open reading frames encoding putative transposases with the presumed piggyBac DDD domains, which are features consistent with autonomous functional transposons. Phylogenetic analysis revealed that AyPLE1.1 and AaPLE1.1 cluster with another noctuid species element, HaPLE1.1, suggesting a common ancestor for the three types of PLEs. This contributes to our understanding of the distribution and evolution of piggyBac in Lepidoptera.     

Molecular evolutionary analysis of the widespread<Emphasis Type="Italic"> piggyBac</Emphasis> transposon family and related "domesticated" sequences

piggyBac is a short inverted-repeat-type DNA transposable element originally isolated from the genome of the moth Trichoplusia ni. It is currently the gene vector of choice for the transformation of various insect species. A few sequences with similarity to piggyBac have previously been identified from organisms such as humans ( Looper), the pufferfish Takifugu rubripes (Pigibaku), Xenopus (Tx), Daphnia (Pokey), and the Oriental fruit fly Bactrocera dorsalis. We have now identified 50 piggyBac-like sequences from publicly available genome sequences and expressed sequence tags (ESTs). This survey allows the first comparative examination of the distinctive piggyBac transposase, suggesting that it might contain a highly divergent DDD domain, comparable to the widespread DDE domain found in many DNA transposases and retroviral integrases which consists of two absolutely conserved aspartic acids separated by about 70 amino acids with a highly conserved glutamic acid about 35 amino acids further away. Many piggyBac-like sequences were found in the genomes of a phylogenetically diverse range of organisms including fungi, plants, insects, crustaceans, urochordates, amphibians, fishes and mammals. Also, several instances of "domestication" of the piggyBac transposase sequence by the host genome for cellular functions were identified. Novel members of the piggyBac family may be useful in genetic engineering of many organisms.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Identification and characterization of piggyBac-like elements in the genome of domesticated silkworm, Bombyx mori

piggyBac is a short inverted terminal repeat (ITR) transposable element originally discovered in Trichoplusia ni. It is currently the preferred vector of choice for enhancer trapping, gene discovery and identifying gene function in insects and mammals. Many piggyBac-like sequences have been found in the genomes of phylogenetically species from fungi to mammals. We have identified 98 piggyBac-like sequences (BmPBLE1-98) from the genome data of domesticated silkworm (Bombyx mori) and 17 fragments from expressed sequence tags (ESTs). Most of the BmPBLE1-98 probably exist as fossils. A total of 21 BmPBLEs are flanked by ITRs and TTAA host dinucleotides, of which 5 contain a single ORF, implying that they may still be active. Interestingly, 16 BmPBLEs have CAC/GTG not CCC/GGG as the characteristic residues of ITRs, which is a surprising phenomenon first observed in the piggyBac families. Phylogenetic analysis indicates that many BmPBLEs have a close relation to mammals, especially to Homo sapiens, only a few being grouped with the T. ni piggyBac element. In addition, horizontal transfer was probably involved in the evolution of the piggyBac-like elements between B. mori and Daphnia pulicaria. The analysis of the BmPBLEs will contribute to our understanding of the characteristic of the piggyBac family and application of piggyBac in a wide range of insect species.     

A new active piggyBac‐like element in Aphis gossypii

Abstract Nine piggyBac‐like elements (PLEs) were identified from the cotton aphid Aphis gossypii Glover. All the PLEs shared high sequence similarity with each other. However, eight of the nine PLEs were unlikely to encode functional transposase due to the existence of disruptive mutations within the coding regions. The other one PLE contained major characteristics of members in the piggyBac family, including TTAA target site duplications, inverted terminal repeats (ITRs), and an open reading frame (ORF) coding for a transposase with a putative DDD domain. This one with an intact transposase ORF was named AgoPLE1.1. The predicted transposase shared 47% similarity with that of Trichoplusia ni piggyBac IFP2. Phylogenetic analyses showed that AgoPLE1.1 was most related to the Heliothis virescens PLE1.1 (HvPLE1.1) element, with 45% and 60% similarity at the nucleotide and amino acid levels, respectively. A functional assay demonstrated that AgoPLE1.1 encoded a functional transposase and was able to cause precise excision in cell cultures. On the other hand, few genomic insertion polymorphisms of AgoPLE1 were observed in the genome of the cotton aphid. These observations suggested that AgoPLE1.1 was a PLE that invaded the cotton aphid genome in recent periods and retained its activity.     

Highly conserved piggyBac elements in noctuid species of Lepidoptera

The piggyBac transposable element was originally discovered in a Trichoplusia ni cell line and nearly identical elements were subsequently discovered in the tephritid fly, Bactrocera dorsalis. This suggested the existence of piggyBac in additional insects and this study shows highly conserved, though not identical, piggyBac sequences in the noctuid species Heliocoverpa armigera, H. zea, and Spodoptera frugiperda, as well as new piggyBac sequences from the T. ni organismal genome. Genomic piggyBac elements could not be unambiguously identified in several other moth species indicating a discontinuous presence of piggyBac in the Lepidoptera. Most sequences have greater than 95% nucleotide identity to the original IFP2 piggyBac, except for a more diverged sequence in S. frugiperda, having approximately 78% identity. Variants of 1.3 and 0.8kb sequences found in both H. armigera and H. zea most likely became established by interbreeding, supporting the notion that the species are conspecific. None of the independent piggyBac sequences isolated from T. ni larval genomes are identical to IFP2, though all have an uninterrupted reading frame with the potential for encoding a functional transposase. The piggyBac sequences from T. ni and the Helicoverpa species, as well as those previously reported from B. dorsalis, all share three common nucleotide substitutions resulting in a single amino acid substitution in the transposase. This suggests that the original IFP2 piggyBac is a related variant of a predecessor element that became widespread. The existence of conserved piggyBac elements, some of which may have been transmitted horizontally between lepidopteran species, raises important considerations for the stability and practical use of piggyBac transformation vectors.     

Analysis of the cis-acting DNA elements required for piggyBac transposable element excision

The terminal DNA sequence requirements for piggyBac transposable element excision were explored using a plasmid-based assay in transfected, cultured insect cells. A donor plasmid containing duplicate 3′piggyBac terminal inverted repeats was constructed that allowed individual nucleotides or groups of nucleotides within one of the 3′ repeats to be mutated. The relative extent of excision using the mutated end versus the wild-type end was then assayed. Removal of even one of the terminal 3′ G nucleotides from the piggyBac inverted repeat, or removal of the dinucleotide AA from the flanking TTAA target site prevents excision of piggyBac at the mutated terminus. Incorporation of an asymmetric TTAC target site at the 3′ end does not prevent excision from the mutated end. Thus, both piggyBac DNA and flanking host DNA appear to play crucial roles in the excision process. Received: 9 July 1996 / Accepted: 6 May 1997     

Germ-line transformation of the Queensland fruit fly, Bactrocera tryoni, using a piggyBac vector in the presence of endogenous piggyBac elements

We report the heritable germ-line transformation of the Queensland fruit fly, Bactrocera tryoni, using a piggyBac vector marked with either the fluorescent protein DsRed or EGFP. A transformation frequency of 5–10% was obtained. Inheritance of the transgenes has remained stable over more than 15 generations despite the presence of endogenous piggyBac sequences in the B. tryoni genome. The sequence of insertion sites shows the usual canonical pattern of piggyBac integraton into TTAA target sites. An investigation of endogenous piggyBac elements in the B. tryoni genome reveals the presence of sequences almost identical to those reported recently for the B. dorsalis complex of fruit flies and two noctuid moths, suggesting a common origin of piggyBac sequences in these species. The availability of transformation protocols for B. tryoni has the potential to deliver improvements in the performance of the Sterile Insect Technique for this pest species.     

Cognate restriction of transposition by piggyBac-like proteins

Mobile genetic elements have been harnessed for gene transfer for a wide variety of applications including generation of stable cell lines, recombinant protein production, creation of transgenic animals, and engineering cell and gene therapy products. The piggyBac transposon family includes transposase or transposase-like proteins from a variety of species including insect, bat and human. Recently, human piggyBac transposable element derived 5 (PGBD5) protein was reported to be able to transpose piggyBac transposons in human cells raising possible safety concerns for piggyBac-mediated gene transfer applications. We evaluated three piggyBac-like proteins across species including piggyBac (insect), piggyBat (bat) and PGBD5 (human) for their ability to mobilize piggyBac transposons in human cells. We observed a lack of cross-species transposition activity. piggyBac and piggyBat activity was restricted to their cognate transposons. PGBD5 was unable to mobilize piggyBac transposons based on excision, colony count and plasmid rescue analysis, and it was unable to bind piggyBac terminal repeats. Within the piggyBac family, we observed a lack of cross-species activity and found that PGBD5 was unable to bind, excise or integrate piggyBac transposons in human cells. Transposition activity appears restricted within species within the piggyBac family of mobile genetic elements.     

Analysis of the cis-acting DNA elements required for piggyBac transposable element excision

The terminal DNA sequence requirements for piggyBac transposable element excision were explored using a plasmid-based assay in transfected, cultured insect cells. A donor plasmid containing duplicate 3′piggyBac terminal inverted repeats was constructed that allowed individual nucleotides or groups of nucleotides within one of the 3′ repeats to be mutated. The relative extent of excision using the mutated end versus the wild-type end was then assayed. Removal of even one of the terminal 3′ G nucleotides from the piggyBac inverted repeat, or removal of the dinucleotide AA from the flanking TTAA target site prevents excision of piggyBac at the mutated terminus. Incorporation of an asymmetric TTAC target site at the 3′ end does not prevent excision from the mutated end. Thus, both piggyBac DNA and flanking host DNA appear to play crucial roles in the excision process.     

Interplasmid transposition demonstrates piggyBac mobility in vertebrate species

The piggyBac transposon is an extremely versatile helper-dependent vector for gene transfer and germ line transformation in a wide range of invertebrate species. Analyses of genome sequencing databases have identified piggyBac homologues among several sequenced animal genomes, including the human genome. In this report we demonstrate that this insect transposon is capable of transposition in primate cells and embryos of the zebrafish, Danio rerio. piggyBac mobility was demonstrated using an interplasmid transposition assay that has consistently predicted the germ line transformation capabilities of this mobile element in several other species. Both transfected COS-7 primate cells and injected zebrafish embryos supported the helper-dependent movement of tagged piggyBac element between plasmids in the characteristic cut-and-paste, TTAA target-site specific manner. These results validate piggyBac as a valuable tool for genetic analysis of vertebrates.     

Transposition of the piggyBac element in embryos of Drosophila melanogaster, Aedes aegypti and Trichoplusia ni.

The Lepidopteran transposable element piggyBac is being recognized as a useful vector for genetic engineering in a variety of insect species. This transposon can mediate transformation in the Dipteran species Ceratitis capitata, and can potentially serve as a versatile vector for transformation of a wide variety of insect species. Using a plasmid-based interplasmid transposition assay, we have demonstrated that this transposon, of the short inverted terminal repeat type, is capable of transposition in embryos of three different insect species, Drosophila melanogaster, the yellow fever mosquito Aedes aegypti, and its host of origin, Trichoplusia ni. This assay can confirm the potential utility of piggyBac as a gene transfer tool in a given insect species, and provides an experimental model for assessing molecular mechanisms of transposon movement. Received: 19 November 1998 / Accepted: 1 March 1999     

Development and use of a piggyBac‐based jumpstarter system in Drosophila suzukii

Spotted wing drosophila, Drosophila suzukii, is an invasive pest that primarily attacks fresh, soft‐skinned fruit. Although others have reported successful integration of marked piggyBac elements into the D. suzukii genome, with a very respectable transgenesis rate of ～16%, here we take this work a step further by creating D. suzukii jumpstarter strains. These were generated through integration of a fluorescent‐marked Minos element carrying a heat shock protein 70‐driven piggyBac transposase gene. We demonstrate that there is a dramatic increase in transformation rates when germline transformation is performed in a transposase‐expressing background. For example, we achieved transformation rates as high as 80% when microinjecting piggyBac‐based plasmids into embryos derived from one of these D. suzukii jumpstarter strains. We also investigate the effect of insert size on transformation efficiency by testing the ability of the most efficient jumpstarter strain to catalyze integration of differently‐sized piggyBac elements. Finally, we demonstrate the ability of a jumpstarter strain to remobilize an already‐integrated piggyBac element to a new location, demonstrating that our jumpstarter strains could be used in conjunction with a piggyBac‐based donor strain for genome‐wide mutagenesis of D. suzukii.     

Isolation and characterisation of a cDNA encoding a zona pellucida protein (ZPB) from the marsupial Trichosurus vulpecula (brushtail possum)

We have cloned a cDNA containing the entire coding sequence of a marsupial (the brushtail possum, Trichosurus vulpecula) zona pellucida protein (ZPB). The open reading frame of 1,581 nt is predicted to encode a ZPB polypeptide of 527 amino acids which contains 20 cysteine residues, 7 potential N‐linked glycosylation sites, a potential N‐terminal signal peptide and a potential C‐terminal trans‐membrane domain, preceded by a furin proteolytic processing signal. Sequence comparisons between possum ZPB and orthologous polypeptides from 7 eutherian species and from Xenopus laevis, reveal the existence of a high degree of sequence similarity, particularly in the central portion of the molecule. Cysteine residues are highly conserved, and all nine species possess potential N‐terminal signal peptide sequences and C‐terminal trans‐membrane domains of approximately the same length. In situ hybridisation revealed that expression of ZPB was restricted to oocytes of primordial and primary follicles of adult possums; no expression was detected in the surrounding granulosa cells. The broad conservation of ZPB sequence, structure and expression over a wide range of mammalian species, revealed by our studies, makes it unlikely that these features account for the different properties of the marsupial and eutherian zona pellucidae. Mol. Reprod. Dev. 52:174–182, 1999. © 1999 Wiley‐Liss, Inc.     

PCR analysis of insertion site specificity, transcription, and structural uniformity of the Lepidopteran transposable element IFP2 in the TN-368 cell genome

The IFP2 element is a unique Lepidopteran transposon that has been associated with spontaneous Baculovirus mutants isolated following passage of the virus in the TN-368 cell line. Independent genomic representatives of IFP2 from TN-368 cells show little sequence divergence, suggesting that IFP2 was recently introduced into this genome and is highly stable. IFP2 is inserted within AT-rich regions of the TN-368 genome and targets TTAA sites. The specificity for TTAA target sites during transposition is not limited to the movement of IFP2 during an active Baculovirus infection, but is a property of its movement in uninfected cells as well. The exact origin of IFP2 remains obscure since it is found in two independently established Trichoplusia ni cell lines but not in three others, and we have not yet identified any IFP2 sequences in either field collected larvae or laboratory colonies.     

The diffusible factor synthase XanB2 is a bifunctional chorismatase that links the shikimate pathway to ubiquinone and xanthomonadins biosynthetic pathways

The diffusible factor synthase XanB2, originally identified in Xanthomonas campestris pv. campestris (Xcc), is highly conserved across a wide range of bacterial species, but its substrate and catalytic mechanism have not yet been investigated. Here, we show that XanB2 is a unique bifunctional chorismatase that hydrolyses chorismate, the end‐product of the shikimate pathway, to produce 3‐hydroxybenzoic acid (3‐HBA) and 4‐HBA. 3‐HBA and 4‐HBA are respectively associated with the yellow pigment xanthomonadin biosynthesis and antioxidant activity in Xcc. We further demonstrate that XanB2 is a structurally novel enzyme with three putative domains. It catalyses 3‐HBA and 4‐HBA biosynthesis via a unique mechanism with the C‐terminal YjgF‐like domain conferring activity for 3‐HBA biosynthesis and the N‐terminal FGFG motif‐containing domain responsible for 4‐HBA biosynthesis. Furthermore, we show that Xcc produces coenzyme Q8 (CoQ8) via a new biosynthetic pathway independent of the key chorismate‐pyruvate lyase UbiC. XanB2 is the alternative source of 4‐HBA for CoQ8 biosynthesis. The similar CoQ8 biosynthetic pathway, xanthomonadin biosynthetic gene cluster and XanB2 homologues are well conserved in the bacterial species within Xanthomonas, Xylella, Xylophilus, Pseudoxanthomonas, Rhodanobacter, Frateuria, Herminiimonas and Variovorax, suggesting that XanB2 may be a conserved metabolic link between the shikimate pathway, ubiquinone and xanthomonadin biosynthetic pathways in diverse bacteria.     

In vivo characterization of a vertebrate ultraconserved enhancer

Genomic sequence comparisons among human, mouse, and pufferfish (Takifugu rubripes (Fugu)) have revealed a set of extremely conserved noncoding sequences. While this high degree of sequence conservation suggests severe evolutionary constraint and predicts a lack of tolerance to change to retain in vivo functionality, such elements have been minimally explored experimentally. In this study, we describe the in-depth characterization of an ancient conserved enhancer, Dc2, located near the dachshund gene, which displays a human-Fugu identity of 84% over 424 basepairs (bp). In addition to this large overall conservation, we find that Dc2 is characterized by the presence of a large block of sequence (144 bp) that is completely identical among human, mouse, chicken, zebrafish, and Fugu. Through the testing of reporter vector constructs in transgenic mice, we observed that the 424-bp Dc2-conserved element is necessary and sufficient for brain tissue enhancer activity. In vivo analyses also revealed that the 144-bp 100% conserved sequence is necessary, but not sufficient, to replicate Dc2 enhancer function. However, the introduction of two separate 16-bp insertions into the highly conserved enhancer core did not cause any detectable modification of its in vivo activity. Our observations indicate that the 144-bp 100% conserved element is tolerant of change at least at the resolution of this transgenic mouse assay and suggest that purifying selection on the Dc2 sequence might not be as strong as we predicted or that some unknown property also constrains this highly conserved enhancer sequence.     

MOLECULAR CLONING AND EXPRESSION OF THE PROLIFERATING CELL NUCLEAR ANTIGEN GENE FROM THE COCCOLITHOPHORID PLEUROCHRYSIS CARTERAE (HAPTOPHYCEAE)1

The gene encoding proliferating cell nuclear antigen (PCNA) was isolated from the marine coccolithophorid microalga Pleurochrysis carterae (Braarud et Fagerland) Christensen (Haptophyceae). Two mRNAs (Pcpcna1 and Pcpcna2) were identified and contained an identical coding region for 222 amino acid residues and an untranslated sequence of 302 base pair (Ut1) and 246 base pair (Ut2), respectively. Comparison between PCR‐derived genomic DNA fragments and cDNA sequences revealed five introns. The coding region of Pcpcna is similar to counterparts in other organisms and contains highly conserved functional domains. Phylogenetic analyses indicated clustering of Pcpcna with pcna in its haptophyte relative Isochrysis galbana Parke. A recombinant fusion protein of Pcpcna, overexpressed in Escherichia coli, was recognized by the PC10 antibody against rat PCNA. Using RT‐PCR and Western blotting, Pcpcna was found to be highly transcribed and translated during the exponential growth phase relative to the stationary growth phase, with a positive correlation between gene expression and growth rate. It can be concluded that the pcna is conserved in this coccolithophorid phytoplankton and that its expression is growth stage related.     

Two different CC‐NBS‐LRR genes are required for Lr10‐mediated leaf rust resistance in tetraploid and hexaploid wheat

Comparative study of disease resistance genes in crop plants and their relatives provides insight on resistance gene function, evolution and diversity. Here, we studied the allelic diversity of the Lr10 leaf rust resistance gene, a CC‐NBS‐LRR coding gene originally isolated from hexaploid wheat, in 20 diploid and tetraploid wheat lines. Besides a gene in the tetraploid wheat variety ‘Altar’ that is identical to the hexaploid wheat Lr10, two additional, functional resistance alleles showing sequence diversity were identified by virus‐induced gene silencing in tetraploid wheat lines. In contrast to most described NBS‐LRR proteins, the N‐terminal CC domain of LR10 was found to be under strong diversifying selection. A second NBS‐LRR gene at the Lr10 locus, RGA2, was shown through silencing to be essential for Lr10 function. Interestingly, RGA2 showed much less sequence diversity than Lr10. These data demonstrate allelic diversity of functional genes at the Lr10 locus in tetraploid wheat, and these new genes can now be analyzed for agronomic relevance. Lr10‐based resistance is highly unusual both in its dependence on two, only distantly, related CC‐NBS‐LRR proteins, as well as in the pattern of diversifying selection in the N‐terminal domain. This indicates a new and complex molecular mechanism of pathogen detection and signal transduction.