Transposition bursts in analysis of repeated mutations in unstable strains of Drosophila melanogaster

The phenomenon of transposition memory was earlier demonstrated for the cut locus and mdg4. This work has been aimed at finding out, in what way the transposition memory can be realized. An unstable stock cmMR17ctMRpN17 was analysed which had high frequency of double cm+ct+ reversions and cmMRctMRpN repeated mutations. A series of five such transpositions could be followed. The ctMRpN17 mutation is a result of insertion at the cut locus mdg4 with the jockey element inserted within it. As seen from in situ hybridization analysis, transitions to the normal phenotype correlate, as a rule, with the excision of mdg4 and the jockey from the cut locus. Analysis of distribution of mdg1, mdg2, mdg3 and jockey on the X-chromosome of unstable revertants and repeated mutants indicated that not only transpositions of mdg4 and jockey, but also those of all mobile elements tested occur. So, we propose that the transposition memory in our genetic system is manifested in the process of transposition bursts.     

The nature of unstable insertion mutations and reversions in the locus cut of Drosophila melanogaster: molecular mechanism of transposition memory

The segment of the locus cut containing the mobile genetic element mdg4 (gypsy) insertions which induce unstable ct^MR2 and ct^MRpN10 mutations has been cloned. Both mutations depend on the insertion of mdg4 into the same sequence, which coincides with that in ct⁶ allele. The ct^MRpN10 mutation differs from ct^MR2 by additional insertion of a novel mobile element jockey into mdg4. Jockey is 2.8 kb long, represented by ˜2–100 copies per genome, very homogeneous and lacks long terminal repeats (LTRs). The excision of mdg4 takes place in stable ct⁺ reversions. On the other hand, a complete single LTR is retained in the case of unstable ct reversions characterized by a high level of reverse directed transpositions of mdg4 into the locus cut. The LTR serves as a guide for reinsertion of mdg4 itself or mdg4 with jockey into the same site of the genome. A possible mechanism of transposition memory (homologous recombination with extrachromosomal circular DNA) is discussed.     

Genomic distribution of copia-like transposable elements in somatic tissues and during development of Drosophila melanogaster

The genomic distribution of elements of the copia, 412, B 104, mdg 1, mdg 4 and 1731 transposon families was compared by the Southern technique in DNA preparations extracted from brains, salivary glands and adult flies of two related Drosophila lines. The copia, 412 and mdg 1 sequences were also probed in DNA from sperm, embryos, and 1st and 2nd instar larvae. The homogeneity of the patterns observed shows that somatic transposition is unlikely to occur frequently. A correlation between mobility and the euchromatic or heterochromatic location of transposable elements is discussed. In addition, an explanation of the variable band intensities of transposable elements in Southern autoradiographs is proposed.     

Fluorescence in situ hybridization analysis of hobo, mdg1 and Dm412 transposable elements reveals genomic instability following the Drosophila melanogaster genome sequencing

The genome of Drosophila melanogaster strain y cn bw sp has been sequenced and the transposable elements insertion sites have been determined. We hybridized fluorescence-labeled probes directed to the hobo transposon, Dm412 and mdg1 retrotransposons to polytene chromosomes and compared the observed sites to those published in the annotated genome sequence. We observed an almost twofold increase in the number of hobo hybridization sites (46 found as compared to 24 annotated sites). There was no evidence that the hobo transposition rate is slowing over the 10-year period. The patterns of Dm412 and mdg1 sites have changed less dramatically since the time of genome sequencing. Three novel Dm412 hybridization sites were detected while 4 out of 30 annotated sites were missing. Only one additional mdg1 site was found, while 1 out of 29 annotated sites has been lost.     

Doc and copia instability in an isogenic Drosophila melanogaster stock

A high degree of heterogeneity and an overall increase in number of insertion sites of the mobile elements Doc and copia were revealed in one substock of an isogenic Drosophila melanogaster stock, while in two other substocks the distribution of copia sites was highly homogenous, but that of Doc sites was again heterogenous. We therefore concluded that copia was unstable in one of the substocks and Doc was unstable in all. Doc instability presumably arose earlier than copia instability. Doc and copia transpositions were directly observed in experiments with one substock. An abundance of copia insertions was revealed in the X chromosome where insertions with deleterious effects are exposed to selection in hemizygous condition. The locations of many other mobile elements (mdg1, mdg2, mdg3, mdg4, 297, B104, H.M.S. Beagle, I, P, BS, FB) were found to be conserved in each substock and did not differ between them, indicating that these mobile elements were stable. This homogeneity is a strong argument against any possibility of inadvertent contamination.     

Simultaneous transposition of different mobile elements: Relation to multiple mutagenesis in Drosophila melanogaster

Summary Several different transposition events occur simultaneously in one and the same germ cell, as we have found by analyzing different genetic systems in Drosophila melanogaster. (i) In unstable ct ^MR2 strains, stable reversions to ct ⁺ and changes in the type of ct mutation, which depend on an excision or transposition of the mobile element mdg4 (Gerasimova 1981; Gerasimova et al. 1984), are frequently accompanied by the appearance of novel mutations in different loci of the X chromosome. Some of these (sn, w, g) seem to be induced by the P-element and copia. (ii) A stable ct ^MR2 reversion to the wild type frequently coexists with an insertion of one to five copies of the P-element in the X-chromosome. Thus, the number of independent transposition events registered by genetic analysis and in situ hybridization may be as great as six. (iii) In two strains with double unstable mutations (cm, ct, and ct, r), double reversions to the wild type occurred at a high rate (80%–97% of total revertants). They frequently coexisted with novel strain-specific mutations. (iv) The stable strain ct ⁶ g²is destabilized by crossing with the MRh12/Cy strain (which contains a number of P-element copies). Both mutations begin to revert to the wild type. Of the revertants 50% have double reversions. Our experiments revealed a high specificity of insertion sites depending on the nature of transposon and the strain genotype. A possible role played by the burst of transposition in the evolution and possible mechanisms of transposition specificity are discussed.     

The <Emphasis Type="Italic">piggyBac</Emphasis> Transposon as a Tool in Genetic Engineering

Transposons are mobile genetic elements that are part of the genomic DNA of numerous organisms and belong to two classes. Unlike class I transposons, class II DNA transposons do not use the stage of RNA synthesis in their transition; they perform it by the cut-and-paste mechanism or with a replicative transposition. The integration of a DNA transposon in a new site results in the duplication of a target sequence on either side of a transposon, and its excision is, as a rule, associated with insertions and deletions. The piggyBac transposon isolated from the Trichoplusia ni moth differs from other mobile elements of its class. Due to its unique ability to leave no traces after excision from an insertion site and to perform successful transposition and transference of large DNA fragments, piggyBac is a convenient tool for the development of gene engineering approaches. The TTAA sequence serves as a target site for transposon integration: insertion in the AT-rich DNA regions is more frequent. The ability of piggyBac to be transferred to a new area independently of the cell apparatus and to restore a DNA site without error after excision lies in the mechanism of its transposition, which is discussed in detail in the present review. Along with other transposons and viruses, the piggyBac transposon is widely used in the transgenesis of various organisms; it also finds application in insertion mutagenesis and gene therapy.     

Passport, a native Tc1 transposon from flatfish, is functionally active in vertebrate cells

The Tc1/mariner family of DNA transposons is widespread across fungal, plant and animal kingdoms, and thought to contribute to the evolution of their host genomes. To date, an active Tc1 transposon has not been identified within the native genome of a vertebrate. We demonstrate that Passport, a native transposon isolated from a fish (Pleuronectes platessa), is active in a variety of vertebrate cells. In transposition assays, we found that the Passport transposon system improved stable cellular transgenesis by 40-fold, has an apparent preference for insertion into genes, and is subject to overproduction inhibition like other Tc1 elements. Passport represents the first vertebrate Tc1 element described as both natively intact and functionally active, and given its restricted phylogenetic distribution, may be contemporaneously active. The Passport transposon system thus complements the available genetic tools for the manipulation of vertebrate genomes, and may provide a unique system for studying the infiltration of vertebrate genomes by Tc1 elements.     

A two-element Enhancer-Inhibitor transposon system in Arabidopsis thaliana

The Enhancer-Inhibitor (En-I), also known as Suppressor-mutator (Spm-dSpm), transposable element system of maize was modified and introduced into Arabidopsis by Agrobacterium tumefaciens transformation. A stable En/Spm transposase source under control of the CaMV 35S promoter mediated frequent transposition of I/dSpm elements. Transposition occurred continuously throughout plant development over at least seven consecutive plant generations after transformation. New insertions were found at both linked and unlinked positions relative to a transposon donor site. The independent transposition frequency was defined as a transposition parameter, which quantified the rate of unique insertion events and ranged from 7.8% to 29.2% in different populations. An increase as well as a decrease in I/dSpm element copy number was seen at the individual plant level, but not at the population level after several plant generations. The continuous, frequent transposition observed for this transposon system makes it an attractive tool for use in gene tagging in Arabidopsis.     

The use of Tn5 transposable elements in a gene trapping strategy for the protozoan Leishmania

The use of transposable elements as a gene-trapping strategy is a powerful tool for gene discovery. Herein we describe the development of a transposable system, based on the bacterial Tn5 transposon, which has been used successfully in Leishmania braziliensis. The transposon carries the neomycin phosphotransferase gene, which is expressed only when inserted in-frame with a Leishmania gene present in the target DNA. Four cosmid clones from a L. braziliensis genomic library were used as targets in transposition reactions and four insertional libraries were constructed and transfected in L. braziliensis. Clones resistant to G418 were selected and analysed by immunofluorescence in order to identify the subcellular localisation of the protein coded by the trapped gene. A definitive subcellular localisation for neomycin phosphotransferase/targeted protein fusion was not obtained in any of the four Leishmania clones investigated. However, the constructed transposable element is highly efficient considering the frequency of insertion in large targets and is therefore a useful tool for functional genetic studies in Leishmania. Our data confirm the utility of the Tn5 transposon system for insertion of sequencing priming sites into target DNA. Furthermore, the high frequency of insertion and even distribution are important in studying genomic regions bearing long and polymorphic repetitive sequences.     

Interplasmid transposition of Drasopbila hobo elements in non-drosophilid insects

A modified hobo element from Drosophila melanogaster was introduced into embryos of the housefly, Musca domestica (family Muscidae) and the Queensland fruitfly, Bactrocera tryoni (family Tephritidae) to assess its ability to transpose. Hobo was capable of transposition in these species and transposition products had all of the hallmarks of hobo transposition products recovered from D. melanogaster, including the movement only of sequences precisely delimited by the inverted terminal repeats of hobo, the creation of an 8 by duplication of the insertion site and an absolute requirement for hobo-encoded transposase. Transposition of hobo into the target gene resulted in a non-random distribution of insertion sites, with 10 of 38 independent insertions into the same nucleotide position. The results indicate that hobo can transpose in heterologous species, further demonstrating the similarty of hobo to Ac (Activator) of Zea mays and Tam3 of Antirrhinum majus. Hobo has excellent potential to act as a gene vector or gene tagging agent in nondrosophilid insects.     

Mod(mdg4)-58.8, isoform of <Emphasis Type="Italic">mod(mdg4)</Emphasis> loci,directly interacts with MTACP1A and MTACP1B proteins of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

As a result of experiments in yeast two-hybrid system, coimmunoprecipitation of proteins from D. melanogaster embryo cell lysate, and immunostaining, it was shown for the first time that Mod(mdg4)-58.8 protein (isoform P), a product of mod(mdg4) locus, directly interacts with mtACP1A and mtACP1B proteins. These proteins are involved in de novo biosynthesis of fatty acids in mitochondria and are required for normal gametogenesis in males and females and, possibly, for the trachea development. This result expands the understanding of the role of mod(mdg4) locus products in the regulation of life activity of the eukaryotic cell.     

Autonomous transposition of gypsy mobile elements and genetic instability in Drosophila melanogaster

Summary The laboratory imitator strain (MS) of Drosophila melanogaster is characterized by an elevated frequency of spontaneous mutation (10^–3–10^–4). Mutations occur in both sexes at premeiotic stages of germ cell development. The increased mutability is a characteristic feature of MS itself, since it appears in the absence of outcrossing. Most of the mutations arising in this strain are unstable: reversions to wild type, high frequency mutation to new mutant states and replicating instability were observed. We have investigated the localization of the transposable genetic elements mdg1, 412, mdg3, gypsy (mdg4), copia and P in the X chromosomes of the MS and in the mutant lines y, ct, sbt derived from it by in situ hybridization. The P element was not found in any of these strains. The distributions of mdg1, 412, mdg3 and copia were identical in the X chromosomes of the MS and its derivatives. However, the sites of hybridization with gypsy differ in the various lines tested. In the polytene chromosomes of MS animals significant variation in location and number of copies of the gypsy element was demonstrated between different larvae; copy numbers as high as 30–40 were observed. These results suggest autonomous transposition of gypsy in the MS genome while several other mobile elements remain stable.     

Site-directed transposon integration in human cells

The Sleeping Beauty (SB) transposon is a promising gene transfer vector that integrates nonspecifically into host cell genomes. Herein, we attempt to direct transposon integration into predetermined DNA sites by coupling a site-specific DNA-binding domain (DBD) to the SB transposase. We engineered fusion proteins comprised of a hyperactive SB transposase (HSB5) joined via a variable-length linker to either end of the polydactyl zinc-finger protein E2C, which binds a unique sequence on human chromosome 17. Although DBD linkage to the C-terminus of SB abolished activity in a human cell transposition assay, the N-terminal addition of the E2C or Gal4 DBD did not. Molecular analyses indicated that these DBD-SB fusion proteins retained DNA-binding specificity for their respective substrate molecules and were capable of mediating bona fide transposition reactions. We also characterized transposon integrations in the presence of the E2C-SB fusion protein to determine its potential to target predefined DNA sites. Our results indicate that fusion protein-mediated tethering can effectively redirect transposon insertion site selection in human cells, but suggest that stable docking of integration complexes may also partially interfere with the cut-and-paste mechanism. These findings illustrate the feasibility of directed transposon integration and highlight potential means for future development.     

Efficient Insertion Mutagenesis Strategy for Bacterial Genomes Involving Electroporation of In Vitro-Assembled DNA Transposition Complexes of Bacteriophage Mu

An efficient insertion mutagenesis strategy for bacterial genomes based on the phage Mu DNA transposition reaction was developed. Incubation of MuA transposase protein with artificial mini-Mu transposon DNA in the absence of divalent cations in vitro resulted in stable but inactive Mu DNA transposition complexes, or transpososomes. Following delivery into bacterial cells by electroporation, the complexes were activated for DNA transposition chemistry after encountering divalent metal ions within the cells. Mini-Mu transposons were integrated into bacterial chromosomes with efficiencies ranging from 10⁴ to 10⁶ CFU/μg of input transposon DNA in the four species tested, i.e., Escherichia coli, Salmonella enterica serovar Typhimurium, Erwinia carotovora, and Yersinia enterocolitica. Efficiency of integration was influenced mostly by the competence status of a given strain or batch of bacteria. An accurate 5-bp target site duplication flanking the transposon, a hallmark of Mu transposition, was generated upon mini-Mu integration into the genome, indicating that a genuine DNA transposition reaction was reproduced within the cells of the bacteria studied. This insertion mutagenesis strategy for microbial genomes may be applicable to a variety of organisms provided that a means to introduce DNA into their cells is available.     

Hvmar1, a mariner‐like element from the tobacco budworm Heliothis virescens,can transpose in Drosophila melanogaster

Transposable elements of the mariner family are widespread and have been found in the genome of plants, animals and insects. However, most of these elements contain multiple inactivating mutations and so far, only three naturally occurring mariner elements are known to be functional. In a previous study, a mariner‐like element called Hvmar1 was discovered in the genome of the tobacco budworm Heliothis virescens. Further analysis of the Hvmar1 nucleotide sequence revealed the presence of 30‐bp imperfect inverted terminal repeats and an intact open reading frame, which is considered to encode a functional transposase. In the present study, we show that the Hvmar1 element is active using interplasmid transposition assays in Drosophila melanogaster embryos. When injected into Drosophila embryos, the helper plasmid produced a transposase that was able to mediate transposition of the Hvmar1 element from a donor to a target plasmid. The transposition efficiency of Hvmar1 in D. melanogaster is approximately 11‐fold lower than that of the well‐known Mos1 mariner transposon. However, this efficiency is comparable to those observed previously with Mos1 in non‐Drosophila insects. We identified 10 independent interplasmid transposition events, albeit the recovery of these events was rare. In each case the Hvmar1 element transposed in a precise manner, with the characteristic TA dinucleotides being duplicated on insertion. Furthermore, two of the target sites identified have been used previously by Mos1 for insertion. The active transposition of Hvmar1 in D. melanogaster provides a basis for examining the mobility of this element in its natural host as well as a starting point for comparative studies with Mos1 and other functional mariner transposons.     

IPB7 transposase behavior in Drosophila melanogaster and Aedes aegypti

Transposons are used in insect science as genetic tools that enable the transformation of insects and the identification and isolation of genes though their ability to insert in or near to them. Four transposons, piggyBac, Mos1, Hermes and Minos are commonly used in insects beyond Drosophila melanogaster with piggyBac, due to its wide host range and frequency of transposition, being the most commonly chosen. The utility of these transposons as genetic tools is directly proportional to their activity since higher transposition rates would be expected to lead to higher transformation frequencies and higher frequencies of insertion throughout the genome. As a consequence there is an ongoing need for hyperactive transposases for use in insect genetics, however these have proven difficult to obtain. IPB7 is a hyperactive mutant of the piggyBac transposase that was identified by a genetic screen performed in yeast, a mammalian codon optimized version of which was then found to be highly active in rodent embryonic stem cells with no apparent deleterious effects. Here we report the activity of IPB7 in D. melanogaster and the mosquito, Aedes aegypti. Somatic transposition assays revealed an increase in IPB7's transposition rate from wild-type piggyBac transposase in D. melanogaster but not Ae. aegypti. However the use of IPB7 in D. melanogaster genetic transformations produced a high rate of sterility and a low transformation rate compared to wild-type transposase. This high rate of sterility was accompanied by significant gonadal atrophy that was also observed in the absence of the piggyBac vector transposon. We conclude that IPB7 has increased activity in the D. melanogaster germ-line but that a component of the sterility associated with its activity is independent of the presence of the piggyBac transposon.