Reduced Germline Mobility of a Mariner Vector Containing Exogenous DNA: Effect of Size or Site?

Germline mobilization of the transposable element mariner is severely inhibited by the insertion of a 4.5- to 11.9-kb fragment of exogenous DNA into a unique SacI site approximately in the middle of the 1286-bp element. In the presence of transposase driven by the germline-specific hsp26-sgs3 promoter, mobilization of the MlwB construct (containing a 11.9-kb insertion) is detected at low frequency. Analysis of a mobilized MlwB element indicated that mobilization is mediated by the mariner transposase. However, transposed MlwB elements are also defective in germline mobilization. Rare, transposase-induced germ-line excision events were also recovered for such vectors. The estimated rate of excision is <0.1% per chromosome per generation. Excision appears to be accompanied by gap repair if a suitable template is available. The data imply that the reduced mobility of mariner vectors with exogenous DNA in the SacI site results from disruption of sequences necessary for efficient mobilization. The relative stability may be a valuable property in the uses of mariner-like elements in genetic engineering of insects of economic importance.     

Insertion and Excision of the Transposable Element Mariner in Drosophila

The transposable element mariner is active in both germline and somatic cells of Drosophila mauritiana. Activity of the element is greatly enhanced in the presence of Mos1, a genetic factor identified as an autonomous copy of mariner. A strain of D. mauritiana containing Mos1 and other copies of mariner was used to initiate a screen for visible mutations. More than 20 mutations were obtained, including alleles of white, yellow and vermilion. Six alleles were characterized at the molecular level, and all were found to contain a mariner element inserted into the affected gene. Four insertions into the white locus were sequenced to determine the exact site of insertion of mariner. There appears to be little sequence specificity requirement for mariner insertion, other than an absolute requirement for the dinucleotide TA, which is duplicated upon insertion. Sequences of phenotypically wild-type germline and somatic revertants obtained from various white alleles, including the previously isolated wpch allele, were obtained using the polymerase chain reaction. Mariner excision is imprecise in both germline and soma, and the most frequent excision events are the same in the two tissues. Mutant derivatives of wpch were also studied, and were found to exhibit a wide range of molecular structures and phenotypes.     

Introduction of the Transposable Element Mariner into the Germline of Drosophila Melanogaster

A chimeric white gene (wpch) and other constructs containing the transposable element mariner from Drosophila mauritiana were introduced into the germline of Drosophila melanogaster using transformation mediated by the P element. In the absence of other mariner elements, the wpch allele is genetically stable in both germ cells and somatic cells, indicating that the peach element (i.e., the particular copy of mariner inserted in the wpch allele) is inactive. However, in the presence of the active element Mos1, the wpch allele reverts, owing to excision of the peach element, yielding eye-color mosaics and a high rate of germline reversion. In strains containing Mos1 virtually every fly is an eye-color mosaic, and the rate of wpch germline reversion ranges from 10 to 25%, depending on temperature. The overall rates of mariner excision and transposition are approximately sixfold greater than the rates in comparable strains of Drosophila simulans. The activity of the Mos1 element is markedly affected by position effects at the site of Mos1 insertion. In low level mosiac lines, dosage effects of Mos1 are apparent in the heavier level of eye-color mosaicism in Mos1 homozygotes than in heterozygotes. However, saturation occurs in high level mosaic lines, and then dosage effects are not observed. A pBluescribe M13+ plasmid containing Mos1 was injected into the pole plasm of D. melanogaster embryos, and the Mos1 element spontaneously integrated into the germline at high efficiency. These transformed strains of D. melanogaster presently contain numerous copies of mariner and may be useful in transposon tagging and other applications.     

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.     

Unexpected stability of mariner transgenes in Drosophila

A number of mariner transformation vectors based on the mauritiana subfamily of transposable elements were introduced into the genome of Drosophila melanogaster and examined for their ability to be mobilized by the mariner transposase. Simple insertion vectors were constructed from single mariner elements into which exogenous DNA ranging in size from 1.3 to 4.5 kb had been inserted; composite vectors were constructed with partial or complete duplications of mariner flanking the exogenous DNA. All of the simple insertion vectors showed levels of somatic and germline excision that were at least 100-fold lower than the baseline level of uninterrupted mariner elements. Although composite vectors with inverted duplications were unable to be mobilized at detectable frequencies, vectors with large direct duplications of mariner could be mobilized. A vector consisting of two virtually complete elements flanking exogenous DNA yielded a frequency of somatic eye-color mosaicism of approximately 10% and a frequency of germline excision of 0.04%. These values are far smaller than those observed for uninterrupted elements. The results imply that efficient mobilization of mariner in vivo requires the presence and proper spacing of sequences internal to the element as well as the inverted repeats.     

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.     

Evolution of the Transposable Element Mariner in Drosophila Species

The distribution of the transposable element mariner was examined in the genus Drosophila. Among the eight species comprising the melanogaster species subgroup, the element is present in D. mauritiana, D. simulans, D. sechellia, D. yakuba and D. teissieri, but it is absent in D. melanogaster, D. erecta and D. orena. Multiple copies of mariner were sequenced from each species in which the element occurs. The inferred phylogeny of the elements and the pattern of divergence were examined in order to evaluate whether horizontal transfer among species or stochastic loss could better account for the discontinuous distribution of the element among the species. The data suggest that the element was present in the ancestral species before the melanogaster subgroup diverged and was lost in the lineage leading to D. melanogaster and the lineage leading to D. erecta and D. orena. This inference is consistent with the finding that mariner also occurs in members of several other species subgroups within the overall melanogaster species group. Within the melanogaster species subgroup, the average divergence of mariner copies between species was lower than the coding region of the alcohol dehydrogenase (Adh) gene. However, the divergence of mariner elements within species was as great as that observed for Adh. We conclude that the relative sequence homogeneity of mariner elements within species is more likely a result of rapid amplification of a few ancestral elements than of concerted evolution. The mariner element may also have had unequal mutation rates in different lineages.     

Mechanisms regulating the copy numbers of six LTR retrotransposons in the genome of Drosophila melanogaster

There has been debate over the mechanisms that control the copy number of transposable elements in the genome of Drosophila melanogaster. Target sites in D. melanogaster populations are occupied at low frequencies, suggesting that there is some form of selection acting against transposable elements. Three main theories have been proposed to explain how selection acts against transposable elements: insertions of a copy of a transposable element are selected against; chromosomal rearrangements caused by ectopic exchange between element copies are selected against; or the process of transposition itself is selected against. The three theories give different predictions for the pattern of transposable element insertions in the chromosomes of D. melanogaster. We analysed the abundance of six LTR (long terminal repeat) retrotransposons on the X and fourth chromosomes of multiple strains of D. melanogaster, which we compare with the predictions of each theory. The data suggest that no one theory can account for the insertion patterns of all six retrotransposons. Comparing our results with earlier work using these transposable element families, we find a significant correlation between studies in the particular model of copy number regulation supported by the proportion of elements on the X for the different transposable element families. This suggests that different retrotransposon families are regulated by different mechanisms.     

Purified mariner (Mos1) transposase catalyzes the integration of marked elements into the germ-line of the yellow fever mosquito, Aedes aegypti

Derivatives of the mariner transposable element, Mos1, from Drosophila mauritiana, can integrate into the germ-line of the yellow fever mosquito, Aedes aegypti. Previously, the transposase required to mobilize Mos1 was provided in trans by a helper plasmid expressing the enzyme under the control of the D. psuedoobscura heat-shock protein 82 promoter. Here we tested whether purified recombinant Mos1 transposase could increase the recovery of Ae. aegypti transformants. Mos1 transposase was injected into white-eyed, kh(w)/kh(w), Ae. aegypti embryos with a Mos1 donor plasmid containing a copy of the wild-type allele of the D. melanogaster cinnabar gene. Transformed mosquitoes were recognized by partial restoration of eye color in the G(1) animals and confirmed by Southern analyses of genomic DNA. At Mos1 transposase concentrations approaching 100 nM, the rate of germ-line transformants arising from independent insertions in G(0) animals was elevated 2-fold compared to that seen in experiments with helper plasmids. Furthermore, the recovery of total G(1) transformants was increased 7.5-fold over the frequency seen with co-injected helper plasmid. Southern blot analyses and gene amplification experiments confirmed the integration of the transposons into the mosquito genome, although not all integrations were of the expected cut-and-paste type transposition. The increased frequency of germ-line integrations obtained with purified transposase will facilitate the generation of Mos1 transgenic mosquitoes and the application of transgenic approaches to the biology of this important vector of multiple pathogens.     

Molecular and Functional Analysis of the Mariner Mutator Element Mos1 in Drosophila

The white-peach allele in Drosophila results from insertion of the transposable element mariner. The particular copy that is inserted in white-peach is an inactive copy referred to as the peach element. The peach element is excised at a high rate in the presence of active copies of mariner located elsewhere in the genome, and the excision of peach in somatic cells is recognized phenotypically by the occurrence of eye-color mosaicism in white-peach flies. Active mariner elements identified by their ability to induce high levels of white-peach mosaicism are denoted Mos (Mosaic) factors. We have sequenced and functionally analyzed the factor Mos1 originally identified in Drosophila mauritiana. The Mos1 element is 1286 base pairs in length, the same length as the peach element. It differs from the peach element in 11 nucleotide positions distributed throughout its length, including four amino acid replacements in the long open reading frame. Analysis of chimeric constructs between Mos1 and peach implies that functionally important differences occur in both the 5' and 3' halves of Mos1. A mariner element identical in sequence to Mos1 yields lower levels of mosaicism in transformants, implying that adjacent flanking sequences have important effects on Mos1 activity. Another mariner element, designated Ma351, isolated from a nonmosaic strain of D. mauritiana, differs from Mos1 in just three nucleotide positions. When introduced into the germline, Ma351 yields various levels of white-peach mosaicism depending on insertion site. These results imply that the activity of mariner elements is determined jointly by their own nucleotide sequences, by the effects of adjacent flanking sequences, and by longer-range position effects.     

Interplasmid transposition of the mariner transposable element in non-drosophilid insects

Plasmid-based transposition assays were performed in developing embryos of the Australian sheep blowfly Lucilia cuprina and the Queensland fruit fly Bactrocera tryoni, using the mariner transposable element from Drosophila mauritiana. Transposition products were recovered that were identical in structure to those recovered from D. melanogaster. Only sequences delimited by the mariner terminal repeats were transposed and all insertions occurred at TA residues, and duplicated these. These are the hallmarks of mariner transpositions observed in the chromosomes of D. melanogaster and D. mauritiana, indicating that the plasmid-based assays are accurate indicators of mariner transposition activity. The recovery of precise transposition products from L. cuprina and B. tryoni demonstrates that mariner should be capable of producing germline transformants in these species. The results obtained from these assays suggests that they will be extremely useful in determining if mariner can transpose in other non-drosophilid insects and for investigating factors that might affect mariner transposition frequency.     

Post-integration stability of piggyBac in Aedes aegypti

The post-integration activity of piggyBac transposable element gene vectors in Aedes aegypti mosquitoes was tested under a variety of conditions. The embryos from five independent transgenic lines of Ae. aegypti, each with a single integrated non-autonomous piggyBac transposable element gene vector, were injected with plasmids containing the piggyBac transposase open-reading frame under the regulatory control of the Drosophila melanogaster hsp70 promoter. No evidence for somatic remobilization was detected in the subsequent adults whereas somatic remobilization was readily detected when similar lines of transgenic D. melanogaster were injected with the same piggyBac transposase-expressing plasmid. Ae. aegypti heterozygotes of piggyBac reporter-containing transgenes and piggyBac transposase-expressing transgenes showed no evidence of somatic and germ-line remobilization based on phenotypic and molecular detection methods. The post-integration mobility properties of piggyBac in Ae. aegypti enhance the utility of this gene vector for certain applications, particularly those where any level of vector remobilization is unacceptable.     

Detection of de novo insertion of the medaka fish transposable element Tol2

Tol2 is a terminal-inverted-repeat transposable element of the medaka fish Oryzias latipes. It is a member of the hAT (hobo/Activator/Tam3) transposable element family that is distributed in a wide range of organisms. We here document direct evidence for de novo insertion of this element. A Tol2 clone marked with the bacterial tetracycline-resistance gene was microinjected into fertilized eggs together with a target plasmid, and the plasmid was recovered from embryos. The screening of plasmid molecules after transformation into Escherichia coli demonstrated transposition of tet into the plasmid and, by inference, precise insertion of Tol2 in medaka fish cells. De novo excision of Tol2 has previously been demonstrated. The present study provides direct evidence that the Tol2 element has the entire activity necessary for cut-and-paste transposition. Some elements of the mariner/Tc1 family, another widespread group, have already been applied to development of gene tagging systems in vertebrates. The Tol2 element of the hAT family, having different features from mariner/Tc1 family elements, also has potential as an alternative gene tagging tool in vertebrates.     

An insertion of Escherichia coli transposable element IS1K into the site immediately before tetracycline-resistance determinant of Bacillus subtilis chromosomal DNA fragment in cloning in E. coli

In cloning in Escherichia coli C600 of a 4.5-kbp HindIII DNA fragment with the tetracycline-resistance determinant (tetBS908) from Bacillus subtilis GSY908 chromosome using a plasmid vector, a 5.2-kbp HindIII DNA fragment was also isolated at a ratio of 2 to 89. The two independently obtained 5.2-kbp fragments were an insertion derivative of the 4.5-kbp fragment and carried E. coli transposable element ISlK, which was inserted at the same site immediately before tetBS908 in the same direction. For the ISlK insertions, the 8-bp sequence CAAATTTT was used as a target, this having no similarity to any published sequences.