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.     

Excision of the Drosophila transposable element mariner: identification and characterization of the Mos factor.

Genetic and molecular evidence presented in this paper demonstrate that the Mos factor for inherited mosaicism is a special copy of the transposable element mariner. Mosaicism observed in the presence of the Mos (Mosaic) factor results from a high frequency of excision of the mariner element from an insertion site near the white-eye gene in Drosophila mauritiana. The Mos factor promotes the excision of mariner elements from genomic insertion sites other than the site in wpch, and it also promotes its own loss from the genome. Putative transpositions of Mos to new genomic sites have also been observed. A copy of mariner present at a particular site in a Mos strain has been shown to be missing in derived strains in which the Mos factor has been lost, and in strains with putative transpositions. We propose that this copy of mariner is identical to the Mos factor.     

Identification of Nucleotide Substitutions Necessary for Trans-Activation of Mariner Transposable Elements in Drosophila: Analysis of Naturally Occurring Elements

Six copies of the mariner element from the genomes of Drosophila mauritiana and Drosophila simulans were chosen at random for DNA sequencing and functional analysis and compared with the highly active element Mos1 and the inactive element peach. All elements were 1286 base pairs in length, but among them there were 18 nucleotide differences. As assayed in Drosophila melanogaster, three of the elements were apparently nonfunctional, two were marginally functional, and one had moderate activity that could be greatly increased depending on the position of the element in the genome. Both molecular (site-directed mutagenesis) and evolutionary (cladistic analysis) techniques were used to analyze the functional effects of nucleotide substitutions. The nucleotide sequence of the element is the primary determinant of function, though the activity level of elements is profoundly influenced by position effects. Cladistic analysis of the sequences has identified a T----A transversion at position 1203 (resulting in a Phe----Leu amino acid replacement in the putative transposase) as being primarily responsible for the low activity of the barely functional elements. Use of the sequences from the more distantly related species, Drosophila yakuba and Drosophila teissieri, as outside reference species, indicates that functional mariner elements are ancestral and argues against their origination by a novel mutation or by recombination among nonfunctional elements.     

Sequence Analysis of Active Mariner Elements in Natural Populations of Drosophila Simulans

Active and inactive mariner elements from natural and laboratory populations of Drosophila simulans were isolated and sequenced in order to assess their nucleotide variability and to compare them with previously isolated mariner elements from the sibling species Drosophila mauritiana and Drosophila sechellia. The active elements of D. simulans are very similar among themselves (average 99.7% nucleotide identity), suggesting that the level of mariner expression in different natural populations is largely determined by position effects, dosage effects and perhaps other factors. Furthermore, the D. simulans elements exhibit nucleotide identities of 98% or greater when compared with mariner elements from the sibling species. Parsimony analysis of mariner elements places active elements from the three species into separate groups and suggests that D. simulans is the species from which mariner elements in D. mauritiana and D. sechellia are most likely derived. This result strongly suggests that the ancestral form of mariner among these species was an active element. The two inactive mariner elements sequenced from D. simulans are very similar to the inactive peach element from D. mauritiana. The similarity may result from introgression between D. simulans and D. mauritiana or from selective constraints imposed by regulatory effects of inactive elements.     

Germline Transformation of Drosophila Virilis with the Transposable Element Mariner

An important goal in molecular genetics has been to identify a transposable element that might serve as an efficient transformation vector in diverse species of insects. The transposable element mariner occurs naturally in a wide variety of insects. Although virtually all mariner elements are nonfunctional, the Mos1 element isolated from Drosophila mauritiana is functional. Mos1 was injected into the pole-cell region of embryos of D. virilis, which last shared a common ancestor with D. mauritiana 40 million years ago. Mos1 PCR fragments were detected in several pools of DNA from progeny of injected animals, and backcross lines were established. Because G(0) lines were pooled, possibly only one transformation event was actually obtained, yielding a minimum frequency of 4%. Mos1 segregated in a Mendelian fashion, demonstrating chromosomal integration. The copy number increased by spontaneous mobilization. In situ hybridization confirmed multiple polymorphic locations of Mos1. Integration results in a characteristic 2-bp TA duplication. One Mos1 element integrated into a tandem array of 370-bp repeats. Some copies may have integrated into heterochromatin, as evidenced by their ability to support PCR amplification despite absence of a signal in Southern and in situ hybridizations.     

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.     

Mariner, Mos and associated aberrant traits in Drosophila mauritiana

A suite of aberrant genetic traits, including increased mutation rate, sex-limited mutation and distorted transmission ratios, was produced among progeny of genetic crosses between two strains of Drosophila mauritiana when a paternally contributed Mos excision factor is placed into a non-Mos genetic background. In the reciprocal cross, involving maternally contributed Mos and Mos associated cytoplasm, the same genetic abnormalities are not observed. Differential effects on mariner excision in germ-line versus somatic tissue are apparent. Because Mos is known to influence the mobility of the mariner transposable element, these traits may be associated with mariner excision and/or transposition.     

Self-inflicted wounds, template-directed gap repair and a recombination hotspot. Effects of the mariner transposase

Aberrant repair products of mariner transposition occur at a frequency of approximately 1/500 per target element per generation. Among 100 such mutations in the nonautonomous element peach, most had aberrations in the 5' end of peach (40 alleles), in the 3' end of peach (11 alleles), or a deletion of peach with or without deletion of flanking genomic DNA (29 alleles). Most mariner mutations can be explained by exonuclease "nibble" and host-mediated repair of the double-stranded gap created by the transposase, in contrast to analogous mutations in the P element. In mariner, mutations in the 5' inverted repeat are smaller and more frequent than those in the 3' inverted repeat, but secondary mutations in target elements with a 5' lesion usually had 3' lesions resembling those normally found at the 5' end. We suggest that the mariner transposase distinguishes between the 5' and 3' ends of the element, and that the 5' end is relatively more protected after strand scission. We also find: (1) that homolog-dependent gap repair is a frequent accompaniment to mariner excision, estimated as 30% of all excision events; and (2) that mariner is a hotspot of recombination in Drosophila females, but only in the presence of functional transposase.     

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.     

Genetic and molecular investigations on the endogenous mobile elements of non-drosophilid fruitflies

A syndrome of abnormal genetic effects, resembling Drosophila hybrid dysgenesis, occurs in Ceratitis capitata when strains of different origin are mated. The pattern of abnormal traits observed appears to be the phenotypic expression of a complex interacting dysgenic system of inducer and suppressor effects; probably more than one system is activated in the crosses. This suggests that different systems of mobile elements occur in different strains and populations of C. capitata. Using a PCR primer specific to the ITR sequence of a deleted element, full length mariner elements were isolated from C. capitata, Ceratitis rosa, and Trirhithrum coffeae. Very high similarities were found in inter- and intraspecific comparisons of the elements. The majority of these elements contained deletions and frame-shifts. However, one clone Ccmar1.18, from C. capitata, was found to possess an uninterrupted ORF coding for 338 amino acids with ∼60% similarity to the Mos1 element of Drosophila mauritiana. Database searches and phylogenetic analyses showed that the mariner elements isolated in the present study are representatives of Robertson's mellifera mariner subfamily. The copy numbers of the elements within each species are very different, ranging from about 10 in T. coffeae to 5000 in C. rosa. This revised version was published online in August 2006 with corrections to the Cover Date.     

Factors affecting transposition of the Himar1 mariner transposon in vitro.

Mariner family transposable elements are widespread in animals, but their regulation is poorly understood, partly because only two are known to be functional. These are particular copies of the Dmmar1 element from Drosophila mauritiana, for example, Mos1, and the consensus sequence of the Himar1 element from the horn fly, Haematobia irritans. An in vitro transposition system was refined to investigate several parameters that influence the transposition of Himar1. Transposition products accumulated linearly over a period of 6 hr. Transposition frequency increased with temperature and was dependent on Mg2+ concentration. Transposition frequency peaked over a narrow range of transposase concentration. The decline at higher concentrations, a phenomenon observed in vivo with Mos1, supports the suggestion that mariners may be regulated in part by "overproduction inhibition." Transposition frequency decreased exponentially with increasing transposon size and was affected by the sequence of the flanking DNA of the donor site. A noticeable bias in target site usage suggests a preference for insertion into bent or bendable DNA sequences rather than any specific nucleotide sequences beyond the TA target site.     

Active mariner transposable elements are widespread in natural populations of Drosophila simulans

The occurrence of active, or autonomous, mariner elements was investigated by crossing white-peach mutant Drosophila simulans females with wild-type males from various geographic origins. From a total of 194 experimental crosses only 17 failed to produce progeny with eye mosaicism (MOS, i.e. pigmented spots in otherwise white-peach eyes). Therefore, active mariner elements inducing somatic excision of the copy inserted at the white locus are abundant in all populations sampled. In the experimental crosses the frequency of mosaic offspring ranged from 0 to 100%, showing that the phenotypic expression is highly variable. The MOS phenotype, measured by the number of spots on the eyes, is quite variable within the progeny of single crosses. Although a difference was observed in the average MOS score (percentage of mosaic flies) between northern and southern populations of France, there was no indication of long range variation between geographic populations. Neither was there a systematic difference between recently collected populations and samples kept several years as isofemale lines.     

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.     

A highly repetitive, mariner-like element in the genome of Hyalophora cecropia.

A highly repetitive DNA element, homologous to the mariner transposon of Drosophila mauritiana was found in the intron of the gene for cecropin A, an antibacterial peptide from the Cecropia moth. The mariner-like elements (MLE) represent a homogeneous population with a copy number of about 1000/genome. Sequencing analysis showed it to be 1255 base pairs long, including 38-base pair terminal inverted repeats. The MLE contains a defective reading frame. Nevertheless, the putative product is clearly homologous to the predicted translation product encoded by mariner. In consonance is also the fact that the inverted repeats are highly conserved between the two elements and that the overall DNA homology is 48%. Since the mariner element is present in several Drosophila species closely related to Drosophila melanogaster and since MLE is present in the lepidopteran Cecropia, a route of horizontal transfer is indicated rather than vertical transmission from a common ancestor. This suggests the possible use of mariner for the construction of an interspecies vector.     

白背飞虱、灰飞虱mariner转座子的初步研究

根据毛里塔尼亚果蝇Drosophila mauritania的mariner转座子序列设计简并引物,以白背飞虱Sogatella furcifera和灰飞虱Laodelphax striatellus基因组DNA为模板,通过PCR反应扩增出了约500 bp的条带,经回收、克隆、测序,获得其核苷酸序列,与毛里塔尼亚果蝇mariner序列相比,核苷酸同源性均为71.3%,氨基酸同源性分别为69.5%和72%,证明白背飞虱和灰飞虱体内确实存在mariner转座子。     

Polymorphisms flanking the mariner integration sites in the rice gall midge (Orseolia oryzae Wood-Mason) genome are biotype-specific.

In an effort to study genome diversity within and between the Indian biotypes of the Asian rice gall midge, Orseolia oryzae, a major insect pest of rice, we made use of mariner transposable element integration site polymorphisms. Using degenerate primers, the design of which is based on mariner sequences, we amplified a ca. 450 bp mariner sequence from the rice gall midge. The mariner sequence showed homology with that of a mariner element isolated from the Hessian fly, Mayetiola destructor, a major dipteran pest of wheat. Southern hybridization, using this mariner fragment as a probe, revealed that the mariner elements are moderately to highly repetitive in the rice gall midge genome. Based on the sequence information of this 450-bp PCR-amplified fragment, outward-directed primers were designed and used in an inverse PCR (iPCR) to amplify the DNA flanking the conserved regions. To study the regions flanking the mariner integration sites, we employed a novel PCR-based approach: a combination of sequence specific amplification polymorphism (SSAP) and amplified fragment length polymorphism (AFLP). The outward-directed mariner-specific primer was used in combination with adapter-specific primers with 1-3 selective nucleotides at their 3' ends. The amplification products were resolved on an agarose gel, Southern-transferred onto nylon membranes, and probed with the iPCR fragment. Results revealed biotype-specific polymorphisms in the regions flanking the mariner integration sites, suggesting that mariner elements in the rice gall midge may be fixed in a biotype-specific manner. The implications of these results are discussed in the context of biotype differentiation.     

Phylogenetic Analysis of Mos1-Like Transposable Elements in the Drosophilidae

We have performed a phylogenetic analysis of 59 mariner elements in 14 Drosophilidae species that are related to the active Drosophila mauritiana Mos1 element. This includes 38 previously described sequences and 21 new sequences amplified by PCR from 10 species. Most of the elements detected are nonfunctional due to several frameshifts and deletions. They have been subdivided into four groups according to specific signatures in the nucleotidic and amino acid sequences. The mean nucleotide diversity is 4.8 ± 0.1% and reflects mainly the divergence of inactive elements over different periods. Although this probably gives rise to occasional homoplasies between distantly related taxa, the elements of each species remain grouped together. Horizontal transfer, reported previously between D. mauritiana and Zaprionus tuberculatus, can be extended to Z. verruca, while the Mos1-like element of Z. indianus belongs to another group. Interpretation of the phylogeny leads to a comparison of the influence of common ancestral sequences and putative horizontal transfers. Received: 31 May 1999 / Accepted: 28 June 1999     

Cloning and characterization of Acmar1, a mariner-like element in the asiatic honey bee,Apis cerana japonica (Hymenoptera,Apocrita)

A mariner-like element was cloned from the genome of the Asiatic honey bee, Apis cerana japonica (Hymenoptera, Apocrita). The (composite) clone, named Acmar1, was 1,378 bp long, and encoded 336 amino acids corresponding to a transposase-like putative polypeptide in a single open reading frame. The D,D(34)D motif, the catalytic domain of the mariner transposase, was present, although there was a deletion of five amino acid residues within it as compared with the active transposase in Drosophila mauritiana. Nineteen-bp-long imperfect inverted terminal repeat-like sequences flanked by TA dinucleotides, the typical target site for mariner insertion, were observed. Southern blot analysis using a fragment covering two-thirds of the Acmar1 transposase coding sequence as a probe indicated the presence of multiple Acmar1-like elements in the genome. Maximum-parsimony phylogenetic analysis based on the transposase amino acid sequences of insect mariner-like elements revealed that Acmar1 is a member of the mellifera subfamily.     

The Transposable Element Mariner Mediates Germline Transformation in Drosophila Melanogaster

A vector for germline transformation in Drosophila melanogaster was constructed using the transposable element mariner. The vector, denoted pMlwB, contains a mariner element disrupted by an insertion containing the wild-type white gene from D. melanogaster, the β-galactosidase gene from Escherichia coli and sequences that enable plasmid replication and selection in E. coli. The white gene is controlled by the promoter of the D. melanogaster gene for heat-shock protein 70, and the β-galactosidase gene is flanked upstream by the promoter of the transposable element P as well as that of mariner. The MlwB element was introduced into the germline of D. melanogaster by co-injection into embryos with an active mariner element, Mos1, which codes for a functional transposase and serves as a helper. Two independent germline insertions were isolated and characterized. The results show that the MlwB element inserted into the genome in a mariner-dependent manner with the termini of the inverted repeats inserted at a TA dinucleotide. Both insertions exhibit an unexpected degree of germline and somatic stability, even in the presence of an active mariner element in the genetic background. These results demonstrate that the mariner transposable element, which is small (1286 bp) and relatively homogeneous in size among different copies, is nevertheless capable of promoting the insertion of the large (13.2 kb) MlwB element. Because of the widespread phylogenetic distribution of mariner among insects, these results suggest that mariner might provide a wide hostrange transformation vector for insects.