Behavior of hobo and P transposons in yellow 2-717 unstable line of Drosophila melanogaster and its derivatives after crossing with a laboratory strain

Using fluorescent in situ hybridization technique (FISH), the frequency of hobo and P mobile elements transpositions on X chromosomes from the y ^2-717 , isolated from the Uman’ population of Drosophila melanogaster, as well as from its phenotypically normal and mutant derivatives, obtained as a result of crosses the males examined with the C(I)DX,ywf/Y females, was evaluated. It was demonstrated that the maximum frequency of hobo transpositions on X chromosomes of the males from derivative strains, subjected to repeated hobo-dysgenic crosses reached a value of 1.2 × 10^?2 per site per X chromosome per generation. The number of hobo copies in male X chromosomes from derivative strains was 3 times higher than in the original initial strain. Furthermore, the “old” hobo sites remained unchanged. In derivative strains, the frequency of hobo insertion was higher than that of excisions. One of the derivative strains, y ^1t-717a1k3-2 , was characterized by high intrastrain instability of hobo element localization. In the y ^2-717a1k3 and y ^1t-717a1k3-2 strains a large inversion, In(1)IB; 13CD, was described. At the absence of the full-sized P element in the strains involved in crosses, maximum frequency of P element transpositions in the derivative strains reached a value of 1.2 × 10^?2 per site per X chromosome per generation.     

Transposition of the hobo element in Drosophila melanogaster somatic cells

Somatic mutation and recombination test on wing cells of Drosophila melanogaster showed that the recombination frequency in the somatic tissues of strains studied correlated with the presence of a full-length copy of the hobo transposable element in the genome. Transposition of hobo in somatic tissue cells at a frequency 3.5 x 10-2 per site per X chromosome was shown by fluorescence in situ hybridization with salivary gland polytene chromosomes of larvae of one of the D. melanogaster strains having a full-length hobo copy.     

The effect of γ-radiation on induction of the hobo element transposition in Drosophila melanogaster

The transposition frequency of the hobo mobile element in four successive generations of Drosophila melanogaster strain y ^2-717 after an acute γ-irradiation with a dose of 30 Gr amounted to 7.5 × 10^?4 per site per genome per generation. Under the same conditions, PCR analysis of the genomic DNA of y ^2-717 flies detected new variants of defective hobo sequence. No changes in the hobo localization and PCR products compared with the control were detected in the case of single irradiation with doses of 3 and 30 Gr. The localizations of hobo element on polytene chromosomes of y ^2-717 strain did not change during 11 generations after five exposures of flies to 30 Gr. Irradiation of a highly unstable D. melanogaster strain y ⁺⁷⁴³ did not increase the number of families with mutant progeny, yet increased the total number of mutant descendants almost twofold, from 5 to 9%.     

The hobo transposable element has transposase-dependent and-independent excision activity in drosophilid species

Mobility of the hobo transposable element was determined for several strains of Drosophila melanogaster and several Drosophila species. Mobility was assessed by use of an in vivo transient assay in the soma of developing embryos, which monitored hobo excision from injected indicator plasmids. Excision was detected in a D. melanogaster strain (cn; ry ⁴²) devoid of endogenous hobo elements only after co-injection of a helper plasmid containing functional hobo transposase under either heat shock or normal promoter regulation. Excision was also detected in D. melanogaster without helper in strains known to contain genomic copies of hobo. In Drosophila species confirmed not to contain hobo, hobo excision occurred at significant rates both in the presence and absence of co-injected helper plasmid. In four of the seven species tested, excision frequencies were two- to fivefold lower in the presence of plasmid-borne hobo. hobo excision donor sites were sequenced in indicator plasmids extracted from D. melanogaster cn; ry ⁴² and D. virilis embryos. In the presence of hobo transposase, the predominant excision sites were identical in both species, having breakpoints at the hobo termini with an inverted duplication of proximal insertion site DNA. However, in the absence of hobo transposase in D. virilis, excision breakpoints were apparently random and occurred distal to the hobo termini. The data indicate that hobo is capable of functioning in the soma during embryogenesis, and that its mobility is unrestricted in drosophilids. Furthermore, drosophilids not containing hobo are able to mobilize hobo, presumably by a hobo-related cross-mobilizing system. The cross-mobilizing system in D. virilis is not functionally identical to hobo with respect to excision sequence specificity.     

Persistent locus-specific instability of yellow 2-717 and Notch Uc-1 in Drosophila melanogaster coincides with hobo multiplication

The presence of multiple copies of the hobo element in unstable yellow and Notch loci in y ^2-717 and Uc-1 Drosophila melanogaster stocks, respectively, was found according to FISH data. Locus-specific instability in these strains is caused by hobo multiplication in the respective loci and its subsequent recombination with neighboring hobo copies rather than its insertion (excision). Original Russian Text L.P. Zakharenko, L.V. Kovalenko, S.Mai, I.K. Zakharov, 2007, published in Tsitologiya, Vol. 49, No. 6, 2007.     

The hobo transposable element has transposase-dependent and-independent excision activity in drosophilid species

Mobility of the hobo transposable element was determined for several strains of Drosophila melanogaster and several Drosophila species. Mobility was assessed by use of an in vivo transient assay in the soma of developing embryos, which monitored hobo excision from injected indicator plasmids. Excision was detected in a D. melanogaster strain (cn; ry ⁴²) devoid of endogenous hobo elements only after co-injection of a helper plasmid containing functional hobo transposase under either heat shock or normal promoter regulation. Excision was also detected in D. melanogaster without helper in strains known to contain genomic copies of hobo. In Drosophila species confirmed not to contain hobo, hobo excision occurred at significant rates both in the presence and absence of co-injected helper plasmid. In four of the seven species tested, excision frequencies were two- to fivefold lower in the presence of plasmid-borne hobo. hobo excision donor sites were sequenced in indicator plasmids extracted from D. melanogaster cn; ry ⁴² and D. virilis embryos. In the presence of hobo transposase, the predominant excision sites were identical in both species, having breakpoints at the hobo termini with an inverted duplication of proximal insertion site DNA. However, in the absence of hobo transposase in D. virilis, excision breakpoints were apparently random and occurred distal to the hobo termini. The data indicate that hobo is capable of functioning in the soma during embryogenesis, and that its mobility is unrestricted in drosophilids. Furthermore, drosophilids not containing hobo are able to mobilize hobo, presumably by a hobo-related cross-mobilizing system. The cross-mobilizing system in D. virilis is not functionally identical to hobo with respect to excision sequence specificity.     

hobo-brothers elements and their time and place for horizontal transfer

Transposable elements (TEs) are ubiquitous components of nearly all genomes studied. These elements are highly variable in copy number, molecular structure and transposition strategies. They can move within and between genomes, thus increasing their copy numbers and avoiding being eliminated by stochastic and deterministic processes. hobo is a class II element isolated from Drosophila melanogaster. Previous phylogenetic analyses have shown that the canonical hobo element from D. melanogaster has a sister group formed by sequences found in D. willistoni (called howilli2) and D. mojavensis (called homo1). In the present study, we investigated 36 Drosophilidae species for sequences similar to howilli2 and homo1 using degenerate primers. Additionally, in silico searches were performed in 21 available Drosophila genomes. The obtained sequences formed a monophyletic sister group with the canonical hobo element; we termed these sequences ‘hobo-brothers’ elements. These elements showed a patch distribution and incongruities with the TE and host species phylogenies, suggesting possible cases of horizontal transfer (HT). Species that possess hobo-brothers sequences are from the New World, mainly Neotropical areas. In addition, the estimated divergence of the sequences found showed that these elements are or were recently active; the large number of HT events observed suggests that these elements could be experiencing an expansion process in Neotropical genomes. A comparison of these results with the literature is discussed with regard to the importance of the time and location of horizontal transposon transfer events.     

Characterization of permissivity for hobo-mediated gonadal dysgenesis in Drosophila melanogaster

The hobo transposon is responsible for one of the three hybrid dysgenic systems that have been described in Drosophila melanogaster. Most studies on the hobo dysgenic system have been carried out using the PM system as a reference. However, these two systems differ significantly. In particular, several studies have failed to find any correlation between the molecular structures of hobo elements, the instability of the transposon and the incidence of gonadal dysgenic (GD) sterility. On the other hand, no study of the ability of females to permit hobo activity in their progeny when they are crossed with males harboring active hobo elements (permissivity) has yet been reported. In order to investigate the parameters involved in hobo permissivity, four E strains were studied with regard to the molecular nature of their hobo sequences and the GD sterility induced by a controlled source of hobo transposase. We show that hobo permissivity varies both within and between E strains. Moreover, permissivity decreases with age in E females. Our results are discussed with respect to similar phenomena that have been described in relation to the reactivity of the IR dysgenic system.     

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.     

Quantitatively Increased Somatic Transposition of Transposable Elements in Drosophila Strains Compromised for RNAi

In Drosophila melanogaster, small RNAs homologous to transposable elements (TEs) are of two types: piRNA (piwi-interacting RNA) with size 23-29nt and siRNA (small interfering RNA) with size 19-22nt. The siRNA pathway is suggested to silence TE activities in somatic tissues based on TE expression profiles, but direct evidence of transposition is lacking. Here we developed an efficient FISH (fluorescence in Situ hybridization) based method for polytene chromosomes from larval salivary glands to reveal new TE insertions. Analysis of the LTR-retrotransposon 297 and the non-LTR retroposon DOC shows that in the argonaut 2 (Ago2) and Dicer 2 (Dcr2) mutant strains, new transposition events are much more frequent than in heterozygous strains or wild type strains. The data demonstrate that the siRNA pathway represses TE transposition in somatic cells. Nevertheless, we found that loss of one functional copy of Ago2 or Dcr2 increases somatic transpositions of the elements at a lower level depending on the genetic background, suggesting a quantitative role for RNAi core components on mutation frequency.     

Unraveling the evolutionary scenario of the hobo element in populations of Drosophila melanogaster and D. simulans in South America using the TPE repeats as markers

Transposable elements (TEs) are nucleotide sequences found in most studied genomes. These elements are highly diversified and have a large variation in nucleotide structure and mechanisms of transposition. hobo is a member of class II, belonging to hAT superfamily, described inDrosophila melanogaster, and it presents in its Open Reading Frame, a repetitive region encoding the amino acids threonine-proline-glutamic acid (TPE), which shows variability in the number of repeats in some regions of the world. Due to this variability some evolutionary scenarios of the hobo element are discussed, such as the scenario of the invasion of hobo element in populations ofD. melanogaster. In the present study, we investigated 22 DNA sequences of D. melanogaster and seven sequences ofD. simulans, both from South America, to check the number of repetitions of TPE, in order to clarify the evolutionary scenario of thehobo element in these populations. Our results showed a monomorphism in populations of both species in South America, with only three TPE repeats. Hence, we discuss and propose an evolutionary scenario of the invasion of the hobo element in populations of D. melanogaster and D. simulans.     

Change in distribution of mobile genetic elements in genome of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>: Cause or consequence of selection for quantitative traits?

The distribution pattern of the hobo transposon and Dm412 retrotransposon hybridization sites on the salivary gland polytene chromosomes from larvae of the Drosophila melanogaster isogenic strain 51 used to analyze the effect of the transposition of transposable elements (TEs) on selection for quantitative traits was studied. It was shown that no more than half of the Dm412 hybridization sites were retained 15 years after isogenization; the frequency of the Dm412 transposition varied from 2.0 × 10⁻⁴ to 8.8 × 10⁻⁵ sites per genome for generation depending on whether the appearance of the same hybridization sites in a part of individuals was considered as independent events or as the manifestation of the appearing sample heterogeneity. The distribution patterns of hobo hybridization sites in two isofemale strains derived from the isogenic strain 51 differed much more markedly; the number of the hobo sites in one of the derivative strains was threefold smaller than in the other one and only some of the sites were common. Within each derivative strain, the TE distribution was uniform, which suggests that inbreeding had no effect on Dm412 activity in this strain. The rates of change in the distribution patterns of various TE in the strain 51 corresponded to their spontaneous transposition rates. Since the isogenic strain accumulates polymorphism in the TE distribution without selection, the TEs are more likely to be the markers of selection events rather than their inducers. Thus, when studying the effects of various environmental factors on TE transposition even in isogenic strains, it is necessary to perform additional close inbreeding to reduce the potential polymorphism.     

Characterization of permissivity for hobo -mediated gonadal dysgenesis in Drosophila melanogaster

The hobo transposon is responsible for one of the three hybrid dysgenic systems that have been described in Drosophila melanogaster. Most studies on the hobo dysgenic system have been carried out using the PM system as a reference. However, these two systems differ significantly. In particular, several studies have failed to find any correlation between the molecular structures of hobo elements, the instability of the transposon and the incidence of gonadal dysgenic (GD) sterility. On the other hand, no study of the ability of females to permit hobo activity in their progeny when they are crossed with males harboring active hobo elements (permissivity) has yet been reported. In order to investigate the parameters involved in hobo permissivity, four E strains were studied with regard to the molecular nature of their hobo sequences and the GD sterility induced by a controlled source of hobo transposase. We show that hobo permissivity varies both within and between E strains. Moreover, permissivity decreases with age in E females. Our results are discussed with respect to similar phenomena that have been described in relation to the reactivity of the IR dysgenic system. Received: 17 August 1998 / Accepted: 17 December 1998     

Pattern of transposable elements distribution on <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> polytene chromosomes before and after quantitative trait selection

The effect of selecting the length of a radial vein on the distribution of hybridization sites for P and hobo transposons and mdg1 and mdg2 retrotransposons on polytene chromosomes of Drosophila melanogaster salivary glands was studied. The pattern of the transposable element (TEs) distribution was polymorphic in both parental and selected strains. The similarity in mdg1 and mdg2 distribution between strains selected in one direction was closer than between strains selected in the opposite direction, but the selected strains were closer to each other than to the parental strain, regardless of direction of selection. No new mdg2 hybridization sites that would be absent from the control were found in the selected strains compared to the control. The number of mdg1 and hobo hybridization sites was more selected in strains in the (+) direction than in the (−) direction. The mobility of hobo copies in the strains correlated with the presence of its full-sized copy in the genome. The polymorphism of all TEs studied except for mdg1 was higher in strains selected in the (+) direction than in the (−) direction. These results suggest that some TEs migrate over the genome independently of selection, whereas the other are markers of evolutionary events rather than their causes.     

A genetic system to detect mitotic recombination between repeated chromosomal sequences in Drosophila Schneider line 2 cells

In order to study mitotic homologous recombination in somatic Drosophila melanogaster cells in vitro and to learn more on the question how recombination is influenced by mutagens, a genetic system was developed where spontaneous and drug-induced recombination could be monitored. Two recombination reporter substrates were stably introduced in multiple copies into the genome of established D. melanogaster Schneider line 2 cells: one plasmid (pSB310) contained the 5′ and 3′ deleted neomycin phosphoribosyltransferase alleles neoL and neoR as direct repeats; the other (pSB485) contained similar deletions (lacZL and lacZR) of the β-galactosidase gene (lacZ). Restoration of a functional neo gene upon mitotic recombination between homologous sequences allowed direct selection for the event, whereas recombination in single cells harbouring the integrated lacZ-based reporter plasmid was detected by histochemical staining or flow cytometric analysis (FACS). The neo-based construct in the clonal transgenic cell line 44CD4 showed a spontaneous recombination frequency of 2.9×10⁻⁴, whereas the 485AD1 cell line harbouring the lacZ-based construct exhibited a frequency of 2.8×10⁻⁴. The alkylating agents EMS and MMS and the clastogen mitomycin C were able to induce recombination in the 485AD1 cell line in a dose-dependent manner. The results obtained from these studies suggest that the transgenic cell lines are potentially useful tools for identifying agents which stimulate direct repeat recombination in somatic Drosophila cells.     

Rearrangements at a hobo element inserted into the first intron of the singed gene in the unstable sn 49 system of Drosophila melanogaster

The cytological structure of the X chromosome and the DNA organisation of the singed locus were examined in five singed bristle mutants of Drosophila melanogaster. These mutants are all derived from the unstable mutant singed-49, isolated from a wild population in the Russian Far East in 1975. Rearrangements were found at a site within the first intron of the singed gene, where a hobo element is inserted in these mutants. One rearrangement, which is associated with a strong bristle phenotype, has an inversion between 2D and the location of singed at 7D, which separates the singed promoter from the singed coding region. Two phenotypically wild-type derivatives have smaller rearrangements within the first intron which do not appear to interfere with singed expression. Two derivatives with bristle phenotypes have more complex rearrangements, and one of them shows a dominant or antimorphic phenotype. DNA blotting and in situ hybridisation experiments show that, in addition to these rearrangements at a hobo element inserted at singed, other hobo elements in these strains have been mobilised. This system is therefore similar to others in which functional hobo elements continue to transpose, resulting in elevated rates of mutation and chromosome rearrangement.     

Polymorphism of <Emphasis Type="Italic">yellow</Emphasis> locus in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> mutants from natural populations

We have studied the molecular characteristics of the yellow locus (y; 1–0.0), which determines the body color of phenotypically wild-type and mutant alleles isolated in different years from geographically distant populations of Drosophila melanogaster. According to the Southern blot, data restriction maps of the yellow locus of all examined strains differ from one another, as well as from Oregon stock. FISH analysis shows that, in the neighborhood of the yellow locus in the X chromosome, neither P nor hobo elements are found in y^1–775 stock, while only hobo is found in these region in y^1–859 and y^1–866 stocks, only the P element is found in y⁺sn⁸⁴⁹ stock, and both elements are found in y^1–719 stock. Thus, all yellow mutants studied are of independent origin. Locus yellow located on the end of X chromosome (region 1A5–8 on the cytologic map) carries significantly more transposon than retrotransposon induced mutations compared to the white locus (region 3C2). It is possible that, at the ends of Drosophila melanogaster chromosomes, transposons are more active than retrotransposons.     

The Reason for the Preservation of High Instability at the yellow Gene in Drosophila melanogaster Strains Isolated from the Natural Population of Uman' during the “Mode for Mutation”

Mobile genetic elements are responsible for most spontaneous mutations in Drosophila melanogaster. The discovered in the 1980s phenomenon of frequent change of the wild-type yellow phenotype for a mutant one, and vice-versa, in strains of D. melanogaster isolated from the Uman' natural population can be, according to our data, explained by repeated inversions and reinversions of the gene regulatory region located between the two copies of the hobo transposon. However, most molecular genetic events accompanying the process can occur without the phenotype change. After several generations, the strains, remaining phenotypically unchanged, can possess different molecular genetic properties with respect to yellow. Using genetically homogenous or isogenic strains for the genetic analysis or for production of the new plant cultivars or animal breeds, geneticists and breeders often face the problem of stability of the strains. In the present study, the mechanism underlying the generation of instability at the yellowlocus of D. melanogaster determined by the hobo-induced genome instability is described.     

Induction of transpositions of hobo-elements in chronically irradiated cells of dysgenetic and non-dysgenetic individuals of Drosophila melanogaster

This paper studies the effect of chronic Γ-radiation of different intensities on the induction of hobo-elements in cells of dysgenetic and non-dysgenetic drosophila species. The level of gonadal atrophy, DNA damage, and mutability of the mini-white locus is estimated. It is shown that the frequency of displacements of the hobo-elements increases with the increase in the chronic irradiation dose, where an essential role belongs to the maternal effect. The level of DNA damage in the cells of embryos and larvae varies and depends on the conditions of induction of hobo-transposons. Analysis of the PCR products showed that chronic irradiation in a certain range of accumulated doses is able to induce formation of new copies of the hobo-elements. At the same time, the structure of deleted hobo-sequences may vary in response to higher doses of irradiation.     

The evolutionary genetics of thehobo transposable element in theDrosophila melanogaster complex

Hobo elements are a family of transposable elements found inDrosophila melanogaster and its three sibling species:D. simulans, D. mauritiana andD. sechellia. Studies inD. melanogaster have shown thathobo may be mobilized, and that the genetic effects of such mobilizations included the general features of hybrid dysgenesis: mutations, chromosomal rearrangements and gonadal dysgenis in F1 individuals. At the evolutionary level somehobo-hybridizing sequences have also been found in the other members of themelanogaster subgroup and in many members of the relatedmontium subgroup. Surveys of older collected strains ofD. melanogaster suggest that completehobo elements were absent prior to 50 years ago and that they have recently been introduced into this species by horizontal transfer. In this paper we review our findings and those of others, in order to precisely describe the geographical distribution and the evolutionary history ofhobo in theD. melanogaster complex. Studies of the DNA sequences reveal a different level of divergence between the groupD. melanogaster, D. simulans andD. mauritiana and the fourth speciesD. sechellia. The hypothesis of multiple transfers in the recent past into theD. melanogaster complex from a common outside source is discussed.