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.     

Cchobo, a hobo-related sequence in Ceratitis capitata

A hobo-related sequence, Cchobo, with high similarity to the Drosophila melanogaster HFL1 and hobo₁₀₈ elements was isolated from the medfly. Thirteen PCR-derived clones, which share 97.9–100% DNA identity, were sequenced, seven of which do not show frame-shift or stop codon mutations in their conceptual translations. The consensus sequence has 99.7% DNA identity with the D. melanogaster hobo element HFL1. In a phylogenetic analysis with other hobo-related elements, Cchobo clusters with the HFL1 and hobo₁₀₈ elements from D. melanogaster and hobo-related elements from D. simulans, D. mauritiana and Mamestra brassicae. These elements may have undergone horizontal transfer in the recent past. The genomic distribution of Cchobo was studied by FISH to mitotic and polytene chromosomes, which revealed that Cchobo is distributed within both the heterochromatin and euchromatin. Intra- and interstrain polymorphisms were detected both at euchromatic and heterochromatic sites. These findings suggest that active copies of the element may be present in the medfly genome.     

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.     

The occurrence of the transposable elementpogo inDrosophila melanogaster

We examined the genomic occurrence of the transposable elementpogo in over 120 strains ofDrosophila melanogaster, from around the world and from different eras. All had multiple copies of a 2.1 kilobase (kb)pogo element, and multiple copies of several size classes between 1.0 and 1.8 kb. There were differences between strains in intensities or presences of deletion-derivative size classes, suggesting current or recent mobility in the species. We were unable to find anypogo-hybridization in eight other species in the genus, in three subgenera, or in the relatedScaptomyza pallida. Thepogo element may be a ‘middle-aged’ element in the genome ofD. melanogaster, having entered the species since its divergence from its sibling species, but long before theP andhobo elements.     

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.     

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.     

Heat shocks do not mobilize mobile elements in genomes ofDrosophila melanogaster inbred lines

Summary Males of three inbred lines ofDrosophila melanogaster were heat-shocked 90 min at 37°C. The progenies from treated and untreated males mated with untreated females of the same line were checked for their chromosomal insertion patterns of various mobile elements by either in situ hybridization or Southern blots. No modification in the pattern of insertion of the elements studied was observed after heat treatment. Hence, heating males of our inbred lines did not mobilize mobile elements, contrary to recent reports on other lines ofDrosophila melanogaster.     

Insertion sites of the transposable elementmariner are fixed in the genome ofDrosophila sechellia

Summary The abundance of the transposable elementmariner differs dramatically in the genomes of the closely related speciesDrosophila simulans, D. mauritiana, D. sechellia, andD. melanogaster. Natural populations ofD. simulans andD. mauritiana have 1–10 and 20–30 copies per diploid genome, respectively, and the insertion sites are polymorphic. The element has not been found inD. melanogaster. In this paper we show thatD. sechellia, a species endemic to the Seychelles Islands, contains only twomariner elements that are at fixed sites in the genome. One element, inserted in chromosome 2R at 51A1–2, contains three deletions and is missing much of the 3 end. The other element, inserted in chromosome 3L at 64A10–11, is the full length of 1286 bp. Although the sequence of the full-length element is polymorphic in populations ofD. sechellia, at least some of the sequences are closely related to elements fromD. simulans andD. mauritiana that are known to be active. However, judging from the progeny of crosses betweenD. sechellia andD. simulans, the biological activity of the full-lengthD. sechellia element appears to be low, either because of the nucleotide sequence of the element or because of its position in the genome, or both.     

Transposable elements behavior following viral genomic stress inDrosophila melanogaster inbred line

To analyze the behavior of endogenous transposable elements under genomic stress, aDrosophila melanogaster inbred line was submitted to three kinds of viral perturbations. First, a retroviral plasmid containing the avian Rous Associated Virus type 2 (RAV-2) previously deleted for the viral envelope coding gene (env) was introduced by P element transformation into theDrosophila genome. An insertion of this avian retroviral sequence was detected byin situ hybridization in site 53C on polytene chromosome arm 2R. Second,Drosophila embryos were injected with RAV-2 particles produced by cell culture after transfection with the retroviral plasmid. Third, theDrosophila melanogaster inbred line was stably infected by the sigma native virus. It appears that neither the offspring of the flies in which the viral DNA was found integrated nor those from the infected sigma flies showed copia or mdgl element mobilization. Injection of the avian RAV-2 particles led, however, to the observation of somatic transpositions of mdgl element on the 2L chromosome, the copia element insertion pattern remaining stable. Thus, endogenous transposable elements show more instability in sublines injected with exogenous viral particles than in a transgenic subline containing a foreign viral insert, all transposable elements not being equally sensitive to such genomic stress. Correspondence to: I. Jouan-Dufournel     

Mobilization of a Hobo-related Sequence in the Genome of Drosophila simulans

The hobo transposable element can occur under three forms in the Drosophila genome: as a complete element (also called canonical), as internally deleted copies, or as hobo-related sequences (relics). Some evidence indicated that canonical elements and internally deleted copies are recent acquisitions of Drosophila genomes, while the “relics” are old components, normally degenerated and immobile. Here we present the characterization of a hobo-related sequence, found in the genome of a hypermutable strain of D. simulans, which insertion into the white locus raised a de novo white mutation. It is a shorter hobo related element presenting, overall, roughly 18% of divergence at the DNA level from the canonical hobo, with many indels that make clear this element is defective. However, its ITRs and flanking regions are extremely conserved. This is the first hobo “relic” showed to be mobilizable. We suggest, and point up some evidences, toward the idea that this sequence could have been mobilized by the canonical element. The presence of a similar “relic” element in D. sechellia allows us to suggest that these elements have been maintained mobilizable since the time of divergence between these species.     

Conservatism of sites of tRNA loci among the linkage groups of severalDrosophila species

Summary The sites of seven tRNA genes (Arg-2, Lys-2, Ser-2b, Ser-7, Thr-3, Thr-4, Val-3b) were studied by in situ hybridization.¹²⁵I-labeled tRNA probes fromDrosophila melanogaster were hybridized to spreads of polytene chromosomes prepared from fourDrosophila species representing different evolutionary lineages (D. melanogaster, Drosophila hydei, Drosophila pseudoobscura, andDrosophila virilis). Most tRNA loci occurred on homologous chromosomal elements of all four species. In some cases the number of hybridization sites within an element varied and sites on nonhomologous elements were found. It was observed that both tRNA ₂ ^Arg and tRNA ₂ ^Lys hybridized to the same site on homologous elements in several species. These data suggest a limited amount of exchange among different linkage groups during the evolution ofDrosophila species.     

Dynamic equilibrium between insertion and excision of P elements in highly inbred lines from an M′ strain of Drosophila melanogaster

Six highly inbred lines of Drosophila melanogaster extracted from an M strain (in the P/M system of hybrid dysgenesis) were studied for the evolution of the number and chromosomal location of complete and defective P elements through generations 52–200. These lines possessed full-sized P elements but differed in their cytotype (M or P). Three lines with P cytotype and full-sized P elements at site 1A had a constant P copy number over generations with low rates of insertion and excision. Three lines with M cytotype and at least one full-sized P element accumulated P copies over the generations and reached a plateau near generation 196, at which rates of transposition and excision were equal to 1.2 × 10^–3 to 3 × 10^–3 events per element per generation. At that time these three lines still presented an M cytotype, produced transposase, and were able to regulate P copy number. The similarity at equilibrium between insertion and excision rates was exactly what was expected from theoretical models for a self-regulated element. The large number of generations necessary to attain the equilibrium in copy number indicates, however, that caution may be de rigueur when testing theoretical models of copy-number containment based on transposition and excision-rate comparison.     

Rates of movement of transposable elements inDrosophila melanogaster

Mobilization rates of nine families of transposable elements (P, hobo, FB, gypsy, 412, copia, blood, 297, andjockey) were estimated by using 182 lines. Lines were started from a completely isogenic population ofDrosophila melanogaster, carrying the markersepia as an indicator of possible contamination, and have been accumulating spontaneous mutations independently for 80 generations of brother-sister (or two double-first-cousin) matings. Transposable element movements have been analyzed in complete genomes by the Southern technique. Mobilization was a rare event, with an average rate of 10^–5 per site per generation. The most active element wasFB. In contrast, the retroelementsgypsy andblood did not move at all. Most changes in restriction patterns were consistent with rearrangements rather than with true transposition. The euchromatic or heterochromatic location of elements was tested by comparing insertion patterns from adults and salivary glands. Certain putative rearrangements involved heterochromatic copies of the retroelements412, copia or297. Clustering of movement across families was observed, suggesting that movement of different families may be non-independent. An association between modified insertion patterns and mutant effects on quantitative traits shows that spontaneous transposition events cause continuous variation.     

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.     

The molecular analysis ofbrown eye color mutations isolated from geographically discrete populations ofDrosophila melanogaster

A large proportion of spontaneous mutations inDrosophila melanogaster strains of laboratory origin are associated with insertions of mobile DNA elements. As a first step toward determining whether spontaneous laboratory mutations are predictive for mutational events occurring in the wild, recessivebrown (bw) eye color mutants were isolated. By inbreeding the progeny of wild-caughtDrosophila melanogaster females,bw mutations were isolated from seven separate geographic sites distributed among Japan, California, Siberia and Hungary. Among a total of 14 mutations studied, no case of transposon mutagenesis was found. At least 4 mutations are associated with small deletions in thebw gene. The remainder are inseparable from wild-typebw by Southern analysis and are presumed to be basepair changes or very small indels. Although only two spontaneousbw mutants of laboratory origin have been analyzed molecularly, one is a mobile element insertion.     

Transposition of mobile elements gypsy (mdg4) and hobo in germ-line and somatic cells of a genetically unstable mutator strain of Drosophila melanogaster.

Summary Using the in situ hybridization technique, we have analysed the distribution of mobile elements in the X chromosomes of male offspring of individual mutator strain (MS) males crossed to attached-X females. The experiments demonstrate varying cytological localization of the mobile elements gypsy (mdg4) and hobo among different individuals. The other mobile elements investigated (mdgl, mdg3, 412, 297, copia, 17.6, Doc, H.M.S. Beagle, Springer, FB) display no changes in insertion sites. Such an experiment is equivalent to analysis of separate gametes of an MS individual. Thus, the ability of gypsy and hobo to transpose in germ-line cells is demonstrated directly. Transpositions occur at premeiotic stages of germ cell development, since they appear in clusters. Analysis of gypsy and hobo transposition events shows that they occur independently. The same experiment demonstrates that gypsy localization varies significantly between different salivary gland cells of an MS individual. Two types of gypsy hybridization sites can be distinguished: permanent sites, common to all cells, and additional ones varying between neighbouring salivary gland cells. These additional sites indicate gypsy transposition in somatic cells of the MS. Transposition of the hobo element in somatic cells has also been observed.     

aubergine mutations in Drosophila melanogaster impair P cytotype determination by telomeric P elements inserted in heterochromatin

Transposable P elements inserted in the heterochromatic Telomeric Associated Sequences on the X chromosome (1A site) of Drosophila melanogaster have a very strong capacity to elicit the P cytotype, a maternally transmitted condition which represses P element transposition and P-induced hybrid dysgenesis. This repressive capacity has previously been shown to be sensitive to mutant alleles of the gene Su(var)205, which encodes HP1 (Heterochromatin Protein 1), thus suggesting a role for chromatin structure in repression. Since an interaction between heterochromatin formation and RNA interference has been reported in various organisms, we tested the effect of mutant alleles of aubergine, a gene that has been shown to play a role in RNA interference in Drosophila, on the repressive properties of telomeric P elements. Seven out of the eight mutant alleles tested clearly impaired the repressive capacities of the two independent telomeric P insertions at 1A analyzed. P repression by P strains whose repressive capacities are not linked to the presence of P copies at 1A were previously found to be insensitive to Su(var)205; here, we show that they are also insensitive to aubergine mutations. These results strongly suggest that both RNA interference and heterochromatin structure are involved in the establishment of the P cytotype elicited by telomeric P elements, and reinforce the hypothesis that different mechanisms for repression of P elements exist which depend on the chromosomal location of the regulatory copies of P.Communicated by G. Reuter     

In situ study of chorion gene amplification in ovarian follicle cells ofDrosophila nasuta

The temporal and spatial pattern of replication of chorion gene clusters in follicle cells during oogenesis inDrosophila melanogaster andDrosophila nasuta was examined by [^3H thymidine autoradiography and byin situ hybridization with chorion gene probes. When pulse labelled with [³H] thymidine, the follicle cells from stage 10–12 ovarian follicles of bothDrosophila melanogaster and,Drosophila nasuta often showed intense labelling at only one or two sites per nucleus.In situ hybridization of chorion gene probes derived fromDrosophila melanogaster with follicle cell nuclei ofDrosophila melanogaster andDrosophila nasuta revealed these discrete [³H] thymidine labelled sites to correspond to the two amplifying chorion gene clusters. It appears, therefore, that in spite of evolutionary divergence, the organization and programme of selective amplification of chorion genes in ovarian follicle cells have remained generally similar in these two species. The endoreplicated and amplified copies of each chorion gene cluster remain closely associated but the two clusters occupy separate sites in follicle cell nucleus.     

Differing amounts of genetic polymorphism in testes and male accessory glands ofDrosophila melanogaster andDrosophila simulans

We surveyed genetic polymorphism by two-dimensional gel electrophoresis of male reproductive tract proteins in 20 isofemale lines each ofDrosophila melanogaster andDrosophila simulans. After classifying 244 such proteins ofDrosophila melanogaster and 271 ofDrosophila simulans by their distribution between testes and accessory glands within the reproductive tract, significant correlations were found between genetic polymorphism and tissue distribution. In both species, gland-specific proteins were significantly more polymorphic than testis-specific proteins, as well as those found in both testes and glands. Simultaneously, inDrosophila simulans, proteins found in roughly equivalent relative abundance in both testes and glands were significantly less variable than gland-specific and testis-specific proteins, as well as those with a quantitative difference in relative abundance between testes and glands. These correlations may reflect general differences in variability between extracellular and intracellular proteins and between proteins with broad as opposed to tissue-specific distributions.We thank the Natural Sciences and Engineering Research Council of Canada for financial support (Grant A0235 to R.S.S.).     

Changes in the chromosomal insertion pattern of the copia element during the process of making chromosomes homozygous in Drosophila melanogaster

In situ hybridization on polytene chromosomes of Drosophila melanogaster was used to compare the insertion patterns of copia and mdgl transposable elements on chromosome 2 in male gametes sampled by two different methods: (i) by crossing the males tested with females from a highly inbred line with known copia and mdgl insertion profiles; (ii) by crossing the same males with females from a marked strain, and analysing the resulting homozygous chromosomes. Crossing of the males with the inbred line led to homogeneous insertion profiles for both the copia and mdgl elements in larvae, thus giving an accurate estimation of the patterns in the two gamete classes of each male. Crossing with the marked strain led, however, to heterogeneity in insertion patterns of the copia transposable element, while no significant polymorphism was observed for mdgl. The use of balancer chromosomes is thus not an adequate way of inferring transposable element insertion patterns of Drosophila males, at least for the copia element. This technique could, however, be powerful for investigating the control of movements of this element.