New regulatory regions of Drosophila 412 retrotransposable element generated by recombination

There are no doubts that transposable elements (TEs) have greatly influenced genomes evolution. They have, however, evolved in different ways throughout mammals, plants, and invertebrates. In mammals they have been shown to be widely present but with low transposition activity; in plants they are responsible for large increases in genome size. In Drosophila, despite their low amount, transposition seems to be higher. Therefore, to understand how these elements have evolved in different genomes and how host genomes have proposed to go around them, are major questions on genome evolution. We analyzed sequences of the retrotransposable elements 412 in natural populations of the Drosophila simulans and D. melanogaster species that greatly differ in their amount of TEs. We identified new subfamilies of this element that were the result of mutation or insertion-deletion process, but also of interfamily recombinations. These new elements were well conserved in the D. simulans natural populations. The new regulatory regions produced by recombination could give rise to new elements able to overcome host control of transposition and, thus, become potential genome invaders.     

Polymorphism in structure of the retrotransposable element 412 in Drosophila simulans and D. melanogaster populations.

The structure of the 412 retrotransposable element was investigated in various natural populations of D. melanogaster and D. simulans by a restriction enzyme analysis. We show that although the canonical structure of the 412 element was the same in both species, a high structural polymorphism existed with various rearranged elements. A 412 family was thus composed of heterogeneous copies of different sizes, with a large proportion of full-size copies. D. simulans had more rearranged copies than D. melanogaster, with some specific copies, such as a 5.6-kb BsrBI fragment, present in all populations of D. simulans. Full-size and rearranged copies were detected in both the euchromatin and the heterochromatin, with many rearranged copies in D. simulans, suggesting a recent mobilization of the 412 element in this species.     

Transposition rate of the 412 retrotransposable element is independent of copy number in natural populations of Drosophila simulans

The transposition and excision rates of the 412 retrotransposable element were estimated in five populations of Drosophila simulans differing in their average 412 copy numbers, which ranged from 2 to 54. The transposition rate was found to equal 1 x 10(-3) to 2 x 10(-3), independently of copy number. No excision was detected. These values eliminate autoregulation as a force maintaining copy number of the 412 element in natural populations of D. simulans.      

Evolutionary stability of the R1 retrotransposable element in the genus Drosophila

R1 is a non-long terminal repeat (non-LTR) retrotransposable element that inserts into a specific sequence of insect 28S ribosomal RNA genes. We have previously shown that this element has been maintained through vertical transmission in the melanogaster species subgroup of Drosophila. To address whether R1 elements have been vertically transmitted for longer periods of evolutionary time, the analysis has been extended to 11 other species from four species groups of the genus Drosophila (melanogaster, obscura, testecea, and repleta). All sequenced elements appeared functional on the basis of the preservation of their open-reading frames and consistently higher rate of substitution at synonymous sites relative to replacement sites. The phylogenetic relationships of the R1 elements from all species analyzed were congruent with the species phylogenies, suggesting that the R1 elements have been vertically transmitted since the inception of the Drosophila genus, an estimated 50-70 Mya. The stable maintenance of R1 through the germ line appears to be the major mechanism for the widespread distribution of these elements in Drosophila. In two species, D. neotestecea of the testecea group and D. takahashii of the melanogaster group, a second family of R1 elements was also present that differed in sequence by 46% and 31%, respectively, from the family that was congruent with the species phylogeny. These second families may represent occasional horizontal transfers or, alternatively, they could reflect the ability of R1 elements to diverge into new families within a species and evolve independently.      

Distribution of Drosophila melanogaster transposable element sequences in species of the obscura group

Fifteen species belonging to the obscura group of the genus Drosophila were screened for sequences homologous to Drosophila melanogaster transposable elements (TEs) as an initial step in the examination of the possible occurrence of TEs at chromosomal inversion breakpoints. Blots of genomic DNAs from species of the obscura group were hybridized at three different stringencies with 14 probes representing the major families of TEs described in D. melanogaster. The probe DNAs included copia, gypsy, 412, 297, mdg1, mdg3, 3S18, F, G, I, jockey, P, hobo, and FB3. D. melanogaster TEs were not well represented in the species of the obscura group analyzed. The TEs that were observed generally exhibited heterogeneous distributions, with the exception of F, gypsy and 412 which were ubiquitous, and 297, G, Sancho 2, hobo and FB which were not detected.by A. Bird     

Insertion polymorphism of retrotransposable elements in populations of the insular, endemic species Drosophila madeirensis

The insertion site numbers of the retrotransposable elements (TE) 412, gypsy and bilbo were determined in individuals of five distinct natural populations of the endemic species Drosophila madeirensis from the island of Madeira. The TE distributions were compared to those of the paleartic, widespread and phylogenetically closely related species, D. subobscura. In situ hybridization and Southern blots showed that in D. madeirensis the number of insertion sites ranged between 10 and 15, three and six, and 35 and 42 for elements 412, gypsy and bilbo, respectively. The corresponding values for D. subobscura were similar. Two of these elements, 412 and gypsy, had very few insertions in the heterochromatin, unlike bilbo, which displayed a high heterochromatic insertion number. The Southern band polymorphism was very high, leading to within-population variation of 97.2%, whatever the population and the TE concerned. Using the polymorphic TE insertion sites as markers to analyse population structure by AMOVA, adapted for RAPD (Randomly Amplified Polymorphic DNA) data, we found small but significant genetic differences between the populations on Madeira. This slight differentiation, coupled with similar copy numbers for each TE between populations, suggests that the D. madeirensis species consists of a single, only slightly subdivided population. These data also show that insular populations and endemic species of Drosophila can have as many copies of TEs as more widespread species.     

Distribution of transposable elements in Drosophila species

We present a global analysis of the distribution of 43 transposable elements (TEs) in 228 species of the Drosophila genus from our data and data from the literature. Data on chromosome localization come from in situ hybridization and presence/absence of the elements from southern analyses. This analysis shows great differences between TE distributions, even among closely related species. Some TEs are distributed according to the phylogeny of their host specie; others do not entirely follow the phylogeny, suggesting horizontal transfers. A higher number of insertion sites for most TEs in the genome of D. melanogaster is observed when compared with that in D. simulans. This suggests either intrinsic differences in genomic characteristics between the two species, or the influence of differing effective population sizes, although biases due to the use of TE probes coming mostly from D. melanogaster and to the way TEs are initially detected in species cannot be ruled out. Data on TEs more specific to the species under consideration are necessary for a better understanding of their distribution in organisms and populations. This revised version was published online in July 2006 with corrections to the Cover Date.     

Transposition induction of the mobile genetic element Dm412 in the Drosophila genome using heat shock]

Males of Drosophila melanogaster isogenic line with oligogene mutation radius incompletus (ri) were exposed to standard heat-shock (SHS: t = 37 degrees C, 90 min) and heavy heat-shock (SHS: three-fold transfer of males from t = 37 degrees C, 2h, t0t = 4 degrees C 1 h, and back). At F1 of the treated males with untreated females of the same isogenic line mass transpositions of MGE Dm412 were found. The new positions of MGE seem to be not random, and 5 "hot sites" of transpositions were detected. The probabilities of transpositions were estimated after SHS and HHS and in control sample. They were, correspondingly, 3.4 x 10(-2), 8.7 x 10(-2) and less than 4.1 x 10(-4) transpositions per genome, per site occupied, per generation. Therefore, as a result of HS treatment, the probabilities of transpositions were two orders of magnitude increased as compared to control, directly at next generation after induction. Comparison of these results with those obtained after step-wise temperature treatment shows that induction is dependent rather of "stressor effect" of temperature treatment than of treatment way used.     

Evolution of the LINE-like I element in the Drosophila melanogaster species subgroup

LINE-like retrotransposons, the so-called I elements, control the system of I-R (inducer-reactive) hybrid dysgenesis in Drosophila melanogaster. I elements are present in many Drosophila species. It has been suggested that active, complete I elements, located at different sites on the chromosomes, invaded natural populations of D. melanogaster recently (1920–1970). But old strains lacking active I elements have only defective I elements located in the chromocenter. We have cloned I elements from D. melanogaster and the melanogaster subgroup. In D. melanogaster, the nucleotide sequences of chromocentral I elements differed from those on chromosome arms by as much as 7%. All the I elements of D. mauritiana and D. sechellia are more closely related to the chromosomal I elements of D. melanogaster than to the chromocentral I elements in any species. No sequence difference was observed in the surveyed region between two chromosomal I elements isolated from D. melanogaster and one from D. simulans. These findings strongly support the idea that the defective chromocentral I elements of D. melanogaster originated before the species diverged and the chromosomal I elements were eliminated. The chromosomal I elements reinvaded natural populations of D. melanogaster recently, and were possibly introduced from D. simulans by horizontal transmission.     

Structure of circular copies of the 412 transposable element present in Drosophila melanogaster tissue culture cells, and isolation of a free 412 long terminal repeat

We have isolated, from Drosophila melanogaster tissue culture cells, extrachromosomal circular forms of the transposable element 412, and have cloned some of them in bacteriophage lambda. A total of 24 clones have been analysed in detail by restriction and heteroduplex mapping. Seventeen clones are virtually identical, and contain complete 412 elements with one copy of the long terminal direct repeat (LTR). The remaining seven clones are all different and contain various rearrangements. Four have deletions, two have some 412 sequence substituted by other DNA and one has both an inversion and a deletion. The clone containing the inversion has two LTRs in inverted orientation and separated by a few thousand bases of 412 DNA. The base sequences of the two LTRs in this clone, and of the LTR in one of the 17 clones containing complete elements are very similar to that of the 481 base-pair LTR of a genomic 412 element. We have found no evidence, in either cloned or uncloned material, for 412 elements with two LTRs as a tandem direct repeat. We have found that there are several "free" 412 LTRs in genomic DNA from D. melanogaster strains Canton S and Oregon R, and from D. melanogaster tissue culture cells. We have cloned and sequenced one of these free LTRs. It is 475 base-pairs long and is flanked by a direct repeat four base-pairs long. This sequence differs from that of the 481 base-pair repeat at 16 places including a ten base deletion.     

Evolution of the transposable element mariner in the Drosophila melanogaster species group

The population biology and molecular evolution of the transposable element mariner has been studied in the eight species of the melanogaster subgroup of the Drosophila subgenus Sophophora. The element occurs in D. simulans, D. mauritiana, D. sechellia, D. teissieri, and D. yakuba, but is not found in D. melanogaster, D. erecta, or D. orena. Sequence comparisons suggest that the mariner element was present in the ancestor of the species subgroup and was lost in some of the lineages. Most species contain both active and inactive mariner elements. A deletion of most of the 3 end characterizes many elements in D. teissieri, but in other species the inactive elements differ from active ones only by simple nucleotide substitutions or small additions/deletions. Active mariner elements from all species are quite similar in nucleotide sequence, although there are some-species-specific differences. Many, but not all, of the inactive elements are also quite closely related. The genome of D. mauritiana contains 20–30 copies of mariner, that of D. simulans 0–10, and that of D. sechellia only two copies (at fixed positions in the genome). The mariner situation in D. sechellia may reflect a reduced effective population size owing to the restricted geographical range of this species and its ecological specialization to the fruit of Morinda citrifolia.     

Evolution of the transposable element Uhu in five species of Hawaiian Drosophila

The complete DNA sequence of three independent isolates of Uhu, a member of the Tc1-like class of transposable elements from D. heteroneura (Uhu-1, Uhu-3, and Uhu-4), has been determined. These isolates have between 95 and 96.4% nucleotide sequence identity indicating that Uhu is well conserved within this species. A comparison of the DNA sequences of Uhu and the D. melanogaster Hb1 transposable element shows that the nucleotide substitution rate for Uhu is comparable to the synonymous rate for the Adh gene in these species. Uhu has been identified in four other species of endemic Hawaiian Drosophila, D. silvestris, D. differens, D. planitibia and D. picticornis, and nine Uhu elements were isolated from genomic libraries of these four species. A 444 base pair region from within the coding region of the Uhu element, with well conserved ends, was amplified by the polymerase chain reaction and used for sequence comparison of elements from different species. The analysis of the sequence similarities between the elements within and between the species shows a grouping of the two pairs of most closely related species (D. heteroneura and D. silvestris, and D. differens and D. planitibia), but shows a much larger variation within the most recently diverged species (D. heteroneura and D. silvestris) than expected. There are extensive nucleotide substitutions and deletions in the Uhu elements from D. picticornis showing that they are degenerating and being lost in this species. These observations indicate that the Uhu element has been transmitted vertically and that transposition may have been activated at the time of formation of each species as it colonized the newly formed islands of the Hawaiian archipelago.     

Distribution of transposable elements in neotropical species of Drosophila

The phylogenetic distribution of transposable families, P, gypsy, hobo, I, and mariner has been analyzed in 33 species of 11 groups of neotropical Drosophila and a Drosophilidae species Zygotrica vittimaculosa, using squash blot and dot blot. Genomic DNA of almost all neotropical species tested hybridized with gypsy probe and some species showed a particularly strong hybridization signal, as D. gaucha, D. virilis, and species of flavopilosa group. The hobo element was restricted to melanogaster group and some strains of D. willistoni. Only D. simulans DNA showed hybridization to mariner probe in all species tested and D. simulans and D. melanogaster showed hybridization with I element probe. P element homologous sequence was present in D. melanogaster and all species and strains of the willistoni and saltans groups tested. The presence of at least one P-homologous sequence was detected in Drosophila mediopunctata. This one was the only P-bearing species of all six tested from the tripunctata group. Four different pairs of primers homologous to segments of the canonical sequence of D. melanogaster's P were used to amplify specific sequences from D. mediopunctata DNA, showing the occurrence of seemingly well-conserved P-homologous sequences. This revised version was published online in July 2006 with corrections to the Cover Date.     

Distribution of xanthurenic acid glucoside in species of the genus Drosophila

Xanthurenic acid 8-O-β-d-glucoside is a side metabolite of the tryptophan-xanthommatin pathway in Drosophila melanogaster, that is accumulated in some eye-color mutants. Since this compound had only been found in this species, we have analyzed 29 other Drosophila species for the occurrence of this compound using cellulose thin-layer chromatography. Xanthurenic acid glucoside was detected in 10 of them (8 of them belong to the Sophophora subgenus). In these species xanthurenic acid glucoside, as well as other metabolites of the final steps of the dihydroxanthommatin pathway (3-hydroxykynurenine, xanthurenic acid and dihydroxanthommatin) were quantitated in order to gain a better understanding of the relationships of the metabolites of this pathway.     

Size matters: non-LTR retrotransposable elements and ectopic recombination in Drosophila

The Drosophila melanogaster genome contains approximately 100 distinct families of transposable elements (TEs). In the euchromatic part of the genome, each family is present in a small number of copies (5-150 copies), with individual copies of TEs often present at very low frequencies in populations. This pattern is likely to reflect a balance between the inflow of TEs by transposition and the removal of TEs by natural selection. The nature of natural selection acting against TEs remains controversial. We provide evidence that selection against chromosome abnormalities caused by ectopic recombination limits the spread of some TEs. We also demonstrate for the first time that some TE families in the Drosophila euchromatin appear to be only marginally affected by purifying selection and contain many copies at high population frequencies. We argue that TEs in these families attain high population frequencies and even reach fixation as a result of low family-wide transposition rates leading to low TE copy numbers and consequently reduced strength of selection acting on individual TE copies. Fixation of TEs in these families should provide an upward pressure on the size of intergenic sequences counterbalancing rapid DNA loss through small deletions. Copy-number-dependent selection on TE families caused by ectopic recombination may also promote diversity among TEs in the Drosophila genome.