The transposable elements of the Drosophila melanogaster euchromatin: a genomics perspective

Background

Transposable elements are found in the genomes of nearly all eukaryotes. The recent completion of the Release 3 euchromatic genomic sequence of Drosophila melanogaster by the Berkeley Drosophila Genome Project has provided precise sequence for the repetitive elements in the Drosophila euchromatin. We have used this genomic sequence to describe the euchromatic transposable elements in the sequenced strain of this species.

Results

We identified 85 known and eight novel families of transposable element varying in copy number from one to 146. A total of 1,572 full and partial transposable elements were identified, comprising 3.86% of the sequence. More than two-thirds of the transposable elements are partial. The density of transposable elements increases an average of 4.7 times in the centromere-proximal regions of each of the major chromosome arms. We found that transposable elements are preferentially found outside genes; only 436 of 1,572 transposable elements are contained within the 61.4 Mb of sequence that is annotated as being transcribed. A large proportion of transposable elements is found nested within other elements of the same or different classes. Lastly, an analysis of structural variation from different families reveals distinct patterns of deletion for elements belonging to different classes.

Conclusions

This analysis represents an initial characterization of the transposable elements in the Release 3 euchromatic genomic sequence of D. melanogaster for which comparison to the transposable elements of other organisms can begin to be made. These data have been made available on the Berkeley Drosophila Genome Project website for future analyses.     

The pogo transposable element family of Drosophila melanogaster.

Summary A 190 by insertion is associated with the white-eosin mutation in Drosophila melanogaster. This insertion is a member of a family of transposable elements, pogo elements, which is of the same class as the P and hobo elements of D. melanogaster. Strains typically have many copies of a 190 by element, 10–15 elements 1.1–1.5 kb in size and several copies of a 2.1 kb element. The smaller elements all appear to be derived from the largest by single internal deletions so that all elements share terminal sequences. They either always insert at the dinucleotide TA and have perfect 21 bp terminal inverse repeats, or have 22 by inverse repeats and produce no duplication upon insertion. Analysis by DNA blotting of their distribution and occupancy of insertion sites in different strains suggests that they may be less mobile than P or hobo. The DNA sequence of the largest element has two long open reading frames on one strand which are joined by splicing as indicated by cDNA analysis. RNAs of this strand are made, whose sizes are similar to the major size classes of elements. A protein predicted by the DNA sequence has significant homology with a human centrosomal-associated protein, CENP-B. Homologous sequences were not detected in other Drosophila species, suggesting that this transposable element family may be restricted to D. melanogaster.     

Transposition of the vertebrate Tol2 transposable element in Drosophila melanogaster

The Tol2 element is a transposon found from a genome of a vertebrate, a small teleost medaka fish. Tol2 encodes a gene for a transposase which is active in vertebrate animals so far tested; for instance, in fish, frog, chicken and mammals, and transgenesis methods using Tol2 have been developed in these model vertebrates. However, it has not been known whether Tol2 can transpose in animals other than vertebrates. Here we report transposition of Tol2 in an invertebrate Drosophila melanogaster. First, we injected a transposon donor plasmid containing a Tol2 construct and mRNA encoding the Tol2 transposase into Drosophila eggs, and found that the Tol2 construct could be excised from the plasmid. Second, we crossed the injected flies, raised the offspring, and found that the Tol2 construct was integrated into the genome of germ cells and transmitted to the next generation. Finally, we constructed a Tol2 construct containing the white gene and injected the transposon donor plasmid and the transposase mRNA into fertilized eggs from the white mutant. We analyzed their offspring, and found that G1 flies with wild type red eyes could be obtained from 35% of the injected fly. We cloned and sequenced 34 integration loci from these lines and showed that these insertions were indeed created through transposition and distributed throughout the genome. Our present study demonstrates that the medaka fish Tol2 transposable element does not require vertebrate-specific host factors for its transposition, and also provides a possibility that Tol2 may be used as a new genetic tool for transgenesis and genome analysis in Drosophila.     

P transposable element in Drosophila melanogaster: horizontal transfer]

The P transposable element family in Drosophila melanogaster is responsible for the syndrome of hybrid dysgenesis which includes chromosomal rearrangements, male recombination, high mutability and temperature sensitive agametic sterility (called gonadal dysgenesis sterility). P element activity is controlled by a complex regulation system, encoded by the elements themselves, which keeps their transposition rate low within the strain bearing P elements and limits copy number by genome. A second regulatory mechanism, which acts on the level of RNA processing, prevents P mobility to somatic cells. The oldest available strains, representing most major geographical regions of the world, exhibited no detectable hybridization to the P-element. In contrast, all recently collected natural populations that were tested carried P-element sequences. The available evidence is consistent with the hypothesis of a worldwide P-element invasion of D. melanogaster during the past 30 years. Timing and direction of the invasion are discussed. The lack of P-element in older strains of Drosophila melanogaster as well as in the species must closely related to Drosophila melanogaster, suggests that P entered the Drosophila melanogaster genome recently, probably by horizontal transfer from an other species. The analysis of P-element elsewhere in the genus Drosophila reveals that several more distantly related species carried transposable elements with sequences quite similar to P. The species with the best-matching P-element is D. willistoni. A P-element from this species was found to match all but one of the 2907 nucleotides of the Drosophila melanogaster P-element. The phylogenic distributions and the likely horizontal transfers of the two other Drosophila transposable elements are discussed.     

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.     

Control of expression of the I factor, a LINE-like transposable element in Drosophila melanogaster.

I factors are LINE-like transposable elements in the genome of Drosophila melanogaster. They normally transpose infrequently but are activated in the germline of female progeny of crosses between males of a strain that contains complete elements, an I or inducer strain and females of a strain that does not, an R or reactive strain. This causes a phenomenon known as I-R hybrid dysgenesis. We have previously shown that the I factor promoter lies between nucleotides 1 and 30. Here we demonstrate that expression of this promoter is regulated by nucleotides 41-186 of the I factor. This sequence can act as an enhancer as it stimulates expression of the hsp7O promoter in ovaries in the absence of heat-shock. Within this region there is a site that is required for promoter activity and that is recognized by a sequence-specific binding protein. We propose that this protein contributes to the enhancer activity of nucleotides 41-186 and that reduced I factor expression in inducer strains is due to titration of this protein or others that interact with it.     

Dynamics of the hobo transposable element in transgenic lines of Drosophila melanogaster

The impact of the hobo transposable element in global reorganization of the Drosophila melanogaster genome has been investigated in transgenic lines generated by injection of hobo elements into the Hikone strain, which lacked them. In the present extensive survey, the chromosomal distribution of hobo insertion sites in the line 28 was found to be homogeneous and similar for all chromosomal arms, except 3L, when compared with other transgenic lines. However, some original features were observed in this line at the genetic and chromosomal levels. Several hotspots of insertion sites were observed on the X, second and third chromosomes. Five sites with a high frequency of hobo insertions were present on the 3L arm in most individuals tested, suggesting the action of selection for hobo element in some sites. The presence of doublets or triplet was also observed, implying that hobo inserts can show local jumps or insertions in preferred regions. This local transposition occurred independently in 11 specific genomic regions in many individuals and generations. The dynamics of this phenomenon were analysed across generations. These results support the use of the hobo system as an important tool in fundamental and applied Drosophila genetics.     

DNA sequence requirements for hobo transposable element transposition in Drosophila melanogaster

We have conducted a structure and functional analysis of the hobo transposable element of Drosophila melanogaster. A minimum of 141 bp of the left (L) end and 65 bp of the right (R) end of the hobo were shown to contain sequences sufficient for transposition. Both ends of hobo contain multiple copies of the motifs GGGTG and GTGGC and we show that the frequency of hobo transposition increases as a function of the copy number of these motifs. The R end of hobo contains a unique 12 bp internal inverted repeat that is identical to the hobo terminal inverted repeats. We show that this internal inverted repeat suppresses transposition activity in a hobo element containing an intact L end and only 475 bp of the R end. In addition to establishing cis-sequences requirements for transposition, we analyzed trans-sequence effects of the hobo transposase. We show a hobo transposase lacking the first 49 amino acids catalyzed hobo transposition at a higher frequency than the full-length transposase suggesting that, similar to the related Ac transposase, residues at the amino end of the transposase reduce transposition. Finally, we compared target site sequences of hobo with those of the related Hermes element and found both transposons have strong preferences for the same insertion sites.     

Behavior of a Drosophila melanogaster transposable element in Saccharomyces cerevisiae.

The Drosophila melanogaster transposable element 412 is transiently unstable in Saccharomyces cerevisiae when present on a freely replicating plasmid. The 412 element undergoes recombination to form two circular molecules, a 412 deletion plasmid and, presumably, a 412 circle. The 412 deletion plasmid contains a single long terminal repeat which most likely is the result of homologous recombination within the long terminal repeats. This recombination occurs at or shortly after transformation and is independent of both the RAD52 gene product and the Flp gene of 2 micron DNA.     

Spread of the autonomous transposable element hobo in the genome of Drosophila melanogaster

The transposable element hobo has been introduced into the previously empty Drosophila melanogaster strain Hikone so that its dynamics can be followed and it can be compared with the P element. Five transformed lines were followed over 58 generations. The results were highly dependent on the culture temperature, the spread of hobo element being more efficient at 25 degrees C. The multiplication of hobo sequences resulted in a change in the features of these lines in the hobo system of hybrid dysgenesis. The number of hobo elements remained low (two to seven copies) and the insertions always corresponded to complete sequences. Our findings suggest that, despite their genetic similarities, P and hobo elements differ in many aspects, such as mobility and regulation mechanisms.      

Sequences homologous to the hobo transposable element in E strains of Drosophila melanogaster

Hobo is one of the three Drosophila melanogaster transposable elements, together with the P and I elements, that seem to have recently invaded the genome of this species. Surveys of the presence of hobo in strains from different geographical and temporal origins have shown that recently collected strains contain complete and deleted elements with high sequence similarity (H strains), but old strains lack hobo elements (E strains). Besides the canonical hobo sequences, both H and E strains show other poorly known hobo-related sequences. In the present work, we analyze the presence, cytogenetic location, and structure of some of these sequences in E strains of D. melanogaster. By in situ hybridization, we found that euchromatic hobo-related sequences were in fixed positions in all six E strains analyzed: 38C in the 2L arm; 42B and 55A in the 2R arm; 79E and 80B in the 3L arm; and 82C, 84C, and 84D in the 3R arm. Sequence comparison shows that some of the hobo-related sequences from Oregon-R and iso-1 strains are similar to the canonical hobo element, but their analysis reveals that they are substantially diverged and rearranged and cannot code for a functional transposase. Our results suggest that these ubiquitous hobo-homologous sequences are immobile and are distantly related to the modern hobo elements from D. melanogaster.     

Intragenomic distribution and stability of transposable elements in euchromatin and heterochromatin of drosophila melanogaster: non-LTR retrotransposon

The intragenomic location of the elements of the I, G, jockey, F, and Doc transposon families has been studied by the Southern blot analysis, in 12 laboratory Drosophila melanogaster stocks. Elements located in euchromatin, heterochromatin, and on the Y chromosome are identified, and their stability has been assessed by comparing the autoradiographs detected in different stocks and analysis of individual flies. Evidence is shown suggesting that preferential location in euchromatin or heterochromatin and the distribution within heterochromatin are distinctive of transposon families. Elements located in heterochromatin can be unstable. These results are discussed in the context of the relationship between transposable elements and the host genome. Received: 21 August 1996 / Accepted: 24 March 1997     

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