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Crossing the cyprinids diploid blunt snout bream Megalobrama amblycephala (BSB) and Carassius auratus red var. (RCC) generated sterile triploid (3nRB) and fertile tetraploid (4nRB) hybrid offspring. Utilizing inverted terminal repeats (ITRs) of transposon Tdr1 from Danio rerio as PCR primer, the results showed that evident change in the number of Tc1-like transposons in 4nRB relative to BSB occurred, whereas such change did not arise in 3nRB compared to BSB. No Tc1-like transposon was found in RCC. A novel transposon was isolated from both BSB and 3nRB and designated as Tma1, which consisted of multiple copies after dot-blot hybridization. Based on the analysis of PCR amplified flanking sequence, characterization of Tma1 indicated that this element flanked by a duplicated TA dinucleotide and harbored an ITR of about 224 bp. Tma1 also harbored an incomplete transposase gene. Another novel transposon designated as Tte1 was detected in 4nRB, which harbored an ITR of roughly 130 bp and consisted of multiple copies, but had no transposase gene. The analysis of PCR amplification and Southern blot hybridization showed that DNAs of 4nRB, which were hybridized to DIG-labeled pTma1, did not give band by PCR with Tma1 primer, on the other hand, 7 of 15 DNA samples from BSB, which were hybridized to DIG-labeled pTte1, did not produce band by PCR with Tte1 primer. These results suggest that Tte1 may be a recent invasion in BSB population and burst in 4nRB offspring. Our data provide clues as to the possible role of transposons as a driving mechanism for genomic evolution.

We studied the occurrence of O-type P elements in at least one species of each subgroup of the saltans group, in order to better understand the phylogenetic relationships among the elements within the saltans group and with those of species belonging to the willistoni group. We found that the O-type subfamily has a patchy distribution within the saltans group (it does not occur in D. neocordata and D. emarginata), low sequence divergence among species of the saltans group as well as in relation to species of the willistoni group, a lower rate of synonymous substitution for coding sequences compared to Adh, and phylogenetic incongruities. These findings suggest that the evolutionary history of the O-type subfamily within the saltans and willistoni groups follows the same model proposed for the canonical subfamily of P elements, i.e., events of horizontal transfer between species of the saltans and willistoni groups.

P elements of two different subfamilies designated as M- and O-type are thought to have invaded host species in the Drosophila obscura group via horizontal transmission from external sources. Sequence comparisons with P elements isolated from other species suggested that the horizontal invasion by the O-type must have been a rather recent event, whereas the M-type invasion should have occurred in the more distant past. To trace the phylogenetic history of O-type elements, additional taxa were screened for the presence of O- and M-type elements using type-specific PCR primers. The phylogeny deduced from the sequence data of a 927-bp section (14 taxa) indicate that O-type elements have undergone longer periods of regular vertical transmission in the lineages of the saltans and willistoni groups of Drosophila. However, starting from a species of the D. willistoni group they were transmitted horizontally into other lineages. First the lineage of the D. affinis subgroup was infected, and finally, in a more recent wave of horizontal spread, species of three different genera were invaded by O-type elements from the D. affinis lineage: Scaptomyza, Lordiphosa, and the sibling species D. bifasciata/D. imaii of the Drosophila obscura subgroup. The O-type elements isolated from these taxa are almost identical (sequence divergence

Up to now, investigations of expression and regulation of P transposable element have been almost exclusively carried out with the Drosophila melanogaster canonical P element. Analyzing eight species of the saltans group, we detected transposase mRNA in germline tissues of D. saltans and D. prosaltans and repressor mRNA in somatic tissues of D. saltans and D. sturtevanti. Sequencing analysis suggested that these transcripts might belong to the canonical subfamily and that they can be transpositionally active only in D. saltans. d N and d S values of Adh and the P element suggested that the sequences found in D. saltans and D. prosaltans might have been present in the ancestor of the saltans subgroup and that the sequence found in D. sturtevanti might have been horizontally transferred from D. saltans.

A phylogenetic analysis of P transposable elements in the Drosophila obscura species group is described. Multiple P sequences from each of 10 species were obtained using PCR primers that flank a conserved region of exon 2 of the transposase gene. In general, the P element phylogeny is congruent with the species phylogeny, indicating that the dominant mode of transmission has been vertical, from generation to generation. One manifestation of this is the distinction of P elements from the Old World obscura and subobscura subgroups from those of the New World affinis subgroup. However, the overall distribution of elements within the obscura species group is not congruent with the phylogenetic relationships of the species themselves. There are at least four distinct subfamilies of P elements, which differ in sequence from each other by as much as 34%, and some individual species carry sequences belonging to different subfamilies. P sequences from D. bifasciata are particularly interesting. These sequences belong to two subfamilies and both are distinct from all other P elements identified in this survey. Several mechanisms are postulated to be involved in determining phylogenetic relationships among P elements in the obscura group. In addition to vertical transmission, these include retention of ancestral polymorphisms and horizontal transfer by an unknown mating-independent mechanism.

RT-PCR was applied to analyze the splicing patterns of P-element-derived mRNAs in Drosophila bifasciata, D. helvetica, and Scaptomyza pallida. D. melanogaster was used as a control. The experiments revealed that P elements are transcribed in all species investigated. However, there are differences in the splicing patterns of IVS3, which has to be removed in order to produce transposase mRNA instead of repressor mRNA. These differences are observed among species as well as between the P element subfamilies, the M and the O type, which coexist in the genomes of D. bifasciata and S. pallida. In D. helvetica M-type transposase mRNA was found in the germline and repressor mRNA in the soma, as has been previously described for the canonical (M-type-related) P element of D. melanogaster. In contrast, in S. pallida only repressor mRNA of M-type elements was detected in all tissues. In D. bifasciata, M-type IVS3, although activated both in the soma and the germline, is never completely excised. Instead, two alternative double-spliced variants occur in which two small introns are removed within the IVS3 region. One of these variants codes for a protein 12 aa longer than the regular transposase. Taking these findings together, transposase production and transpositional activity of M-type elements seem to be limited to D. helvetica and D. melanogaster, whereas M-type elements have become immobile in D. bifasciata and S. pallida. Unlike the M type, the splicing of O-type transcripts in D. bifasciata and S. pallida follows the classical rules of tissue-specific P element regulation: transposase mRNA is produced exclusively in the germline whereas repressor mRNA is formed in somatic cells. Thus O-type elements are thought to be still transpositionally active in both species. This finding is in accordance with the postulated recent transfer of O-type elements between the gene pools of D. bifasciata and S. pallida. In addition, we were able to show that the IVS3 double-spliced variants of both P element types are produced regularily in all species of the genus Drosophila investigated so far, but not in S. pallida.

To analyze the behavior of endogenous transposable elements under genomic stress, a Drosophila 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 the Drosophila genome. An insertion of this avian retroviral sequence was detected by in 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, the Drosophila 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 mdg1 element mobilization. Injection of the avian RAV-2 particles led, however, to the observation of somatic transpositions of mdg1 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.

We report the cloning and analysis of a sample representative of all P-elements from Scaptomyza pallida. We have compared four independent stocks of this species, using Southern blot and in situ hybridization experiments to examine the number, structure, and distribution of P-elements. All four stocks give similar results: they contain about 15 P-elements including three to four full-length elements as well as shorter, deleted elements. All elements are divergent from one another and most of them appear to be immobile since they are located at identical positions in the genomes of independent stocks. These data indicate that P-elements are old components of the S. pallida genome. Moreover, the presence of P-sequences in species closely related to S. pallida suggests that they have had a long evolutionary history in the Scaptomyza genus. We have also found that most P-elements of S. pallida are located in the pericentromeric heterochromatin. This corroborates other studies which show that in the course of their evolution, transposable elements tend to accumulate into pericentromeric heterochromatin, where they become immobile and noncoding.

Two P-elements (bif1 and bif2) were isolated from a genomic library of Drosophila bifasciata. Both elements are internally deleted and have lost the coding capacity for a functional transposase. One of the elements (bif2) contains an insert consisting of a repetitive sequence. The terminal inverted repeats and the segments necessary for passive mobility are well conserved. Element bif2 has retained rudiments of the coding sequence of exon 0 and exon 3, but the reading frame is destroyed by insertions and deletions. The comparison of the D. bifasciata P-elements with P-elements of Drosophila melanogaster and Drosophila nebulosa reveals that the two latter sequences are more similar to each other than either of them is to the D. bifasciata elements. This finding contradicts the phylogenetic relationship of the species and can be taken as an indirect but unequivocal evidence for recent horizontal gene transfer from a relative of D. nebulosa to the gene pool of D. melanogaster. The P-elements of D. bifasciata are phylogenetically ancient and have evolved independently for about 50 million years. A higher substitution rate at the third codon position as well as a predominance of conservative replacements at the amino acid level indicates that the P-elements of D. bifasciata have been under selective constraint over a long period and that immunobilization has occurred only recently.

The P-element-mediated gene transfer system was used to introduce Drosophila teissieri 5S genes into the Drosophila melanogaster genome. Eight transformed D. melanogaster strains that carry D. teissieri 5S mini-clusters consisting of 9-21 adjacent 5S units were characterized. No genetic exchanges between D. melanogaster and D. teissieri 5S clusters were detected over a 2-year survey of the eight strains. The occurrence of small rearrangements within the D. melanogaster 5S cluster was demonstrated in one of the transformed strains.

This report describes the distribution of P-element sequences among members of the closely related willistoni and saltans species groups of the subgenus Sophophora. Gel-blotting analyses showed that many, but not all, species from each of these groups possess sequences with homology to the P transposable element of Drosophila melanogaster, a sophophoran species belonging to the melanogaster species group. Furthermore, P-homologous fragments are present in lower numbers in willistoni- and saltans-group species than in D. melanogaster P strains, and, in some species of those two groups, exhibit species-characteristic hybridization patterns. On the basis of these results, it is proposed that P elements have had a long evolutionary history in the willistoni and saltans lineages.