Induction of M2-macrophages by tumour cells and tumour growth promotion by M2-macrophages: a quid pro quo in pancreatic cancer.

INTRODUCTION: More effective therapies are required to improve survival of pancreatic cancer. Possible immunologic targets include tumour associated macrophages (TAMs), generally consisting of M1- and M2-macrophages. We have analysed the impact of TAMS on pancreatic cancer in a syngeneic orthotopic murine model. METHODS: 6606PDA murine pancreatic cancer cells were orthotopically injected into C57BL6 mice. Tumour growth was monitored using MRI. Macrophages were depleted by clodronate liposomes. Tumours including microvessel density were evaluated using immunohistochemistry, immunofluorescence and/or cytometric beads assays. Naïve macrophages were generated employing peritoneal macrophages. In vitro experiments included culturing of macrophages in tumour supernatants as well as tumour cells cultured in macrophage supernatants using arginase as well as Griess assays. RESULTS: Clodronate treatment depleted macrophages by 80% in livers (p = 0.0051) and by 60% in pancreatic tumours (p = 0.0169). MRI revealed tumour growth inhibition from 221.8 mm(3) to 92.3 mm(3) (p = 0.0216). Micro vessel densities were decreased by 44% (p = 0.0315). Yet, MCP-1-, IL-4- and IL-10-levels within pancreatic tumours were unchanged. 6606PDA culture supernatants led to a shift from naïve macrophages towards an M2-phenotype after a 36 h treatment (p < 0.0001), reducing M1-macrophages at the same time (p < 0.037). In vivo, M2-macrophages represented 85% of all TAMs (p < 0.0001). Finally, culture supernatants of M2-macrophages induced tumour growth in vitro by 63.2% (p = 0.0034). CONCLUSIONS: This quid pro quo of tumour cells and M2-macrophages could serve as a new target for future immunotherapies that interrupt tumour promoting activities of TAMs and change the iNOS-arginase balance towards their tumoricidal capacities.

Macrophages promote tumour growth and liver metastasis in an orthotopic syngeneic mouse model of colon cancer.

PURPOSE: Tumour-associated macrophages have been shown to promote proliferation, angiogenesis and metastasis in several carcinomas. The effect on colon cancer has not yet been clarified. Furthermore, Kupffer cells in the liver might initiate the formation of metastases by directly binding tumour cells. METHODS: An orthotopic syngeneic mouse model of colon cancer as well as a liver metastases model has been studied, using murine CT-26 colon cancer cells in Balb/c-mice. Macrophages were depleted in both models by clodronate liposomes. Tumour sizes and metastases were determined using 7-Tesla MRI. The macrophage and vascular density in the orthotopic tumours as well as the Kupffer cell density in the livers were evaluated using immunohistochemistry. RESULTS: Animals in the macrophage-depleted group displayed significantly smaller primary tumours (37 ± 20 mm(3)) compared to the control group (683 ± 389 mm(3), p = 0.0072). None of the mice in the depleted group showed liver or peritoneal metastases, whereas four of six control mice displayed liver and five out of six mice peritoneal metastases. The vascular density was significantly lower in the macrophage-depleted group (p = 0.0043). In the liver metastases model, animals of the Kupffer cell-depleted group (14.3 ± 7.7) showed significantly less liver metastases than mice of the two control groups (PBS liposomes, 118.5 ± 28.2, p = 0.0117; NaCl, 81.7 ± 23.2, p = 0.0266). The number of liver metastases correlated directly with the Kupffer cell density (p = 0.0221). CONCLUSION: Macrophages promote tumour growth, angiogenesis and metastases in this orthotopic syngeneic mouse model. Kupffer cells enhance the formation of metastases in the liver.

Activity tracker-based intervention to increase physical activity in patients with type 2 diabetes and healthy individuals: study protocol for a randomized controlled trial.

BACKGROUND: One relevant strategy to prevent the onset and progression of type 2 diabetes mellitus (T2DM) focuses on increasing physical activity. The use of activity trackers by patients could enable objective measurement of their regular physical activity in daily life and promote physical activity through the use of a tracker-based intervention. This trial aims to answer three research questions: (1) Is the use of activity trackers suitable for longitudinal assessment of physical activity in everyday life? (2) Does the use of a tracker-based intervention lead to sustainable improvements in the physical activity of healthy individuals and in people with T2DM? (3) Does the accompanying digital motivational intervention lead to sustainable improvements in physical activity for participants using the tracker-based device? METHODS: The planned study is a randomized controlled trial focused on 1642 participants with and without T2DM for 9 months with regard to their physical activity behavior. Subjects allocated to an intervention group will wear an activity tracker. Half of the subjects in the intervention group will also receive an additional digital motivational intervention. Subjects allocated to the control group will not receive any intervention. The primary outcome is the amount of moderate and vigorous physical activity in minutes and the number of steps per week measured continuously with the activity tracker and assessed by questionnaires at four time points. Secondary endpoints are medical parameters measured at the same four time points. The collected data will be analyzed using inferential statistics and explorative data-mining techniques. DISCUSSION: The trial uses an interdisciplinary approach with a team including sports psychologists, sports scientists, health scientists, health care professionals, physicians, and computer scientists. It also involves the processing and analysis of large amounts of data collected with activity trackers. These factors represent particular strengths as well as challenges in the study. TRIAL REGISTRATION: The trial is registered at the World Health Organization International Clinical Trials Registry Platform via the German Clinical Studies Trial Register (DRKS), DRKS00027064 . Registered on 11 November 2021.

Foxp3(+) T cells expressing RORγt represent a stable regulatory T-cell effector lineage with enhanced suppressive capacity during intestinal inflammation.

Foxp3 (forkhead box P3 transcription factor)-expressing regulatory T cells (Tregs) are essential for immunological tolerance, best illustrated by uncontrolled effector T-cell responses and autoimmunity upon loss of Foxp3 expression. Tregs can adopt specific effector phenotypes upon activation, reflecting the diversity of functional demands in the different tissues of the body. Here, we report that Foxp3(+)CD4(+) T cells coexpressing retinoic acid-related orphan receptor-γt (RORγt), the master transcription factor for T helper type 17 (Th17) cells, represent a stable effector Treg lineage. Transcriptomic and epigenetic profiling revealed that Foxp3(+)RORγt(+) T cells display signatures of both Tregs and Th17 cells, although the degree of similarity was higher to Foxp3(+)RORγt(-) Tregs than to Foxp3(-)RORγt(+) T cells. Importantly, Foxp3(+)RORγt(+) T cells were significantly demethylated at Treg-specific epigenetic signature genes such as Foxp3, Ctla-4, Gitr, Eos, and Helios, suggesting that these cells have a stable regulatory rather than inflammatory function. Indeed, adoptive transfer of Foxp3(+)RORγt(+) T cells in the T-cell transfer colitis model confirmed their Treg function and lineage stability in vivo, and revealed an enhanced suppressive capacity as compared with Foxp3(+)RORγt(-) Tregs. Thus, our data suggest that RORγt expression in Tregs contributes to an optimal suppressive capacity during gut-specific immune responses, rendering Foxp3(+)RORγt(+) T cells as an important effector Treg subset in the intestinal system.

Different evolutionary behaviour of P element subfamilies: M-type and O-type elements in Drosophila bifasciata and D. imaii.

Distribution and variation of two P-element subfamilies designated M-type and O-type elements were investigated in Drosophila bifasciata (Db) and its relatives. PCR screening revealed that full-sized and internally deleted elements of both types occur in three geographic Db strains and in the related species, D. imaii (Di). Molecular analyses indicate differences in the evolutionary behaviour of the two P-element types. Internally deleted M-type elements fall into two size classes present in all three Db strains. In contrast, internally deleted O-type elements vary between the strains in number and length. With respect to genomic location, M-type elements seem to be restricted to conserved euchromatic sites, whereas the positions of O-type elements appear to be geographically variable. In one strain of Db (Italy), O-type elements seem to accumulate in the heterochromatin. Sequencing of a 397-bp segment shows intra- and interspecific divergence of M-type elements. In a 452-bp segment of the O-type elements, no substitutions were found, neither within nor between species. This finding suggests recent introgression of O-type elements via hybridization between Db and Di. Sequence identity and variation in chromosomal locations among different copies imply that O-type elements are transpositionally active. For M-type elements, genomic mobility cannot be proved. In a survey of several other taxa, no O-type-related sequences were detected so far. Therefore, the origin of the O-type subfamily remains unknown, whereas the source of M-type elements can be traced back to the genus Scaptomyza.

Structure and expression of clustered P element homologues in Drosophila subobscura and Drosophila guanche.

Sequence relationships and functional aspects were analysed in the P element homologues of Drosophila subobscura (Ds) and D. guanche (Dg). In both species, the P homologues are clustered at a single genomic position. They lack the characteristic terminal structures of actively transposing P elements, but they have the coding capacity for a 66-kDa 'repressor-like' protein. Two different types of cluster units (G-type and A-type) can be distinguished. The A-type unit, which is present in multiple copies, is transcribed in adult flies. In contrast, the G-type unit has a much lower copy number and is apparently not expressed. In Dg, the isolated G-type sequence carries a 420-bp insertion in the promoter region, which is probably responsible for inactivation. Sequence comparisons of different cluster units show that differentiation of the two types precedes the lineage split of these species. Substitution rates of the deduced proteins reveal two distinct subregions: high variability at the N terminus and strong sequence conservation in the rest of the protein. The variable region contains motifs characteristic of DNA-binding proteins. Adaptive diversification of the cluster units towards specific binding properties might be a plausible explanation for variability in the N-termini. Both unit types have lost the weak promoter region characteristic of P transposons. In the A-type unit, a new promoter has been formed which is apparently composed of parts of insertion sequences derived from two different mobile elements.(ABSTRACT TRUNCATED AT 250 WORDS)

Transcription and splicing patterns of M- and O-type P elements in drosophila bifasciata, D. helvetica, and scaptomyza pallida

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.

Regulatory T cells promote a protective Th17-associated immune response to intestinal bacterial infection with C. rodentium.

Intestinal infection with the mouse pathogen Citrobacter rodentium induces a strong local Th17 response in the colon. Although this inflammatory immune response helps to clear the pathogen, it also induces inflammation-associated pathology in the gut and thus, has to be tightly controlled. In this project, we therefore studied the impact of Foxp3(+) regulatory T cells (Treg) on the infectious and inflammatory processes elicited by the bacterial pathogen C. rodentium. Surprisingly, we found that depletion of Treg by diphtheria toxin in the Foxp3(DTR) (DEREG) mouse model resulted in impaired bacterial clearance in the colon, exacerbated body weight loss, and increased systemic dissemination of bacteria. Consistent with the enhanced susceptibility to infection, we found that the colonic Th17-associated T-cell response was impaired in Treg-depleted mice, suggesting that the presence of Treg is crucial for the establishment of a functional Th17 response after the infection in the gut. As a consequence of the impaired Th17 response, we also observed less inflammation-associated pathology in the colons of Treg-depleted mice. Interestingly, anti-interleukin (IL)-2 treatment of infected Treg-depleted mice restored Th17 induction, indicating that Treg support the induction of a protective Th17 response during intestinal bacterial infection by consumption of local IL-2.

Loss of Survivin influences liver regeneration and is associated with impaired Aurora B function.

The chromosomal passenger complex (CPC) acts as a key regulator of mitosis, preventing asymmetric segregation of chromosomal material into daughter cells. The CPC is composed of three non-enzymatic components termed Survivin, the inner centromere protein (INCENP) and Borealin, and an enzymatic component, Aurora B kinase. Survivin is necessary for the appropriate separation of sister chromatids during mitosis and is involved in liver regeneration, but its role in regenerative processes is incompletely elucidated. Whether Survivin, which is classified as an inhibitor of apoptosis protein (IAP) based on domain composition, also has a role in apoptosis is controversial. The present study examined the in vivo effects of Survivin ablation in the liver and during liver regeneration after 70% hepatectomy in a hepatocyte-specific knockout mouse model. The absence of Survivin caused a reduction in the number of hepatocytes in the liver, together with an increase in cell volume, macronucleation and polyploidy, but no changes in apoptosis. During liver regeneration, mitosis of hepatocytes was associated with mislocalization of the members of the CPC, which were no longer detectable at the centromere despite an unchanged protein amount. Furthermore, the loss of survivin in regenerating hepatocytes was associated with reduced levels of phosphorylated Histone H3 at serine 28 and abolished phosphorylation of CENP-A and Hec1 at serine 55, which is a consequence of decreased Aurora B kinase activity. These data indicate that Survivin expression determines hepatocyte number during liver development and liver regeneration. Lack of Survivin causes mislocalization of the CPC members in combination with reduced Aurora B activity, leading to impaired phosphorylation of its centromeric target proteins and inappropriate cytokinesis.

Influence of tris(4-chlorophenyl)methanol, non-ortho PCB 77 and gamma-hexachlorocyclohexane on human sperm function in vitro.

In recent years, there has been growing concern that environmental pollutants in general, and organochlorines in particular, adversely affect male fertility. Therefore, we investigated the effects of tris(4-chlorophenyl)methanol (TCPM), non-ortho PCB 77 and gamma-hexachlorocyclohexane (gamma-HCH, lindane) on human sperm functions in vitro. Human spermatozoa from healthy donors were washed in human tubular fluid medium containing 1% human serum albumin, filtered through glass wool and exposed to different concentrations of TCPM, PCB 77 or gamma-HCH. After incubation for 5 h at 37 degrees C and 5% CO(2), sperm vitality and the percentage of living acrosome-reacted spermatozoa were examined using triple stain technique. Total sperm motility was evaluated by computer-assisted sperm analysis (Stroemberg-Mika) after 5 h. For TCPM, total motility was additionally measured after 18 and 40 h. Different concentrations of PCB 77 and gamma-HCH did not alter the percentage of spontaneous living acrosome-reacted spermatozoa, vitality and total motility. TCPM dose-dependently altered sperm motility, vitality and acrosome reaction. The percentage of living acrosome-reacted spermatozoa was increased at overtly toxic concentrations. Therefore, it is suggested that unspecific acrosomal loss has been induced by degenerative processes. In conclusion, even high concentrations of PCB 77 and gamma-HCH did not affect human sperm functions in vitro. Only very high cytotoxic TCPM concentrations modulated spontaneous acrosome reaction and total motility. Therefore, in vivo effects on human sperm function seem to be unlikely. However, individual susceptibility has to be considered and little is known about additive and possible synergistic effects as other environmental pollutants with similar potencies have been found in the human male and female reproductive tract.

Repeated horizontal transfer of P transposons between Scaptomyza pallida and Drosophila bifasciata.

Two distinct P element subfamilies, designated M-type and O-type, reside in the genome of D. bifasciata. PCR-screening of 65 Drosophila species revealed that only D. bifasciata and its closest relative D. imaii possess O-type elements. Outside the genus, O-type elements were detected in Scaptomyza pallida. Restriction analyses show that the general structure of the O-type elements from S. pallida and D. bifasciata is the same. Sequence divergence turned out to be extremely low (0.43%). These results suggest that the O-type subfamily of D. bifasciata has been received by horizontal transfer from an external source, most probably from the genus Scaptomyza, as has been previously suspected for the M-type family. Since the sequence divergence between M-type elements from S. pallida and D. bifasciata is eighteen-fold higher than that between O-type elements, two independent intergeneric transfer events have to be postulated. In order to re-examine the taxonomic status of S. pallida, a partial sequence (489 bp) of the Adh gene was analysed. The data clearly prove that S. pallida has to be placed far outside the D. obscura group.

A new P element subfamily from Drosophila tristis, D. ambigua, and D. obscura.

A new P element subfamily, designated T-type, was found in the genomes of the three closely related species Drosophila ambigua, Drosophila obscura, and Drosophila tristis. The subfamily comprises both full-sized and internally deleted P elements. The T-type element of D. ambigua is longer than the canonical P elements owing to a 300-bp insertion in the 3' noncoding region. Tandemly arranged T-type elements were detected in D. ambigua and D. tristis. The overall structure of T-type elements resembles that of the Drosophila melanogaster P element and the termini are formed by perfect inverted repeats of 33 bp. However, none of the elements studied so far have intact reading frames. Sequence comparisons with other P element subfamilies from the obscura group indicate that the T-type elements are most closely related to the terminally truncated P homologues of Drosophila guanche and Drosophila subobscura. Therefore they can be considered as the lineage-specific P transposons of the obscura group. Furthermore, this finding indicates that the clustered P homologues of D. guanche and D. subobscura must be derived from transpositionally active P elements rather than from an immobile genomic sequence.

Ancient and recent horizontal invasions of drosophilids by P elements.

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 <1%). In contrast, no such striking similarities are observed among M-type elements. Nevertheless, the sequence phylogeny of M-type elements is also not in accordance with the phylogeny of their host species, suggesting earlier horizontal transfer events. The results imply that P elements cross species barriers more frequently than previously thought but require a particular genomic environment and thus seem to be confined to a rather narrow spectrum of host species. Consequently, different P element types acquired by successive horizontal transmission events often coexist within the same genome.

Repetitive sequences in the genome of Anemone blanda: identification of tandem arrays and of dispersed repeats.

A tandemly repetitive sequence family (AbS1) and a repetitive sequence (Hd) forming part of a larger dispersed element (dorf-1) of Anemone blanda were characterised. The AbS1 satellite sequence family is located in all 4',6-diamidino-2-phenylindole (DAPI) positive intercalary heterochromatic bands and in the DAPI positive heterochromatic terminal region of chromosome 3, while the dispersed Hd homologous sequences are preferentially associated with euchromatic chromosome regions. The major component of the AbS1 satellite is AbS1-H1 with a basic repeat unit of 1640 bp; a minor fraction (AbS1-H5) consists of 320 bp units. A subsection of the AbS1-H1 repeat unit exhibits homologies to the 25S rRNA gene of flowering plants suggesting that the 1.64 kb satellite was generated by amplification of a precursor satellite and/or single copy sequence together with an rDNA fragment. The rDNA homologous region is considered to evolve at a rate similar to pseudogenes and thus the age of this satellite DNA fraction can be roughly estimated as about 27 million years. The dispersed repeated sequence Hd (about 1300 bp) is associated with the 8 kb element dorf-1. A. blanda dorf-1 constitutes about 0.2% of the genome (3 x 10(4) copies), is bounded by identical long terminal repeats, and exhibits partial homology to the Lilium gypsy-type element del1, but has yet to be confirmed as a retrotransposon. In contrast to the AbS1 satellite sequence family, Hd homologous sequences were found not only in A. apennina, the closest relative of A. blanda, but also in A. nemorosa and A. ranunculoides indicating that a progenitor sequence of dorf-1 was present in a common ancestor before speciation occurred.

P-related sequences in Drosophila bifasciata: a molecular clue to the understanding of P-element evolution in the genus Drosophila.

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 phylogenetic position of Drosophila eskoi deduced from P element and Adh sequence data.

PCR screening with primers specific for the T-, M-, and O-type P element subfamilies was performed to investigate the interspecific distribution in 18 species and to reconstruct the phylogenetic history of the various types within the obscura species group. T-type elements occur in D. ambigua, D. tristis, D. obscura, D. subsilvestris, and D. eskoi. In the genomes of D. subobscura, D. madeirensis, and D. guanche they are present in the form of terminally truncated T-type derivatives. The wide distribution suggests that the T-type subfamily had a long evolutionary history in the obscura lineage. In contrast, the patchy occurrence of M- and O-type elements can be ascribed to four independent events of horizontal invasion of different lineages. The cladogenesis of the obscura group was investigated using a partial sequence of the Adh gene as a marker. In contrast to earlier findings, the position of D. eskoi had to be revised. D. eskoi appears as the closest relative of the D. ambigua clade, whereas D. tsukubaensis is the sister taxon of the species pair D. bifasciata/D. imaii. This result is in good accordance with the P element data, where high sequence similarity (95%) was found among the T-type elements of D. eskoi and those of D. ambigua and D. tristis.

P-element homologous sequences are tandemly repeated in the genome of Drosophila guanche.

In Drosophila guanche, P-homologous sequences were found to be located in a tandem repetitive array (copy number: 20-50) at a single genomic site. The cytological position on the polytene chromosomes was determined by in situ hybridization (chromosome O: 85C). Sequencing of one complete repeat unit (3.25 kilobases) revealed high sequence similarity between the central coding region comprising exons 0 to 2 and the corresponding section of the Drosophila melanogaster P element. The rest of the sequence has diverged considerably. Exon 3 has no coding function and the inverted repeats have disappeared. The P homologues of D. guanche apparently have lost their mobility but have retained the coding capacity for a protein similar to the 66-kDa P-element repressor of D. melanogaster. Divergence between different repeat units indicates early amplification of the sequence at this particular genomic site. The presence of a common P-element site at 85C in Drosophila subobscura, Drosophila madeirensis, and D. guanche suggests that clustering of the sequence at this location took place before the phylogenetic radiation of the three species.

Two distinct P element subfamilies in the genome of Drosophila bifasciata.

The genome of Drosophila bifasciata harbours two distinct subfamilies of P-homologous sequences, designated M-type and O-type elements based on similarities to P element sequences from other species. Both subfamilies have some general features in common: they are of similar length (M-type: 2935 bp, O-type: 2986 bp), are flanked by direct repeats of 8 bp (the presumptive target sequence), contain terminal inverted repeats, and have a coding region consisting of four exons. The splice sites are at homologous positions and the exons have the coding capacity for proteins of 753 amino acids (M-type) and 757 amino acids (O-type). It seems likely that both types of element represent functional transposons. The nucleotide divergence of the two P element subfamilies is high (31%). The main structural difference is observed in the terminal inverted repeats. Whereas the termini of M-type elements consists of 31 bp inverted repeats, the inverted repeats of the O-type elements are interrupted by non-complementary stretches of DNA, 12 bp at the 5' end and 14 bp at the 3' end. This peculiarity is shared by all members of the O-type subfamily. Comparison with other P element sequences indicates incongruities between the phylogenies of the species and the P transposons. M-type and O-type elements apparently have no common origin in the D. bifasciata lineage. The M-type sequence seems to be most closely related to the P element from Scaptomyza pallida and thus could be considered as a more recent invader of the D. bifasciata gene pool. The origin of the O-type elements cannot be unequivocally deduced from the present data.(ABSTRACT TRUNCATED AT 250 WORDS)

The evolutionary life history of P transposons: from horizontal invaders to domesticated neogenes.

P elements, a family of DNA transposons, are known as aggressive intruders into the hitherto uninfected gene pool of Drosophila melanogaster. Invading through horizontal transmission from an external source they managed to spread rapidly through natural populations within a few decades. Owing to their propensity for rapid propagation within genomes as well as within populations, they are considered as the classic example of selfish DNA, causing havoc in a genomic environment permissive for transpositional activity. Tracing the fate of P transposons on an evolutionary scale we describe different stages in their evolutionary life history. Starting from horizontal transfer events, which now appear to be rather a common phenomenon, the initial transpositional burst in the new host is slowed down by the accumulation of defective copies as well as host-directed epigenetic silencing. This leads to the loss of mobility and, finally, to molecular erosion by random mutations. Possible escape routes from genomic extinction are the reactivation within the original host genome by recombination or suspension of the repressing regime, horizontal emigration to a virgin gene pool, or genomic integration and acquisition of a novel function as a domesticated host gene.

Identification of a complete P-element in the genome of Drosophila bifasciata.

A full-size P-element (IbifM3) was isolated from a genomic library of Drosophila bifasciata. The sequence has a length of 2935 bp and is flanked by 8 bp duplications of the target site. The termini are formed by 31 bp inverted repeats. The four exons have intact reading frames and possess the coding capacity for a protein of 753 amino acids and a molecular weight of 86.4 kd. The sections of the D. melanogaster transposase presumed to be functionally important (three leucine zippers and a helix turn helix motif) are conserved in the D. bifasciata P-element. Copy number and genomic distribution resemble the situation in true P-strains of D. melanogaster. Both findings support the idea that IbifM3 represents an active transposon. The sequence comparison between the P-elements of D. bifasciata, D. melanogaster and Scaptomyza pallida reveals relationships not in accordance with the phylogeny of the species. This result suggests a further case of horizontal transmission involving mobile elements in the genus Drosophila.