A P Element Containing Suppressor of Hairy-Wing Binding Regions Has Novel Properties for Mutagenesis in Drosophila Melanogaster

P elements are widely used as insertional mutagens to tag genes, facilitating molecular cloning and analyses. We modified a P element so that it carried two copies of the suppressor of Hairy-wing [su(Hw)] binding regions isolated from the gypsy transposable element. This transposon was mobilized, and the genetic consequences of its insertion were analyzed. Gene expression can be altered by the su(Hw) protein as a result of blocking the interaction between enhancer/silencer elements and their promoter. These effects can occur over long distances and are general. Therefore, a composite transposon (SUPor-P for suppressor-P element) combines the mutagenic efficacy of the gypsy element with the controllable transposition of P elements. We show that, compared to standard P elements, this composite transposon causes an expanded repertoire of mutations and produces alleles that are suppressed by su(Hw) mutations. The large number of heterochromatic insertions obtained is unusual compared to other insertional mutagenesis procedures, indicating that the SUPor-P transposon may be useful for studying the structural and functional properties of heterochromatin.     

The Maternally Inherited Regulation of P Elements in Drosophila Melanogaster Can Be Elicited by Two P Copies at Cytological Site 1a on the X Chromosome

Two P elements, inserted at the cytological site 1A on an X chromosome from an Drosophila melanogaster natural population (Lerik, USSR), were isolated by genetic methods to determine if they are sufficient to cause the P cytotype, the cellular condition that regulates the P family of transposable element. The resulting "Lerik P(1A)" line (abbreviated "Lk-P(1A)") carries only one P element in situ hybridization site but genomic Southern analysis indicates that this site contains two, probably full length, P copies separated by at least one EcoRI cleavage site. Because the Lk-P(1A) line shows some transposase activity, at least one of these two P elements is autonomous. The Lk-P(1A) line fully represses germline P element activity as judged by the GD sterility and snw hypermutability assays; this result shows that the P cytotype can be elicited by only two P element copies. However, the Lk-P(1A) line does not fully repress delta 2-3(99B) transposase activity in the soma, although it fully represses delta 2-3(99B) transposase activity in the germline (delta 2-3(99B) is an in vitro modified P element that produces a high level of transposase activity in both the germline and the soma). The germline regulatory properties of the Lk-P(1A) line are maternally transmitted, even when the delta 2-3(99B) element is used as the source of transposase. By contrast, the partial regulation of delta 2-3(99B) somatic activity is chromosomally inherited. These results suggest that the regulatory P elements of the Lk-P(1A) line are inserted near a germline-specific enhancer.     

Studies on the Rate and Site-Specificity of P Element Transposition

A single genetically marked P element can be efficiently mobilized to insertionally mutagenize the Drosophila genome. We have investigated how the structure of the starting element and its location along the X chromosome influenced the rate and location of mutations recovered. The structure of two P[rosy+] elements strongly affected mobilization by the autonomous "Jumpstarter-1" element. Their average transposition rates differed more than 12-fold, while their initial chromosomal location had a smaller effect. The lethal and sterile mutations induced by mobilizing a P[rosy+] element from position 1F were compared with those identified previously using a P[neoR] element at position 9C. With one possible exception, insertion hotspots for one element were frequently also targets of the other transposon. These experiments suggested that the genomic location of a P element does not usually influence its target sites on nonhomologous chromosomes. During the course of these experiments, Y-linked insertions expressing rosy+ were recovered, suggesting that marked P elements can sometimes insert and function at heterochromatic sites.     

High-frequency P element loss in Drosophila is homolog dependent

P transposable elements in Drosophila melanogaster can undergo precise loss at a rate exceeding 13% per generation. The process is similar to gene conversion in its requirement for a homolog that is wild type at the insertion site and in its reduced frequency when pairing between the homologs is inhibited. However, it differs from classical gene conversion by its high frequency, its requirement for P transposase, its unidirectionality, and its occurrence in somatic and premeiotic cells. Our results suggest a model of P element transposition in which jumps occur by a "cut-and-paste" mechanism but are followed by double-strand gap repair to restore the P element at the donor site. The results also suggest a technique for site-directed mutagenesis in Drosophila.     

Excision of one of two defective P elements as the cause of alternate mutational events (sn+ and sne) of the singed bristle allele snw in Drosophila melanogaster

The X-linked singed locus is concerned with the bristle phenotype and female sterility, and is known as a hot spot of P element insertion. A moderate allele of singed, singed-weak (snw) (Engels, 1979; 1984) is inserted with P elements. It is used as an index of P element activity, since it mutates at a high frequency to either a more extreme allele, singed-extreme (sne), or to a phenotype that is equivalent to the wild type (sn+) when an autonomous P element exists. We show here that snw is inserted with two defective P elements in reverse orientation, and the two alternate mutational events (sn+ and sne) are caused by the excision of one or the other of the P elements present in the singed gene. It is interesting that sn+ and sne are inserted with a single P element in the same position, but show very different phenotypes. The insertional sites of P elements in the singed locus possibly contain an unidentified repetitive sequence, which is repeated dozens of times per haploid genome of the wild-type strain Canton-S.     

A simulation of P element horizontal transfer in Drosophila

Experimental data suggest that the P transposable element has invaded the Drosophila melanogaster genome after a horizontal transfer from the phylogenetically distant species Drosophila willistoni. The differences between P element phylogeny and that of the Drosophila genus could in part be explained by horizontal transfers. In vivo experiments show that P elements are able to transpose in the genomes of other Drosophila species. This suggests that horizontal transmission of P elements could have taken place in many species of this genus. The regulation, transposition, and deleterious effects of the P element in D. melanogaster were formalized and integrated in a global model to produce a simulation program that simulates a P element invasion. The simulations show that our knowledge of the P element in D. melanogaster can explain its behavior in the Drosophila genus. The equilibrium state of the invaded population of a new species depends on its ability to repair damage caused by P element activity. If repair is efficient, the equilibrium state tends to be of the P type state, in which case the element could subsequently invade other populations of the species. Conversely, the equilibrium state is of the M′ type state when the ability to repair damage is low. The invasion of the P element into other populations of this new species can then only occur by genetic drift and it is likely to be lost. The success of a P element invasion into a new species thus greatly depends on its ability to produce dysgenic crosses. This revised version was published online in August 2006 with corrections to the Cover Date.     

Distribution and conservation of mobile elements in the genus Drosophila

Essentially nothing is known of the origin, mode of transmission, and evolution of mobile elements within the genus Drosophila. To better understand the evolutionary history of these mobile elements, we examined the distribution and conservation of homologues to the P, I, gypsy, copia, and F elements in 34 Drosophila species from three subgenera. Probes specific for each element were prepared from D. melanogaster and hybridized to genomic DNA. Filters were washed under conditions of increasing stringency to estimate the similarity between D. melanogaster sequences and their homologues in other species. The I element homologues show the most limited distribution of all elements tested, being restricted to the melanogaster species group. The P elements are found in many members of the subgenus Sophophora but, with the notable exception of D. nasuta, are not found in the other two subgenera. Copia-, gypsy-, and F-element homologues are widespread in the genus, but their similarity to the D. melanogaster probe differs markedly between species. The distribution of copia and P elements and the conservation of the gypsy and P elements is inconsistent with a model that postulates a single ancient origin for each type of element followed by mating-dependent transmission. The data can be explained by horizontal transmission of mobile elements between reproductively isolated species.      

P-Element-Induced Male Recombination Can Be Produced in Drosophila Melanogaster by Combining End-Deficient Elements in Trans

Male recombination, not normally present in Drosophila melanogaster, can be produced at high rates when target P elements at homologous sites are combined in the presence of transposase protein. We have produced a set of elements by in situ deletion of a particular insertion and have found elements that have deletions stretching into either end. Elements were tested in pairs to see whether they complement each other in their ability to induce recombination. The combination of elements that are deficient for the same end produces very little recombination, but the combination of a right-end and a left-end element can generate recombination values higher than given by two complete P[CaSpeR] elements at homologous sites. This strongly suggests that ``hybrid' P elements, containing ends from two different elements, can be recognized by transposase protein. We have also examined genotypes containing a normal and an end-deficient element and found that they yield reasonably high levels of recombination. We interpret the resultant gametes from such genotypes as showing that the majority of events in this genotype derive from the association of complementary ends from the same element, whereas the complementary ends from elements in trans associate in only a minority of cases.     

The Relationship between P Elements and Male Recombination in Drosophila Melanogaster

P element dysgenesis associated male recombination in Drosophila was examined with a selective system focused upon 5% of the standard female genetic map divided into eight recombination segments. We found no correspondence between P element mobilization events and recombination in males in the intervals monitored. We defined two adjacent short genetic and molecular regions, one devoid of male recombination and the other acting as a "hot spot" for exchange in the absence of supporting P element insertion and excision activity. These data suggest that, even in the presence of mobilizing P elements, transposase may be active at non-P element sites, and that the genome may harbor sequences ranging from highly responsive to completely unresponsive to transposase action. A viewpoint is presented wherein P elements, with sequences that bind transposase, serve to focus the recombination action of transposase to encompass a region of DNA radiating outward from the initial binding site. We suggest that this region is measured in terms of chromosomal segments rather than limited to P element sequences.     

Preferential insertion of P elements into genes expressed in the germ-line of Drosophila melanogaster

Summary ADrosophila transposable element, the P element, containing the bacterial gene encoding -galactosi-dase is widely used to search for tissue-specific enhancers. Thislac-Z-containing P element (P-lacZ ry ⁺) can be moved around the genome by a number of techniques. When it comes to lie close to a tissue-specific enhancer, blue staining results in particular tissues of the fly. Many different patterns of expression have already been obtained with the long-term aim of cloning the nearby genes that these enhancers normally regulate. Whilst analysing a set of flies containing theseP-lacZ inserts for sex-specific expression in the adult, a preference was noticed for insertion into regions of DNA generating expression of -galactosidase in the male gonad. Since the transposition events generating these flies occurred in the male germ-line, it seemed possible that there was preferential insertion of the element into DNA which was being transcribed. To test this, transpositions were generated of the sameP-lacZ ry ⁺ in both the male and female germ-lines. The results are compatible with the above hypothesis. This finding has important implications for the type of enhancers likely to be found by this method, and may also be relevant to those using P elements as mutagens in Drosophila and for the study of the mechanism of P transposition.     

Morphological criteria for recognising homology in isolated skeletal elements: comparison of traditional and morphometric approaches in conodonts

Abstract: Accurate hypotheses of primary homology are fundamental to many aspects of the systematics and palaeobiology of fossils. They are particularly critical for conodonts: virtually all areas of conodont research are underpinned by homology, yet the majority of conodont taxa are found only as disarticulated skeletal elements, and hypotheses of element homology are inferred from morphological comparisons with complete skeletons. This can cause problems in taxa where more than one location within the conodont skeleton is occupied by elements with similar morphology. In such cases, morphological comparisons can yield equivocal or erroneous hypotheses of homology of isolated elements. The Eramosa Lagerstätte of Ontario (Silurian, Wenlock) preserves both isolated skeletal elements and articulated conodont skeletons. The latter provide a topological context within which to test hypotheses of element homology and allow blind testing of qualitative discrimination of elements. When applied to P₁ and P₂ elements of Wurmiella excavata, this revealed inaccuracy and inconsistency in distinguishing these P element types. Standardised morphometric protocols were used to further test the efficacy of those characters used in traditional qualitative identification of P element homology, revealing that, individually, none of these characters provides an effective discriminator between P element types. Principal components and discriminant function analyses of ten ‘traditional’ morphological variables combined can distinguish P₁ from P₂ elements with a similar success rate to expert identification. Eigenshape and elliptic Fourier analyses of element outlines proved less effective at capturing shape differences that allowed for discrimination between P₁ and P₂ elements. Analysis of both traditional and outline data demonstrates that in some individuals P₁ and P₂ elements are morphologically distinct from one another, while in others they are almost indistinguishable. These results demonstrate that although qualitative assessments of homology can be prone to error, especially when undertaken by inexperienced researchers, the morphometric and analytical protocols used here provide effective additional tool for discriminating morphologically similar but non‐homologous elements. These methods thus hold promise of broad application to other conodont taxa where identification of element homology in collections of isolated specimens is problematic.     

Transposable DNA elements and life history traits

As an initial study of the influence of transposable DNA elements on life history traits, and as a model system for estimating the impact of somatic genetic damage on longevity, the effect of P DNA element movement in somatic cells on adult lifespan was measured in Drosophila melanogaster males. Lifespan was significantly reduced in males that contained the somatically active P[ry⁺ 2–3](99B) element and 17, 4, 3, but not just a single P element. Furthermore, there appears to be a direct correlation between the number of transposing P elements and the amount of lifespan reduction. This reduction in lifespan observed in males with somatically active P elements is probably due to genetic damage in embryos, larvae and pupae from P-element excisions and insertions, leading to changes in gene structure and regulation, chromosome breakage, and subsequent cell death in adults. This hypothesis is supported in this study by a significant increase in recessive sex-linked lethal mutations in the same males that had reduced lifespans and by the previous observation of chromosome breakage in somatic cells of similar males. The evolutionary implications of these results are discussed, including the possible influence of somatic DNA transpositions on fitness and other life history traits.     

Analysis of P element transposase protein-DNA interactions during the early stages of transposition

P elements are a family of transposable elements found in Drosophila that move by using a cut-and-paste mechanism and that encode a transposase protein that uses GTP as a cofactor for transposition. Here we used atomic force microscopy to visualize the initial interaction of transposase protein with P element DNA. The transposase first binds to one of the two P element ends, in the presence or absence of GTP, prior to synapsis. In the absence of GTP, these complexes remain stable but do not proceed to synapsis. In the presence of GTP or nonhydrolyzable GTP analogs, synapsis happens rapidly, whereas DNA cleavage is slow. Both atomic force microscopy and standard biochemical methods have been used to show that the P element transposase exists as a pre-formed tetramer that initially binds to either one of the two P element ends in the absence of GTP prior to synapsis. This initial single end binding may explain some of the aberrant P element-induced rearrangements observed in vivo, such as hybrid end insertion. The allosteric effect of GTP in promoting synapsis by P element transposase may be to orient a second site-specific DNA binding domain in the tetramer allowing recognition of a second high affinity transposase-binding site at the other transposon end.     

Maternal inheritance of P cytotype in Drosophila melanogaster: a “pre-P cytotype” is strictly extra-chromosomally transmitted

In Drosophila melanogaster, transposition of the P element is under the control of a cellular state known as cytotype. The P cytotype represses P transposition whereas the M cytotype is permissive for transposition. In the long-term, the P cytotype is determined by chromosomal P elements but over a small number of generations it is maternally inherited. In order to analyse the nature of this maternal inheritance, we tested whether a maternal component can be transmitted without chromosomal P elements. We used a stable determinant of P cytotype, linked to the presence of two P elements at the tip of the X chromosome (IA site) in a genome devoid of other P elements. We measured P repression capacity using two different assays: gonadal dysgenic sterility (GD) and P-lacZ transgene repression. We show that zygotes derived from a P cytotype female (heterozygous for P (1A)/balancer devoid of P copies) and which inherit no chromosomal P elements from the mother, have, however, maternally received a P-type extra-chromosomal component: this component is insufficient to specify the P cytotype if the zygote formed does not carry chromosomal P elements but can promote P cytotype determination if regulatory P elements have been introduced paternally. We refer to this strictly extra-chromosomally inherited state as the “pre-P cytotype”. In addition, we show that a zygote that has the pre-P cytotype but which has not inherited any chromosomal P elements, does not transmit the pre-P cytotype to the following generation. The nature of the molecular determinants of the pre-P cytotype is discussed.     

Establishment and maintenance of the P cytotype associated with telomeric P elements in Drosophila melanogaster

P elements inserted near the left telomere of the X chromosome are associated with the P cytotype, a maternally transmitted condition that strongly regulates the activity of the P transposon family in some strains of Drosophila. The regulatory abilities of two such elements, TP5 and TP6, are stable in homozygous stocks over many generations. However, these regulatory abilities are attenuated when the telomeric P elements are transmitted through heterozygous females, and they are utterly lost when the elements are transmitted through males. Paternally transmitted telomeric P elements reacquire regulatory ability when they pass through a female germ line. This reacquisition is enhanced if the females in which it occurs came from mothers who carried a telomeric P element. The enhancement has two components: (1). a strictly maternal effect that is transmitted to the females independently of the mother's telomeric P element ("presetting" or the "pre-P cytotype") and (2). a zygotic effect associated with inheritance of the mother's telomeric P element. One telomeric P element can enhance the reacquisition of another's regulatory ability. When X chromosomes that carry telomeric P elements are extracted through males and made homozygous by using a balancer chromosome, most of the resulting stocks develop strong regulatory abilities in a few generations. However, some of the stocks do not attain the regulatory ability of the original population.     

The Regulatory Properties of Autonomous Subtelomeric P Elements Are Sensitive to a Suppressor of Variegation in Drosophila Melanogaster

Genetic recombination was used in Drosophila melanogaster to isolate P elements, inserted at the telomeres of X chromosomes (cytological site 1A) from natural populations, in a genetic background devoid of other P elements. We show that complete maternally inherited P repression in the germline (P cytotype) can be elicited by only two autonomous P elements at 1A and that a single element at this site has partial regulatory properties. The analysis of the surrounding chromosomal regions of the P elements at 1A shows that in all cases these elements are flanked by Telomeric Associated Sequences, tandemly repetitive noncoding sequences that have properties of heterochromatin. In addition, we show that the regulatory properties of P elements at 1A can be inhibited by some of the mutant alleles of the Su(var)205 gene and by a deficiency of this gene. However, the regulatory properties of reference P strains (Harwich and Texas 007) are not impaired by Su(var)205 mutations. Su(var)205 encodes Heterochromatin Protein 1 (HP1). These results suggest that the HP1 dosage effect on the P element properties is site-dependent and could involve the structure of the chromatin.     

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 by (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 consist of 31 by inverted repeats, the inverted repeats of the O-type elements are interrupted by non-complementary stretches of DNA, 12 by at the 5 end and 14 by 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. The sequence comparison also provides new insights into conserved domains with possible implications for the function of P transposons.     

NfCR1, the first non-LTR retrotransposon characterized in the Australian lungfish genome, Neoceratodus forsteri, shows similarities to CR1-like elements

The genomes of lungfish, together with those of some urodele amphibians, are the largest of all vertebrate genomes. It has been assumed that the bulk of the DNA making up these large genomes has been derived from repeat elements, like the noncoding DNA of those genomes that have been sequenced (e.g., human). In an attempt to characterize repeat sequences in the lungfish genome, we have isolated, by restriction enzyme digestion of genomic DNA, sequences of a repeat element in Neoceratodus forsteri, the most primitive of the living lungfishes. The fragments sequenced from the EcoRI and BglII digests were used to perform genome walking PCR in order to obtain the full sequence of the repeat element. This element shares homology with the non-LTR (LINE) element, Chicken Repeat 1 (CR1), described for several vertebrates and some invertebrates; we have called it N. forsteri CR1 (NfCR1). NfCR1 shares all the domains of other CR1 elements but it also has several unique features that suggest it may no longer be active in the lungfish genome. It occurs in both full-length and 5'-truncated versions and in its present "inactive" form represents approximately 0.05% of the lungfish genome.