An in vitro study to improve understanding of how P\T-DNA interactions occur to carry out the different chemical steps of the P\T transposition reaction: P\T protein contains two DNA-binding sites and the active oligomeric form of the transposase protein is at least a dimer.

P\T protein requires both 5' and 3' P-element termini for efficient donor DNA cleavage to occur. P\T protein creates a staggered cleavage at the P-element termini; the 3' cleavage site is at the end of the P-element, while the 5' cleavage site is 17bp within the P-element 31bp inverted repeats.

Hrb27C plays a functional role in P\T IVS3 splicing inhibition.

Influence of gamma irradiation on P-element excision and excision-site repair mechanism is directly tested by embryonic somatic excision assays. Frequency of precise or near precise excision of P{Δ2-3} increases with gamma ray does, a positive interaction between gamma irradiation and P-element activity is concluded.

P\T contains a non-canonical GTP-binding domain that is critical for its ability to mediate transposition in Drosophila cells.

A model of the accumulation of recombinants in the germline through the action of mitotic recombination is determined. The basic model is refined by considering hybrid element insertions and exploration of the stochastic effects through a computer simulation of the time course of P-element-induced recombination in spermatogenesis. These models lead to a number of predictions related to the distribution of progeny, which can be tested against observations.

P{SR} has been identified in a Q population. Populations containing such strong repressor elements may have a selective advantage over populations which rely on maternally transmitted P cytotype or P{KP}-induced weak levels of repression, due to repression of hybrid dysgenesis.

The KP repressor protein binds to multiple sites on the ends of P-element DNA, unlike the full length transposase protein. These sites include the high affinity transposase binding site and, at the highest concentrations tested, the terminal inverted repeats. The DNA binding domain is localised to the N-terminal 98 amino acids and contains a CCHC sequence, a potential metal-binding motif. The KP repressor protein can dimerise and contains two protein-protein interaction regions and this dimerisation is essential for high affinity DNA binding.

Codon usage in the Dwil\P-element\T gene is more similar to the codon usage of D.willistoni protein-coding genes (Dwil\Adh, Dwil\Sod and Dwil\per) than P\T gene is to the codon usage of D.melanogaster protein-coding genes (Adh, Sod and per). This observation supports the contention that the P-element has a longer evolutionary history in D.willistoni than in D.melanogaster and therefore is adding evidence supporting the hypothesis of horizontal transfer from D.willistoni to D.melanogaster.

Experimental results from expressing sense and antisense constructs from an Hsp70 promoter suggests that in nature P-element activity could be regulated by P-encoded polypeptides and by antisense P-element RNAs although evidence for naturally occurring antisense P-element RNA has not yet been obtained.

Mutation of the KP-element demonstrates that the leucine zipper of the KP polypeptide is important for P-element regulation. This supports the multimer-poisoning model of P-element repression because leucine zipper motifs are involved in protein-protein interactions.

The autoregulatory nature of P cytotype in the germline is brought about through a combination of transcriptional repression and alteration of P-element IVS3 splicing. The transcriptional regulatory effects of P-cytotype are not restricted to the P-element promoter suggesting a general mechanism of repression.

P\T induces male recombination additively and without a requirement for P element insertion or excision.

P element mobilizations occur during the meiotic cell cycle: using non-disjunction to produce patroclinous daughters with both sister X-chromatids, approximately 4% of dysgenic male gametes were seen to have transposon perturbations of meiotic origin, and the proportion of gametes containing lesions of premeiotic origin was estimated at 32%. Results of the analysis are consistent with the gap-repair model of P element transposition.

The P cytotype has maternal transmission of repressor that causes reduced expression of transposase promoter. Two classes of repressor exist that have discrete structural characteristics: type I are large repressors that exist of P-element sequences through exon 2 and the first 9 nucleotides of the 2-3 intron and type II repressors are small elements that delete exons 2 and 3. The type I repressor can repress cytotype- dependent alleles and P-element mobility in somatic and germline tissues.

P{Ins1a} sequence consists of 0.6kb core P-element, P\T^core and 1.15kb KP elements, P\T^KP and P\T^KP'. 5' and 3' deletions of P{Ins1a} are inserted in the Zw promoter region and provide some Zw gene activity. In vitro transcription analysis of P\T sequences activating Zw-Act5C transcriptional gene reveals three distinct cis-acting regions, one in P\T^core and two in the P\T^KP and P\T^KP' elements, that are required for overexpression. Putative transcriptional regulatory proteins, identified in gel retardation assays, bind to each of the cis-acting regions.

The splicing of the third intron of the P-element was assayed in pole cells: not all pole cells were capable of splicing the third intron. Almost all pole cells that have the splicing activity penetrate the gonad and differentiate into primordial germ cells.

P cytotype repression of P{lacZ} expression is observed in the germline. The intensity of repression is stronger than in somatic tissues and the repression has a maternal effect that is restricted to the germline. The thermosensitivity of P{lacZ} repression parallels thermosensitivity of the P cytotype.

Mobilization of the 66kD repressor element to new positions in the genome can show substantial maternal effect repression in the germline, indicating that genomic position is an important determinant of maternal P cytotype. Maternal repression requires a very specific time and location of repressor expression or genomic position of a repressor element may affect more than just repressor protein production.

Individual P elements isolated from wild type strains show distinct profiles of repression and suppression abilities which may be mediated by P-encoded polypeptides or by antisense P RNAs initiated from external genomic promoters. Repression of gonadal dysgenic sterility may operate through a maternal effect while effects on sn^w mutability seem to be zygotic.

P-element transposition mechanisms can be biochemically studied using an in vitro reaction system, transfer of P-element DNA from a donor to target plasmid. Transposition events can occur in the presence of partially purified P\T. A 3'-hydroxyl group on the P-element terminus is required for transposition.

The exon sequence located 12 to 31bp from the 5' splice site of the P\T ORF2-ORF3 intron is required to inhibit splicing of this intron in somatic tissue.

P\T represses transcription from the P-element promoter in vitro.

All P-element-Ecol\lacZ insertions are repressed in a P background, a reduction in the number of transcripts. The repression occurs in all tissues at all developmental stages. Results suggest that P trans-acting products can exert a direct repression on the P\T promoter transcription.

The 20 nucleotide sequence required for the somatic inhibition of splicing of the ORF2-ORF3 intron of the P-element is also capable of inhibiting the splicing of this intron in HeLa cell nuclear extracts.

No correspondence between P-element mobilisation events and recombination in males is found. Definition of 2 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, suggests that transposase may be active at non-P-element sites and that the genome may harbour sequences ranging from highly responsive to completely unresponsive to transposase action.

Modified P-element derivatives that encode only the 66kD P-element protein display zygotic repression of transposase activity in both the germline and soma. The production of the 66kD repressor at high levels during oogenesis may be responsible for the maternal inheritance of the P cytotype.

P{Δ2-3}, in conjunction with P{CaSpeR}, induces recombination in the male germ line. Recombination appears to be premeiotic in a high proportion of cases. P{Δ2-3}-P{CaSpeR} combination also elevates the incidence of somatic recombination.

Insertion of a complex transposon carrying P\T^KP, P\T^KP' and P\T^core into the 5' region of Zw does not alter the transcription start site or length of RNA transcripts but increases the amount of mRNA.

The structure of P{Ins1} comprises of two defective P-elements (P\T^KP and P\T^KP') that encompass a third defective P-element (P\T^core), the core sequences. A complex transposon comprising of two defective P-elements (P\T^KP and P\T^KP') that encompass a third defective P-element (P\T^core) is studied.

The 190bp ORF2-ORF3 intron retains its germ line specificity when placed, as part of a 240bp P-element fragment, into the context of a different gene, in this case Ecol\lacZ reporter gene.