Inheritance of P-element regulation in Drosophila melanogaster.

The ability to repress P-element-induced gonadal dysgenesis was studied in 14 wild-type strains of D. melanogaster derived from populations in the central and eastern United States. Females from each of these strains had a high ability to repress gonadal dysgenesis in their daughters. Reciprocal hybrids produced by crossing each of the wild-type strains with an M strain demonstrated that repression ability was determined by a complex mixture of chromosomal and cytoplasmic factors. Cytoplasmic transmission of repression ability was observed in all 14 strains and chromosomal transmission was observed in 12 of them. Genomic Southern blots indicated that four of the strains possessed a particular type of P element, called KP, which has been proposed to account for the chromosomal transmission of repression ability. However, in this study several of the strains that lacked KP elements exhibited as much chromosomal transmission of repression ability as the strains that had KP elements, suggesting that other kinds of P elements may be involved.     

The Drosophila P-element KP repressor protein dimerizes and interacts with multiple sites on P-element DNA.

Drosophila P elements are mobile DNA elements that encode an 87-kDa transposase enzyme and transpositional repressor proteins. One of these repressor proteins is the 207-amino-acid KP protein which is encoded by a naturally occurring P element with an internal deletion. To study the molecular mechanisms by which KP represses transposition, the protein was expressed, purified, and characterized. We show that the KP 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, an 11-bp transpositional enhancer, and, at the highest concentrations tested, the terminal 31-hp inverted repeats. The DNA binding domain was localized to the N-terminal 98 amino acids and contains a CCHC sequence, a potential metal binding motif. We also demonstrate that the KP repressor protein can dimerize and contains two protein-protein interaction regions and that this dimerization is essential for high-affinity DNA binding.     

Identification of novel Drosophila meiotic genes recovered in a P-element screen.

The segregation of homologous chromosomes from one another is the essence of meiosis. In many organisms, accurate segregation is ensured by the formation of chiasmata resulting from crossing over. Drosophila melanogaster females use this type of recombination-based system, but they also have mechanisms for segregating achiasmate chromosomes with high fidelity. We describe a P-element mutagenesis and screen in a sensitized genetic background to detect mutations that impair meiotic chromosome pairing, recombination, or segregation. Our screen identified two new recombination-deficient mutations: mei-P22, which fully eliminates meiotic recombination, and mei-P26, which decreases meiotic exchange by 70% in a polar fashion. We also recovered an unusual allele of the ncd gene, whose wild-type product is required for proper structure and function of the meiotic spindle. However, the screen yielded primarily mutants specifically defective in the segregation of achiasmate chromosomes. Although most of these are alleles of previously undescribed genes, five were in the known genes alphaTubulin67C, CycE, push, and Trl. The five mutations in known genes produce novel phenotypes for those genes.     

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.     

In vitro spermatogenesis in Drosophila

Summary In vitro spermatogenesis of isolated single spermatocyte cysts of Drosophila hydei was studied by microscopic observations and time-lapse cinematography. Cysts of spermatocytes isolated during diplotene develop as far as the coiling stage of spermatid differentiation. The existence of an interphase between meiosis I and meiosis II is, for the first time, documented. Meiosis, Nebenkern formation, and elongation of spermatids occur just as in D. melanogaster; however, an individualization cone, as described for D. melanogaster, can not be detected.     

Genetics of P-element transposition into Drosophila melanogaster centric heterochromatin

Heterochromatin is a major component of higher eukaryotic genomes, but progress in understanding the molecular structure and composition of heterochromatin has lagged behind the production of relatively complete euchromatic genome sequences. The introduction of single-copy molecular-genetic entry points can greatly facilitate structure and sequence analysis of heterochromatic regions that are rich in repeated DNA. In this study, we report the isolation of 502 new P-element insertions into Drosophila melanogaster centric heterochromatin, generated in nine different genetic screens that relied on mosaic silencing (position-effect variegation, or PEV) of the yellow gene present in the transposon. The highest frequencies of recovery of variegating insertions were observed when centric insertions were used as the source for mobilization. We propose that the increased recovery of variegating insertions from heterochromatic starting sites may result from the physical proximity of different heterochromatic regions in germline nuclei or from the association of mobilizing elements with heterochromatin proteins. High frequencies of variegating insertions were also recovered when a potent suppressor of PEV (an extra Y chromosome) was present in both the mobilization and selection generations, presumably due to the effects of chromatin structure on P-element mobilization, insertion, and phenotypic selection. Finally, fewer variegating insertions were recovered after mobilization in females, in comparison to males, which may reflect differences in heterochromatin structure in the female and male germlines. FISH localization of a subset of the insertions confirmed that 98% of the variegating lines contain heterochromatic insertions and that these schemes produce a broader distribution of insertion sites. The results of these schemes have identified the most efficient methods for generating centric heterochromatin P insertions. In addition, the large collection of insertions produced by these screens provides molecular-genetic entry points for mapping, sequencing, and functional analysis of Drosophila heterochromatin.     

Efficient recovery of centric heterochromatin P-element insertions in Drosophila melanogaster

Approximately one-third of the human and Drosophila melanogaster genomes are heterochromatic, yet we know very little about the structure and function of this enigmatic component of eukaryotic genomes. To facilitate molecular and cytological analysis of heterochromatin we introduced a yellow(+) (y(+))-marked P element into centric heterochromatin by screening for variegated phenotypes, that is, mosaic gene inactivation. We recovered >110 P insertions with variegated yellow expression from approximately 3500 total mobilization events. FISH analysis of 71 of these insertions showed that 69 (97%) were in the centric heterochromatin, rather than telomeres or euchromatin. High-resolution banding analysis showed a wide but nonuniform distribution of insertions within centric heterochromatin; variegated insertions were predominantly recovered near regions of satellite DNA. We successfully used inverse PCR to clone and sequence the flanking DNA for approximately 63% of the insertions. BLAST analysis of the flanks demonstrated that either most of the variegated insertions could not be placed on the genomic scaffold, and thus may be inserted within novel DNA sequence, or that the flanking DNA hit multiple sites on the scaffold, due to insertions within different transposons. Taken together these data suggest that screening for yellow variegation is a very efficient method for recovering centric insertions and that a large-scale screen for variegated yellow P insertions will provide important tools for detailed analysis of centric heterochromatin structure and function.     

Three-partner conversion induced by the P-element transposase in Drosophila melanogaster

Conversion of one P-derived transposon into another has already been shown to occur with a measurable frequency. However, the mechanism responsible for such replacements has remained controversial. We previously proposed a mechanism involving three partners. We assumed that after excision of the P-element inserted at the target site, the double-strand break was repaired using, first, the homologous P sequences on the sister chromatid, and second, a remote template, the donor P-derived transposon. However, two other mechanisms have been proposed. The first involves two partners only, the broken end and the remote template, while the second involves transposition of the donor into the target P-element, followed by a double recombination event. Here we describe the conversion of a defective P-element using as a remote template an enhancer-trap element that is itself unable to transpose because it lacks 21 bp at its 5' end. This result makes it possible to exclude the possibility that this conversion event occurred after transposition. The new allele was molecularly and genetically characterized. The occurrence of a polymorphism at position 33 of the P-element sequence and of an imperfect copy of the template on the 3' side of the converted transposon confirmed that the sister chromatid was absolutely necessary as a partner for repair. Our results show that targeting of a marked P-element is possible, even when this element is unable to transpose. This provides a means of improving recovery of conversion events by eliminating unwanted transpositions catalyzed by the P transposase.     

Customized microinjection glass capillary needles for P-element transformations in Drosophila melanogaster

Here we describe how to generate customized microinjection needles from glass capillary tubes. Controls demonstrate the range of variables and effects on needle tip shape using a standard Flaming/Brown micropipet needle puller. Needles generated with two-cycle pulls provide a wider range of needle shapes in a predictable fashion. We used the needle puller's ramp function for multiple-cycle programs to determine the useful range of heat settings inherent to the glass capillary tube. This articlefocuses primarily on the preparation of injection needles utilized for P-element-mediated germ-line transformation in Drosophila melanogaster that do not require the dechorionation of the egg. However, these types of needles can be usefulfor numerous other types of injections, such as RNA interference, homologous recombination mutagenesis, morpholinos, transient gene regulation, drug delivery, and the transfer of cytoplasmic factors that are useful in a wide range of biological systems ranging from plants to vertebrates. Using our standard needle, we correlate the survival of injected D. melanogaster embryos with transformation efficiencies and plasmid construct characteristics.     

Studying Drosophila embryogenesis with P-lacZ enhancer trap lines

Summary Embryos of 171 Drosophila lines carrying a P-lacZ insertion on the second or third chromosome were analyzed regarding their pattern of lacZ expression. All lines were selected from a larger screen of about 4000 lines (Bier et al. 1989). Tissue specificity and time of onset of lacZ expression was documented for each line. Thereby, a comprehensive list of markers for the various tissue and cell types of the Drosophila embryo could be assembled. With the help of several P-lacZ lines the development of a number of structures was studied which so far had been described only insufficiently or not at all. In particular, the embryonic origin and early development of the oenocytes, imaginal discs, histoblasts, fat body, dorsal vessel, and perineurial cells was analyzed. Several previously unknown cell types associated with the dorsal vessel, trachea, and epidermis were discovered. By combining data regarding the origin of the different mesodermally derived organs it was possible to generate in some detail a fate map of the mesoderm of the stage 11 Drosophila embryo. Offprint requests to: V. Hartenstein     

P-element homing is facilitated by engrailed polycomb-group response elements in Drosophila melanogaster

P-element vectors are commonly used to make transgenic Drosophila and generally insert in the genome in a nonselective manner. However, when specific fragments of regulatory DNA from a few Drosophila genes are incorporated into P-transposons, they cause the vectors to be inserted near the gene from which the DNA fragment was derived. This is called P-element homing. We mapped the minimal DNA fragment that could mediate homing to the engrailed/invected region of the genome. A 1.6 kb fragment of engrailed regulatory DNA that contains two Polycomb-group response elements (PREs) was sufficient for homing. We made flies that contain a 1.5 kb deletion of engrailed DNA (en(Δ1.5)) in situ, including the PREs and the majority of the fragment that mediates homing. Remarkably, homing still occurs onto the en(Δ1. 5) chromosome. In addition to homing to en, P[en] inserts near Polycomb group target genes at an increased frequency compared to P[EPgy2], a vector used to generate 18,214 insertions for the Drosophila gene disruption project. We suggest that homing is mediated by interactions between multiple proteins bound to the homing fragment and proteins bound to multiple areas of the engrailed/invected chromatin domain. Chromatin structure may also play a role in homing.     

P-element transposition in Drosophila melanogaster: influence of size and arrangement in pairs

In Drosophila melanogaster, several factors have been suggested to influence the rates of P-element transposition and excision, including position effects, size and structure of the elements and differences in transposase source. We have investigated the effect of the size of the starting P-element on the rates of excision and transposition. Four transgenes localized at the same insertion site on the X chromosome and which differ by the number of copies of an internal repeated sequence, were studied. Transgenes with sizes ranging from 11 kb to 22 kb excise at similar rates, and size does not correlate with the differences in transposition rate between them. We also studied the behavior of double P-elements, located at the same site and arranged in various configurations: nested, contiguous or separated by a few base pairs, in the same or reverse orientation. These double P-elements display different mobilities depending on the arrangement of the two transgenes. Transposition and excision rates were also studied for an insertion bearing four transgenes in very close proximity. Our results suggest that several neighboring elements could excise together. We also propose a new model to explain the formation of all the double P-elements we describe. Received: 7 October 1999 / Accepted: 28 December 1999     

Distinct P-element excision products in somatic and germline cells of Drosophila melanogaster

The footprints remaining following somatic P-element excision from the Drosophila white locus were recovered and characterized. Two different types of footprints were observed. Over 75% of the footprints were short, composed of 4 or 7 nucleotides of the P-element inverted terminal repeat, and were similar to those found in a previously described plasmid excision assay. The remaining footprints were composed of 14-18 nucleotides of both inverted terminal repeats. These large footprints were indistinguishable from those recovered following germline P-element excision. Enhanced expression of the Drosophila homologue of the Ku70 protein did not affect the structure of the somatic footprints. Therefore, this protein is not a limiting factor for double-strand break repair by nonhomologous end-joining in Drosophila somatic cells.