P Element Insertions and Rearrangements at the Singed Locus of Drosophila Melanogaster

DNA from the singed gene of Drosophila melanogaster was isolated using an inversion between a previously cloned P element at cytological location 17C and the hypermutable allele singed-weak. Five out of nine singed mutants examined have alterations in their DNA maps in this region. The singed locus is a hotspot for mutation during P-M hybrid dysgenesis, and we have analyzed 22 mutations induced by P-M hybrid dysgenesis. All 22 have a P element inserted within a 700-bp region. The precise positions of 10 P element insertions were determined and they define 4 sites within a 100-bp interval. During P-M hybrid dysgenesis, the singed-weak allele is destabilized, producing two classes of phenotypically altered derivatives at high frequency. In singed-weak, two defective P elements are present in a "head-to-head" or inverse tandem arrangement. Excision of one element results in a more extreme singed bristle phenotype while excision of the other leads to a wild-type bristle phenotype.     

The P cytotype in Drosophila melanogaster: a maternally transmitted regulatory state of the germ line associated with telomeric P elements

The incomplete P elements TP5 and TP6 are inserted in the TAS repeats near the left telomere of the Drosophila melanogaster X chromosome. These telomeric P elements repress P-induced gonadal dysgenesis and germ-line hypermutability in both sexes. However, their capacity to repress hypermutability is lost when they are transmitted patroclinously in a cross. TP5 and TP6 do not repress P-element activity in somatic cells, nor do they alter the somatic or germ-line phenotypes of P-insertion alleles. In the germ line, these elements suppress the phenotype of a P-insertion allele of the singed gene that is evoked by other P elements, presumably because these other elements encode repressor polypeptides. This suppression is more effective when the telomeric P elements are inherited maternally. Regulation by telomeric P elements parallels that of the P cytotype, a state that represses P-element activity in some strains of Drosophila. This state exists only in the germ line and is maternally transmitted along with the P elements themselves. Regulation by known repressor P polypeptides is not restricted to the germ line and does not require maternal transmission of the relevant P elements. Regulation by telomeric P elements appears to be epistatic to regulation by repressor P polypeptides.     

Y-Linked male sterile mutations induced by P element in Drosophila melanogaster.

The Y chromosome in Drosophila melanogaster is composed of highly repetitive sequences and is essential only in the male germ line. We employed P-element insertional mutagenesis to induce male sterile mutations in the Y chromosome. By using a combination of two modifiers of position effect variegation, adding an extra Y chromosome and increasing temperature, we isolated 61 P(ry+) elements in the Y chromosome. Six of these Y-linked insertions (approximately 10%) induced male sterile mutations that are mapped to two genes on the long and one on the short arms of the Y chromosome. These mutations are revertible to the wild type in a cell-autonomous and germ-line-dependent manner, consistent with previously defined Y-linked gene functions. Phenotypes associated with these P-induced mutations are similar to those resulting from deletions of the Y chromosome regions corresponding to the male fertility genes. Three alleles of the kl-3 gene on the Y long arm result in loss of the axonemal outer dynein arms in the spermatid tail, while three ks-2 alleles on the Y short arm induce defects at early postmeiotic stages. The recovery of the ms(Y) mutations induced by single P-element insertions will facilitate our effort to understand the structural and functional properties of the Y chromosome.     

Effects of Single P-Element Insertions on Bristle Number and Viability in Drosophila Melanogaster

Single P-element mutagenesis was used to construct 1094 lines with P[lArB] inserts on all three major chromosomes in an isogenic background previously free of P elements. The effects of insertions on bristle number and on viability were assessed by comparison to 392 control lines. The variance and effects of P-element inserts on bristle number and viability were larger than those inferred from spontaneous mutations. The distributions of effects on bristle number were symmetrical and highly leptokurtic, such that a few inserts with large effects caused most of the increase in variance. The distribution of effects on viability were negatively skewed and platykurtic. On average, the effects of P-element insertions on bristle number were partly recessive and on viability were completely recessive. P-element inserts with large effects on bristle number tended to have reduced viability, but the correlation between the absolute value of the effects on bristle number and on viability was not strong. Fifty P-element inserts tagging quantitative trait loci (QTLs) with large effects on bristle number were mapped cytogenetically. Two P-element-induced scabrous alleles and five extramacrochaetae alleles were generated. Single P-element mutagenesis is a powerful method for identifying QTLs at the level of genetic locus.     

Testing the palindromic target site model for DNA transposon insertion using the Drosophila melanogaster P-element

Understanding the molecular mechanisms that influence transposable element target site preferences is a fundamental challenge in functional and evolutionary genomics. Large-scale transposon insertion projects provide excellent material to study target site preferences in the absence of confounding effects of post-insertion evolutionary change. Growing evidence from a wide variety of prokaryotes and eukaryotes indicates that DNA transposons recognize staggered-cut palindromic target site motifs (TSMs). Here, we use over 10 000 accurately mapped P-element insertions in the Drosophila melanogaster genome to test predictions of the staggered-cut palindromic target site model for DNA transposon insertion. We provide evidence that the P-element targets a 14-bp palindromic motif that can be identified at the primary sequence level, which predicts the local spacing, hotspots and strand orientation of P-element insertions. Intriguingly, we find that the although P-element destroys the complete 14-bp target site upon insertion, the terminal three nucleotides of the P-element inverted repeats complement and restore the original TSM, suggesting a mechanistic link between transposon target sites and their terminal inverted repeats. Finally, we discuss how the staggered-cut palindromic target site model can be used to assess the accuracy of genome mappings for annotated P-element insertions.     

Visible mutations induced by P-M hybrid dysgenesis in Drosophila melanogaster result predominantly from P element insertions.

This study supplied no evidence that P-M hybrid dysgenesis is a general release mechanisms for transposon movement. Newly induced mutations (23 singed, three yellow, and one white) were generated by hybrid dysgenesis and were molecularly analyzed for the presence or absence of P element insertions. Only one dysgenically-induced insertion mutation out of 27 analyzed was the result of a non-P insert; this frequency is not statistically above the non-dysgenic control level. Thus, it appears that individual transposable elements families are independently regulated.     

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.     

Superunstable systems in Drosophila melanogaster. Analysis of mutations in signed and cut loci

The lines of the M'-cytotype characterized by a long-term instability (which was shown to be conditioned by transpositions of the new mobile element, Stalker) were hybridized with the P-line. This resulted in the appearance of a number of superunstable mutations at the yellow, white, singed, ocelliless and some other loci. The authors analyzed four independently obtained families of superunstable mutations at the singed locus. A wide spectrum of derivatives and high frequency of mutations were demonstrated, as well as the regularities of allelic transitions. Besides this, mutagenesis at the cut locus was observed in the chromosomes carrying sn mutations with frequency of 5.05 x 10(4). By means of the blot analysis it has been shown that most of ct mutations are intragenic deficiencies, ranging from 1.3 to 3 Kb, whose appearance is, conceivably, attributed to the inaccuracy of the insertion excision (the insertion is present but fails to alter the phenotype) at the cut locus of the chromosomes with the superunstable sn-alleles. In the lines with the sn- and ct-mutations the transpositions of the P-element and the Stalker were found, which indicates their involvement in mutagenesis. The authors discuss possible effects of inserting the complicated constructions, based on the combinations of P-element and the Stalker, on the induction of superinstability.     

Morewright (Mwr), a New Meiotic Mutant of Drosophila Melanogaster Affecting Nonexchange Chromosome Segregation

A new meiotic mutation, morewright (mwr) was identified by screening for new mutations that act as dominant enhancers of the dosage-sensitive Drosophila melanogaster female meiotic mutant, nod(DTW). mwr is a recessive meiotic mutant, specifically impairing the segregation of nonexchange chromosomes. Cytological evidence suggests that the meitoic defect in mwr/mwr females is in homologue recognition because the chromosomes appear to be misaligned on an intact spindle. The mwr mutation was recovered during a screen of random P-element insertions on a chromosome with a single insertion located at 50C. The P-element insertion is a recessive female-sterile mutation. While excision of the P element from the mwr-bearing chromosome partially relieves the female sterility, the excisions retain the dominant nod(DTW)-enhancing activity. The mwr meiotic phenotype maps very close to the female-sterile P insertion. Thus the mwr locus appears to encode a function required for partner recognition in meiosis, although its relationship to the neighboring female-sterile mutation remains to be elucidated.     

Telomeric P elements associated with cytotype regulation of the P transposon family in Drosophila melanogaster

P elements inserted at the left end of the Drosophila X chromosome were isolated genetically from wild-type P strains. Stocks carrying these elements were tested for repression of P-strain-induced gonadal dysgenesis in females and for repression of transposase-catalyzed P-element excision in males and females. Both traits were repressed by stocks carrying either complete or incomplete P elements inserted near the telomere of the X chromosome in cytological region 1A, but not by stocks carrying only nontelomeric X-linked P elements. All three of the telomeric P elements that were analyzed at the molecular level were inserted in one of the 1.8-kb telomere-associated sequence (TAS) repeats near the end of the X chromosome. Stocks with these telomeric P elements strongly repressed P-element excision induced in the male germline by a P strain or by the transposase-producing transgenes H(hsp/CP)2, H(hsp/CP)3, a combination of these two transgenes, and P(ry(+), delta2-3)99B. For H(hsp/CP)2 and P(ry(+), delta2-3)99B, the repression was also effective when the flies were subjected to heat-shock treatments. However, these stocks did not repress the somatic transposase activity of P(ry(+), delta2-3)99B. Repression of transposase activity in the germline required maternal transmission of the telomeric P elements themselves. Paternal transmission of these elements, or maternal transmission of the cytoplasm from carriers, both were insufficient to repress transposase activity. Collectively, these findings indicate that the regulatory abilities of telomeric P elements are similar to those of the P cytotype.     

Cloning and characterization of avfA and omtB genes involved in aflatoxin biosynthesis in three Aspergillus species

A collection of lethal and semi-lethal P-element insertions in the 70CD region of chromosome 3 of Drosophila melanogaster was used to investigate genes and gene arrangements by a combination of genetic, cytological, functional and molecular methods. The 12 lethal insertions studied fall into seven complementation groups of six genes. Lethal phases, expression patterns and other phenotypic aspects of these genes were determined. The genes and additional available sequences were placed on cloned genomic DNA fragments and arranged in an EcoRI map of 150kb that covers approximately the bands 70C7-8 to 70D1. Determination of deficiency breakpoints links the genetic, physical and molecular data. The sequences adjacent to seven independent P-element insertions were established after plasmid rescue or polymerase chain reaction. Similarity searches allowed the assignment of the P-element insertions to known mutations, expressed sequence tags, sequence tagged sites, or homologous genes of other species. Among these were identified a putative transacylase, a putative cell cycle gene, and the gene responsible for the dominant Polycomb-suppressor phenotype of devenir. The genomic sequence of the l(3)70Ca/b gene reveals a novel heat shock protein (hsc70Cb). l(3)70Da was identified as a member of the CDC48/PEX1 ATPase family and its coding sequence was determined.     

Cytogenetic analysis of the cSOD microregion in Drosophila melanogaster

This report describes the genetic organization of a euchromatic region on the third chromosome of Drosophila melanogaster extending cytologically from 68A2 to C1, an interval comprising 10 or 11 polytene chromosome bands. The gene for cytoplasmic superoxide dismutase (cSOD) maps within this interval, as does low xanthine dehydrogenase (lxd).--Recessive lethal mutations were generated within the region by ethyl methanesulfonate mutagenesis and by hybrid dysgenesis. These lethals fall into 11 functional groups, which were partially ordered by complementation with deletions having breakpoints within the region. The distribution of dysgenesis-induced mutations in the region is highly nonrandom, the majority being within a single group. The mutability of this gene is comparable to that of singed (sn), a documented "hot-spot" for P-element insertion.--One of the EMS-induced lethals, l-108, fulfills biochemical criteria expected of a hypomorphic allele of cSOD. To our knowledge this is the first such allele recovered of this gene, and it should prove very useful in an analysis of the in vivo function of cytoplasmic SOD. Indeed, it has been demonstrated that cSOD is almost certainly a vital gene.     

P-Element Insertion Alleles of Essential Genes on the Third Chromosome of Drosophila Melanogaster: Correlation of Physical and Cytogenetic Maps in Chromosomal Region 86e-87f

We have established a collection of 2460 lethal or semi-lethal mutant lines using a procedure thought to insert single P elements into vital genes on the third chromosome of Drosophila melanogaster. More than 1200 randomly selected lines were examined by in situ hybridization and 90% found to contain single insertions at sites that mark 89% of all lettered subdivisions of the Bridges' map. A set of chromosomal deficiencies that collectively uncover ~25% of the euchromatin of chromosome 3 reveal lethal mutations in 468 lines corresponding to 145 complementation groups. We undertook a detailed analysis of the cytogenetic interval 86E-87F and identified 87 P-element-induced mutations falling into 38 complementation groups, 16 of which correspond to previously known genes. Twenty-one of these 38 complementation groups have at least one allele that has a P-element insertion at a position consistent with the cytogenetics of the locus. We have rescued P elements and flanking chromosomal sequences from the 86E-87F region in 35 lines with either lethal or genetically silent P insertions, and used these as probes to identify cosmids and P1 clones from the Drosophila genome projects. This has tied together the physical and genetic maps and has linked 44 previously identified cosmid contigs into seven ``supercontigs' that span the interval. STS data for sequences flanking one side of the P-element insertions in 49 lines has identified insertions in the αγ element at 87C, two known transposable elements, and the open reading frames of seven putative single copy genes. These correspond to five known genes in this interval, and two genes identified by the homology of their predicted products to known proteins from other organisms.     

Molecular characterization of the Rpt1/p48B ATPase subunit of the <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> 26S proteasome

The function and the molecular properties of the Rpt1/p48B ATPase subunit of the regulatory particle of the Drosophila melanogaster 26S proteasome have been studied by analyzing three mutant Drosophila stocks in which P-element insertions occurred in the 5′-non-translated region of the Rpt1/p48B gene. These P-element insertions resulted in larval lethality during the second instar larval phase. Since the Rpt1/p48B gene resides within a long intron of an annotated, but uncharacterized Drosophila gene (CG17985), the second instar larval lethality may be a consequence of a combined damage to two independent genes. To analyze the phenotypic effect of the mutations affecting the Rpt1/p48B gene alone, imprecise P-element excision mutants were selected. One of them, the pupal lethal P1 mutation, is a hypomorphic allele of the Rpt1/p48B gene, in which the displacement of two essential regulatory sequences of the gene occurred due to the insertion of a 32 bp residual P-element sequence. This mutation caused a 30-fold drop in the cellular concentration of the Rpt1/p48B mRNA. The decline in the cellular Rpt1/p48B protein concentration induced serious damage in the assembly of the 26S proteasomes, the accumulation of multiubiquitinated proteins, a change in the phosphorylation pattern of the subunit and depletion of this ATPase protein from the chromatin.