An inverse PCR screen for the detection of P element insertions in cloned genomic intervals in Drosophila melanogaster.

Abstract

We developed a screening approach that utilizes an inverse polymerase chain reaction (PCR) to detect P element insertions in or near previously cloned genes in Drosophila melanogaster. We used this approach in a large scale genetic screen in which P elements were mobilized from sites on the X chromosome to new autosomal locations. Mutagenized flies were combined in pools, and our screening approach was used to generate probes corresponding to the sequences flanking each site of insertion. These probes then were used for hybridization to cloned genomic intervals, allowing individuals carrying insertions in them to be detected. We used the same approach to perform repeated rounds of sib-selection to generate stable insertion lines. We screened 16,100 insert bearing individuals and recovered 11 insertions in five intervals containing genes encoding members of the kinesin superfamily in Drosophila melanogaster. In addition, we recovered an insertion in the region including the Larval Serum Protein-2 gene. Examination by Southern hybridization confirms that the lines we recovered represent genuine insertions in the corresponding genomic intervals. Our data indicates that this approach will be very efficient both for P element mutagenesis of new genomic regions and for detection and recovery of "local" P element transposition events. In addition, our data constitutes a survey of preferred P element insertion sites in the Drosophila genome and suggests that insertion sites that are mutable at a rate of approximately 10(-4) are distributed every 40-50 kb.

The roX genes encode redundant male-specific lethal transcripts required for targeting of the MSL complex

“…Eight P‐elements cytologically mapped to the 10C region were obtained from the Bloomington Drosophila Stock Center and tested for proximity to roX2 by inverse PCR and hybridization to overlapping cosmids spanning the roX2 region (Dalby et al ., 1995). Two lethal insertions were found near roX2 : P1547 disrupts RpII215 and is 9.8 kb distal to roX2 ; and P1877 is an insertion in CT4038, 6.8 kb distal to roX2 (Figure 1).…”

Genetic Analysis of Myoblast Fusion: blown fuse Is Required for Progression Beyond the Prefusion Complex

“…Doberstein, manuscript in preparation), which maps close to blow (Heitzler et al, 1993). We isolated genomic DNA flanking the scraps 3427 P element by the inverse polymerase chain reaction (Dalby et al, 1995) and used that DNA to screen a genomic DNA library in DASH at high stringency. cDNAs were isolated from a 9-12-h embryo gt11 library (Zinn et al, 1988) using the genomic phage inserts as probes.…”

The Drosophila tango gene encodes a bHLH-PAS protein that is orthologous to mammalian Arnt and controls CNS midline and tracheal development

“…The eye color changes can signify alterations in the copy number and/or positions of the P[w + ; lacW] element with respect to its original site within the genome. An inverse PCR screen was performed to identify P-element insertions into the tgo gene according to the method of Dalby et al (1995) with modifications by Bob Nelson (personal communication). Identification of the genomic DNA sites flanking the novel P-element insertions was achieved by plasmid rescue and subsequent hybridization and DNA sequence analyses.…”

“…Mutations in tgo were generated using a two-step strategy. The first step involved hopping P-elements from the adjacent neu gene into the tgo gene (Dalby et al, 1995;Tower et al, 1992; see Materials and Methods). The P-element mutant tgo strain was then used to screen for EMS-induced tgo mutations.…”

“…It is expected that some of these strains will have novel P-element insertions. Flies from 120 distinct lines that had altered eye color were screened using an inverse PCR/Southern blot procedure (Dalby et al, 1995). Three lines (R65, R91, and R94), all with red eyes, were found to have novel P-element insertions in or near the tgo locus (Fig.…”