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. 2003 Sep 16;100(19):10860-5.
doi: 10.1073/pnas.1832753100. Epub 2003 Sep 5.

Mapping Drosophila mutations with molecularly defined P element insertions

R Grace Zhai  1 , P Robin Hiesinger, Tong-Wey Koh, Patrik Verstreken, Karen L Schulze, Yu Cao, Hamed Jafar-Nejad, Koenraad K Norga, Hongling Pan, Vafa Bayat, Michael P Greenbaum, Hugo J Bellen
Affiliations

Mapping Drosophila mutations with molecularly defined P element insertions

R Grace Zhai et al. Proc Natl Acad Sci U S A. .

Abstract

The isolation of chemically induced mutations in forward genetic screens is one of the hallmarks of Drosophila genetics. However, mapping the corresponding loci and identifying the molecular lesions associated with these mutations are often difficult and labor-intensive. Two mapping methods are most often used in flies: meiotic recombination mapping with marked chromosomes and deficiency mapping. The availability of the fly genome sequence allows the establishment and usage of molecular markers. Single-nucleotide polymorphisms have therefore recently been used to map several genes. Here we show that thousands of molecularly mapped P element insertions in fly strains that are publicly available provide a powerful alternative method to single-nucleotide polymorphism mapping. We present a strategy that allows mapping of lethal mutations, as well as viable mutations with visible phenotypes, with minimal resources. The most important unknown in using recombination rates to map at high resolution is how accurately recombination data correlate with molecular maps in small intervals. We therefore surveyed distortions of recombination rates in intervals <500 kb. We document the extent of distortions between the recombination and molecular maps and describe the required steps to map with an accuracy of <50 kb. Finally, we describe a recently developed method to determine molecular lesions in 50-kb intervals by using a heteroduplex DNA mutation detection system. Our data show that this mapping approach is inexpensive, efficient, and precise, and that it significantly broadens the application of P elements in Drosophila.

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Figures

Fig. 1.
Fig. 1.
Schematic outline of the principle of P insertion mapping. (A) The three-step mapping procedure: rough and fine mapping are based on meiotic recombination mapping by using molecularly defined P element insertions. Mutation detection is based on temperature gradient capillary electrophoresis (TGCE), sequencing, and complementation tests with candidate genes. (B) Crossing scheme of rough and fine mapping. Mutant chromosomes are indicated by open bars, P insertion-containing chromosomes are in pink, and the balancer chromosomes are in black. The mutation sites (red stars) are marked with either 1 or 2 to indicate the different alleles. Note that all flies are in a w– background, meaning the P insertions are the only source of w+. Shown in the gray box are P and F1 crosses. Shown in the green box are the nonrecombinant offspring. Shown in the yellow box are the possible recombination events, which are color-coded and labeled A, B, and C, corresponding to the F1 female, where the three types of recombination events are marked likewise. (C) Calculation of mapping positions. PMD, projected molecular distance in base pairs; MD, molecular distance in base pairs; RD, recombination distance in cM. Note that the PMP can be calculated by using P1 (as shown) or P2 (PMDc).
Fig. 2.
Fig. 2.
Fine mapping results for 15 representative complementation groups. Panels represent mapping experiments, and cytological positions of each region are listed next to each panel label. The x axis is the distance between the P insertions in kilobases. The first P insertion site is normalized to 0, and the relative insertion sites of the other P elements are listed on the x axis. The number of flies counted is indicated above each P insertion (white-eyed flies/total). The red crosses mark the PMP by using pairs of P insertions, and their intervals (in kilobases) are indicated at the bottom. The y axis represents the RR (in cM/megabases) of each segment between two neighboring P insertions. Identified genes bearing the mutations are indicated by red bars on the x axis, and their relative genomic regions are listed in the red box above the gene in examples (A–H).
Fig. 3.
Fig. 3.
Molecular identification of lethal mutations by TGCE (SpectruMedix Reveal) and sequencing. TGCE was performed in a 96-well format with controls and all alleles in adjacent wells. Results of TGCE and sequencing are shown for two alleles of one complementation group that was fine-mapped with P insertion mapping. The DNA for sequencing and TGCE are from the same preparation of heterozygous flies (mutant chromosome over isogenized chromosome). Controls are homozygous for the isogenized chromosome. The same primers were used for TGCE and sequencing. The heteroduplexes in C and G could clearly be differentiated as double peaks when compared with the control traces in A and E. Sequencing of the heterozygous DNA revealed G-to-A transitions for both cases shown in D and H (forward and complement of reverse sequence shown). These transitions can appear as N, A, or G in the heterozygous sequence, depending on the intensity of the partial signals.
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References

    1. Lindsley, D. L. & Zimm, G. G. (1992) The Genome of Drosophila melanogaster (Academic, San Diego).
    1. The FlyBase Consortium (2003) Nucleic Acids Res. 31, 172–175. - PMC - PubMed
    1. Adams, M. D., Celniker, S. E., Holt, R. A., Evans, C. A., Gocayne, J. D., Amanatides, P. G., Scherer, S. E., Li, P. W., Hoskins, R. A., Galle, R. F., et al. (2000) Science 287, 2185–2195. - PubMed
    1. Berger, J., Suzuki, T., Senti, K. A., Stubbs, J., Schaffner, G. & Dickson, B. J. (2001) Nat. Genet. 29, 475–481. - PubMed
    1. Martin, S. G., Dobi, K. C. & St. Johnston, D. (August 30, 2001) Genome Biol. 2, research0036.1–0036.12. - PMC - PubMed

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