Zhai R Grace, Hiesinger P Robin, Koh Tong-Wey, Verstreken Patrik, Schulze Karen L, Cao Yu, Jafar-Nejad Hamed, Norga Koenraad K, Pan Hongling, Bayat Vafa, Greenbaum Michael P, Bellen Hugo J
Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX 77030, USA.
Proc Natl Acad Sci U S A. 2003 Sep 16;100(19):10860-5. doi: 10.1073/pnas.1832753100. Epub 2003 Sep 5.
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.
在正向遗传学筛选中分离化学诱导的突变是果蝇遗传学的标志之一。然而，定位相应的基因座并鉴定与这些突变相关的分子损伤通常既困难又耗费人力。果蝇中最常使用的两种定位方法是：利用标记染色体进行减数分裂重组定位和缺失定位。果蝇基因组序列的可用性使得分子标记的建立和使用成为可能。因此，单核苷酸多态性最近已被用于定位多个基因。在这里，我们表明，公开可用的果蝇品系中数千个经分子定位的P元件插入提供了一种替代单核苷酸多态性定位的强大方法。我们提出了一种策略，该策略允许以最少的资源定位致死突变以及具有可见表型的存活突变。在使用重组率进行高分辨率定位时，最重要的未知因素是在小间隔内重组数据与分子图谱的准确相关性如何。因此，我们调查了<500 kb间隔内重组率的偏差。我们记录了重组图谱与分子图谱之间的偏差程度，并描述了以<50 kb的精度进行定位所需的步骤。最后，我们描述了一种最近开发的方法，通过使用异源双链DNA突变检测系统在50 kb间隔内确定分子损伤。我们的数据表明，这种定位方法成本低、效率高且精确，并且它显著拓宽了P元件在果蝇中的应用。
