Nature screen: an efficient method for screening natural populations of Drosophila for targeted P-element insertions.
The efficiency of molecular techniques is making it increasingly necessary to rely on reverse genetics to understand the function of genes. Tissue-specific libraries allow one to identify numerous genes that can be cloned, sequenced, and mapped and whose temporal and tissue-specific pattern of expression are well characterized but whose function remains unknown. In such cases, it is desirable to generate targeted mutations to examine the phenotype of loss-of-function lesions. Here we describe a method for identifying naturally occurring variants of Drosophila melanogaster with specific genes tagged by a nearby P element. Imprecise P-element excision can then be used to generate a series of small deletions in or near the gene. In the method described here, large numbers of wild-caught males were crossed to balancer females, and inserts were identified in pooled samples by the polymerase chain reaction with one primer from each target gene and one primer from the P-element terminal repeat. We present the calculations for the probability of successfully tagging a gene and show that it is greatly improved by simultaneously screening inserts into several genes. If a large natural population is available, a nature screen is faster and easier than inducing P-element transposition in the laboratory, but the resulting lines, being genetically heterogeneous, may require more subsequent work to isolate. Using this method to screen the genomes of approximately 10,400 males, we found P-element inserts in close proximity to 3 of 10 genes that were screened.
Clark
A G
AG
Department of Biology, Pennsylvania State University, University Park 16802.
Silveria
S
S
Meyers
W
W
Langley
C H
CH
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004251:DNA Transposable Elements; D004331:Drosophila melanogaster / Q000235:genetics*; D005069:Evaluation Studies as Topic; D005260:Female; D017353:Gene Deletion; D005821:Genetic Techniques; D005828:Genetics, Population; D008297:Male; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D012091:Repetitive Sequences, Nucleic Acid; D016324:Sequence Tagged Sites
D017931:DNA Primers; D004251:DNA Transposable Elements