Ballinger, Dennis G. and Benzer, Seymour (1989) Targeted gene mutations in Drosophila. Proceedings of the National Academy of Sciences of the United States of America, 86 (23). pp. 9402-9406. ISSN 0027-8424 http://resolver.caltech.edu/CaltechAUTHORS:20120510-091736046
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A cloned gene can be of interest because of its expression in a particular tissue or at a certain developmental stage, or because of homology to an interesting gene from another organism. In Drosophila its location in the genome is readily determined by in situ hybridization to the banded larval salivary gland polytene chromosomes, but it is more difficult to isolate mutations that may reveal its function. This paper describes a general method for detecting transposable element insertions into the gene in question. This "reverse genetics" then offers the possibility of observing a consequent mutant phenotype, providing a key to the normal function of the gene. The sensitivity of the polymerase chain reaction makes it possible to detect the occurrence of a single appropriate P-element transposon insertion among a population of mutagenized flies. This is accomplished by the use of oligonucleotide primers--one a sequence from within the cloned gene and the other homologous to the terminal sequence of the P-element DNA--to prime synthesis into the DNA flanking an insertion site. A segment of DNA, bounded by the two primers, will be a target for amplification only in a fly in which a P-element has inserted within about 2 kilobases of the gene primer. This technique has been used to detect P-element insertions near a gene expressed in the Drosophila compound eye. Potential problems with the technique and possible refinements in the screen are discussed. In principle, it could be utilized to detect insertion of a foreign element into any gene for which at least a partial sequence is known and could be extended to other organisms.
|Additional Information:||© 1989 National Academy of Sciences. Contributed by Seymour Benzer, August 14, 1989. We thank A. Kamb for suggesting the possible use of PCR to detect insertions and K. Mecklenburg for the sequence of the 4A11 cDNA; this work would not have been possible without their generosity. We also thank J. Chang, E. Eichenberger, and R. Young for excellent technical assistance; L. Searles for the vermilion P-insertion strain and gene sequence, and C. Zuker for suggesting the use of larval DNA in the final stages of screening. We thank N. Bonini, A. Kamb, and P. Sternberg for animated discussions throughout these studies, and the members of our laboratory for helpful comments on the manuscript. This work was supported by grants from the National Science Foundation (DCB-8409366), the National Institutes of Health (GM 40499), and the Lucille P. Markey Charitable Trust. D.G.B. was supported by a postdoctoral fellowship from the National Institutes of Health and by a Myron A. Bantrell Fellowship.|
|Subject Keywords:||cDNA; P element; polymerase chain reaction; photoreceptor|
|Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Jason Perez|
|Deposited On:||10 May 2012 20:05|
|Last Modified:||10 May 2012 20:05|
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