Hamilton, Bruce A. and Palazzolo, Michael J. and Chang, Joo H. and VijayRaghavan, K. and Mayeda, Carol A. and Whitney, Michael A. and Meyerowitz, Eliott M. (1991) Large Scale Screen for Transposon Insertions into Cloned Genes. Proceedings of the National Academy of Sciences of the United States of America, 88 (7). pp. 2731-2735. ISSN 0027-8424. http://resolver.caltech.edu/CaltechAUTHORS:HAMpnas91
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We describe a method of screening for transposon insertions in or near Drosophila loci that correspond to cloned DNA sequences. We mobilize a modified P element transposon that carries a bacterial plasmid origin of replication and a drug-resistance marker. The genomic sequences flanking each transposon insertion site can then be rescued as a plasmid in Escherichia coli. Libraries of such plasmids, representing pools of transposon-mutagenized individuals, are used as hybridization probes against cloned sequences to determine whether a transposon has inserted next to a particular site in the genome. The number of loci that can be screened simultaneously by this procedure is quite large. We have screened an array of cDNA clones representing almost 700 distinct loci against libraries representing 760 mutagenized flies, and we obtained hybridization signals to 7 different cDNAs. Three of these events have been analyzed in detail and represent genuine insertions near genomic sequences that correspond to the cDNAs.
|Additional Information:||Copyright © 1991 by National Academy of Sciences Communicated by Normam H. Horowitz, December 14, 1990 (received for review August 31, 1990). We thank D. L. Lindsley and J. Merriam for fly stocks; E. B. Lewis and S. E. Celniker for fly stocks, useful discussions, and comments on the manuscript; D. G. Ballinger for the P element oligodeoxynucleotide, fly stocks, and discussions; U. VijayRaghavan, S. Halsell, and T. Jack for practical advice; A. van der Bliek for shi; and H. D. Lipshitz, C. H. Martin, and members of the Meyerowitz laboratory for comments on drafts of this paper. B.A.H. was supported in part by a U.S. Public Health Service Predoctoral National Research Service Award (T32 GM07616); M.J.P. is a Lucille P. Markey Scholar; K.V.R. was supported by a Rockefeller Foundation Biotechnology Career Fellowship. This work was supported by a scholar's grant from the Lucille P. Markey Charitable Trust (M.J.P.) and U.S. Public Health Service Program Project Grant GM40499 (E.M.M.). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.|
|Subject Keywords:||Drosophila, P element, reverse genetics, plasmid rescue|
|Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Archive Administrator|
|Deposited On:||12 Sep 2005|
|Last Modified:||14 Nov 2014 19:18|
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