Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1991 Apr 1;88(7):2731–2735. doi: 10.1073/pnas.88.7.2731

Large scale screen for transposon insertions into cloned genes.

B A Hamilton 1, M J Palazzolo 1, J H Chang 1, K VijayRaghavan 1, C A Mayeda 1, M A Whitney 1, E M Meyerowitz 1
PMCID: PMC51312  PMID: 1849274

Abstract

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.

Full text

PDF
2734

Images in this article

2732Image
on p.2732
2733Image
on p.2733
2733Image
on p.2733
2734Image
on p.2734

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ballinger D. G., Benzer S. Targeted gene mutations in Drosophila. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9402–9406. doi: 10.1073/pnas.86.23.9402. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bender W., Spierer P., Hogness D. S. Chromosomal walking and jumping to isolate DNA from the Ace and rosy loci and the bithorax complex in Drosophila melanogaster. J Mol Biol. 1983 Jul 25;168(1):17–33. doi: 10.1016/s0022-2836(83)80320-9. [DOI] [PubMed] [Google Scholar]
  3. Bier E., Vaessin H., Shepherd S., Lee K., McCall K., Barbel S., Ackerman L., Carretto R., Uemura T., Grell E. Searching for pattern and mutation in the Drosophila genome with a P-lacZ vector. Genes Dev. 1989 Sep;3(9):1273–1287. doi: 10.1101/gad.3.9.1273. [DOI] [PubMed] [Google Scholar]
  4. Daniels S. B., McCarron M., Love C., Chovnick A. Dysgenesis-induced instability of rosy locus transformation in Drosophila melanogaster: analysis of excision events and the selective recovery of control element deletions. Genetics. 1985 Jan;109(1):95–117. doi: 10.1093/genetics/109.1.95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dower W. J., Miller J. F., Ragsdale C. W. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 1988 Jul 11;16(13):6127–6145. doi: 10.1093/nar/16.13.6127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Feinberg A. P., Vogelstein B. "A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity". Addendum. Anal Biochem. 1984 Feb;137(1):266–267. doi: 10.1016/0003-2697(84)90381-6. [DOI] [PubMed] [Google Scholar]
  7. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  8. Hyde D. R., Mecklenburg K. L., Pollock J. A., Vihtelic T. S., Benzer S. Twenty Drosophila visual system cDNA clones: one is a homolog of human arrestin. Proc Natl Acad Sci U S A. 1990 Feb;87(3):1008–1012. doi: 10.1073/pnas.87.3.1008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kaiser K., Goodwin S. F. "Site-selected" transposon mutagenesis of Drosophila. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1686–1690. doi: 10.1073/pnas.87.5.1686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Katz F., Moats W., Jan Y. N. A carbohydrate epitope expressed uniquely on the cell surface of Drosophila neurons is altered in the mutant nac (neurally altered carbohydrate). EMBO J. 1988 Nov;7(11):3471–3477. doi: 10.1002/j.1460-2075.1988.tb03222.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kim H. S., Smithies O. Recombinant fragment assay for gene targetting based on the polymerase chain reaction. Nucleic Acids Res. 1988 Sep 26;16(18):8887–8903. doi: 10.1093/nar/16.18.8887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. O'Kane C. J., Gehring W. J. Detection in situ of genomic regulatory elements in Drosophila. Proc Natl Acad Sci U S A. 1987 Dec;84(24):9123–9127. doi: 10.1073/pnas.84.24.9123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Palazzolo M. J., Hamilton B. A., Ding D. L., Martin C. H., Mead D. A., Mierendorf R. C., Raghavan K. V., Meyerowitz E. M., Lipshitz H. D. Phage lambda cDNA cloning vectors for subtractive hybridization, fusion-protein synthesis and Cre-loxP automatic plasmid subcloning. Gene. 1990 Mar 30;88(1):25–36. doi: 10.1016/0378-1119(90)90056-w. [DOI] [PubMed] [Google Scholar]
  14. Palazzolo M. J., Hyde D. R., VijayRaghavan K., Mecklenburg K., Benzer S., Meyerowitz E. Use of a new strategy to isolate and characterize 436 Drosophila cDNA clones corresponding to RNAs detected in adult heads but not in early embryos. Neuron. 1989 Oct;3(4):527–539. doi: 10.1016/0896-6273(89)90211-0. [DOI] [PubMed] [Google Scholar]
  15. Palazzolo M. J., Meyerowitz E. M. A family of lambda phage cDNA cloning vectors, lambda SWAJ, allowing the amplification of RNA sequences. Gene. 1987;52(2-3):197–206. doi: 10.1016/0378-1119(87)90046-1. [DOI] [PubMed] [Google Scholar]
  16. Perucho M., Hanahan D., Wigler M. Genetic and physical linkage of exogenous sequences in transformed cells. Cell. 1980 Nov;22(1 Pt 1):309–317. doi: 10.1016/0092-8674(80)90178-6. [DOI] [PubMed] [Google Scholar]
  17. Robertson H. M., Preston C. R., Phillis R. W., Johnson-Schlitz D. M., Benz W. K., Engels W. R. A stable genomic source of P element transposase in Drosophila melanogaster. Genetics. 1988 Mar;118(3):461–470. doi: 10.1093/genetics/118.3.461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  19. Saiki R. K., Scharf S., Faloona F., Mullis K. B., Horn G. T., Erlich H. A., Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350–1354. doi: 10.1126/science.2999980. [DOI] [PubMed] [Google Scholar]
  20. Salz H. K., Cline T. W., Schedl P. Functional changes associated with structural alterations induced by mobilization of a P element inserted in the Sex-lethal gene of Drosophila. Genetics. 1987 Oct;117(2):221–231. doi: 10.1093/genetics/117.2.221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Steller H., Pirrotta V. P transposons controlled by the heat shock promoter. Mol Cell Biol. 1986 May;6(5):1640–1649. doi: 10.1128/mcb.6.5.1640. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Van Vactor D., Jr, Krantz D. E., Reinke R., Zipursky S. L. Analysis of mutants in chaoptin, a photoreceptor cell-specific glycoprotein in Drosophila, reveals its role in cellular morphogenesis. Cell. 1988 Jan 29;52(2):281–290. doi: 10.1016/0092-8674(88)90517-x. [DOI] [PubMed] [Google Scholar]
  24. Voelker R. A., Greenleaf A. L., Gyurkovics H., Wisely G. B., Huang S. M., Searles L. L. Frequent Imprecise Excision among Reversions of a P Element-Caused Lethal Mutation in Drosophila. Genetics. 1984 Jun;107(2):279–294. doi: 10.1093/genetics/107.2.279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wilson C., Pearson R. K., Bellen H. J., O'Kane C. J., Grossniklaus U., Gehring W. J. P-element-mediated enhancer detection: an efficient method for isolating and characterizing developmentally regulated genes in Drosophila. Genes Dev. 1989 Sep;3(9):1301–1313. doi: 10.1101/gad.3.9.1301. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES