Skip to main content
Genetics logoLink to Genetics
. 1998 Jul;149(3):1427–1434. doi: 10.1093/genetics/149.3.1427

Molecular screening for P-element insertions in a large genomic region of Drosophila melanogaster using polymerase chain reaction mediated by the vectorette.

H Eggert 1, K Bergemann 1, H Saumweber 1
PMCID: PMC1460217  PMID: 9649531

Abstract

As an alternative to existing methods for the detection of new insertions during a transposon mutagenesis, we adapted the method of vectorette ligation to genomic restriction fragments followed by PCR to obtain genomic sequences flanking the transposon. By combining flies containing a defined genomic transposon with an excess of flies containing unrelated insertion sites, we demonstrate the specificity and sensitivity of the procedure in the detection of integration events. This method was applied in a transposon-tagging screen for BJ1, the Drosophila homolog of the vertebrate gene Regulator of Chromosome Condensation (RCCI). Genetic mobilization of a single genomic P element was used to generate preferentially new local insertions from which integrations into a genomic region surrounding the BJ1 gene were screened. Flies harboring new insertions were phenotypically selected on the basis of the zeste1-dependent transvection of white. We detected a single transposition to a 13-kb region close to the BJ1 gene among 6650 progeny that were analyzed. Southern analysis of the homozygous line confirmed the integration 3 kb downstream of BJ1.

Full Text

The Full Text of this article is available as a PDF (169.9 KB).

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. Cooley L., Kelley R., Spradling A. Insertional mutagenesis of the Drosophila genome with single P elements. Science. 1988 Mar 4;239(4844):1121–1128. doi: 10.1126/science.2830671. [DOI] [PubMed] [Google Scholar]
  3. Dalby B., Pereira A. J., Goldstein L. S. An inverse PCR screen for the detection of P element insertions in cloned genomic intervals in Drosophila melanogaster. Genetics. 1995 Feb;139(2):757–766. doi: 10.1093/genetics/139.2.757. [DOI] [PMC free article] [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. Delattre M., Anxolabéhère D., Coen D. Prevalence of localized rearrangements vs. transpositions among events induced by Drosophila P element transposase on a P transgene. Genetics. 1995 Dec;141(4):1407–1424. doi: 10.1093/genetics/141.4.1407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Engels W. R. P elements in Drosophila. Curr Top Microbiol Immunol. 1996;204:103–123. doi: 10.1007/978-3-642-79795-8_5. [DOI] [PubMed] [Google Scholar]
  7. Frasch M. The maternally expressed Drosophila gene encoding the chromatin-binding protein BJ1 is a homolog of the vertebrate gene Regulator of Chromatin Condensation, RCC1. EMBO J. 1991 May;10(5):1225–1236. doi: 10.1002/j.1460-2075.1991.tb08064.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gehring W. J., Klemenz R., Weber U., Kloter U. Functional analysis of the white gene of Drosophila by P-factor-mediated transformation. EMBO J. 1984 Sep;3(9):2077–2085. doi: 10.1002/j.1460-2075.1984.tb02094.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gubb D., Roote J., McGill S., Shelton M., Ashburner M. Interactions between WHITE Genes Carried by a Large Transposing Element and the ZESTE Allele in DROSOPHILA MELANOGASTER. Genetics. 1986 Mar;112(3):551–575. doi: 10.1093/genetics/112.3.551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hamilton B. A., Palazzolo M. J., Chang J. H., VijayRaghavan K., Mayeda C. A., Whitney M. A., Meyerowitz E. M. Large scale screen for transposon insertions into cloned genes. Proc Natl Acad Sci U S A. 1991 Apr 1;88(7):2731–2735. doi: 10.1073/pnas.88.7.2731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hazelrigg T., Levis R., Rubin G. M. Transformation of white locus DNA in drosophila: dosage compensation, zeste interaction, and position effects. Cell. 1984 Feb;36(2):469–481. doi: 10.1016/0092-8674(84)90240-x. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Littleton J. T., Stern M., Schulze K., Perin M., Bellen H. J. Mutational analysis of Drosophila synaptotagmin demonstrates its essential role in Ca(2+)-activated neurotransmitter release. Cell. 1993 Sep 24;74(6):1125–1134. doi: 10.1016/0092-8674(93)90733-7. [DOI] [PubMed] [Google Scholar]
  14. Ohtsubo M., Kai R., Furuno N., Sekiguchi T., Sekiguchi M., Hayashida H., Kuma K., Miyata T., Fukushige S., Murotsu T. Isolation and characterization of the active cDNA of the human cell cycle gene (RCC1) involved in the regulation of onset of chromosome condensation. Genes Dev. 1987 Aug;1(6):585–593. doi: 10.1101/gad.1.6.585. [DOI] [PubMed] [Google Scholar]
  15. Riley J., Butler R., Ogilvie D., Finniear R., Jenner D., Powell S., Anand R., Smith J. C., Markham A. F. A novel, rapid method for the isolation of terminal sequences from yeast artificial chromosome (YAC) clones. Nucleic Acids Res. 1990 May 25;18(10):2887–2890. doi: 10.1093/nar/18.10.2887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Shore D., Langowski J., Baldwin R. L. DNA flexibility studied by covalent closure of short fragments into circles. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4833–4837. doi: 10.1073/pnas.78.8.4833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Spradling A. C., Stern D. M., Kiss I., Roote J., Laverty T., Rubin G. M. Gene disruptions using P transposable elements: an integral component of the Drosophila genome project. Proc Natl Acad Sci U S A. 1995 Nov 21;92(24):10824–10830. doi: 10.1073/pnas.92.24.10824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Tower J., Karpen G. H., Craig N., Spradling A. C. Preferential transposition of Drosophila P elements to nearby chromosomal sites. Genetics. 1993 Feb;133(2):347–359. doi: 10.1093/genetics/133.2.347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Valdes J. M., Tagle D. A., Collins F. S. Island rescue PCR: a rapid and efficient method for isolating transcribed sequences from yeast artificial chromosomes and cosmids. Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5377–5381. doi: 10.1073/pnas.91.12.5377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Wu C. T., Goldberg M. L. The Drosophila zeste gene and transvection. Trends Genet. 1989 Jun;5(6):189–194. doi: 10.1016/0168-9525(89)90074-7. [DOI] [PubMed] [Google Scholar]
  23. Zhang P., Spradling A. C. Efficient and dispersed local P element transposition from Drosophila females. Genetics. 1993 Feb;133(2):361–373. doi: 10.1093/genetics/133.2.361. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES