Abstract
P element-induced gene conversion has been previously used to modify the white gene of Drosophila melanogaster in a directed fashion. The applicability of this approach of gene targeting in Drosophila melanogaster, however, has not been analyzed quantitatively for other genes. We took advantage of the P element-induced forked allele, f(hd), which was used as a target, and we constructed a vector containing a modified forked fragment for converting f(hd). Conversion frequencies were analyzed for this locus as well as for an alternative white allele, w(eh812). Combination of both P element-induced mutant genes allowed the simultaneous analysis of conversion frequencies under identical genetic, developmental, and environmental conditions. This paper demonstrates that gene conversion through P element-induced gap repair can be applied with similar success rates at the forked locus and in the white gene. The average conversion frequency at forked was 0.29%, and that at white was 0.17%. These frequencies indicate that in vivo gene targeting in Drosophila melanogaster should be applicable for other genes in this species at manageable rates. We also confirmed the homolog dependence of reversions at the forked locus, indicating that P elements transpose via a cut-and-paste mechanism. In a different experiment, we attempted conversion with a modified forked allele containing the su(Hw) binding site. Despite an increased sample size, there were no conversion events with this template. One interpretation (under investigation) is that the binding of the su(Hw) product prevents double-strand break repair.
Full Text
The Full Text of this article is available as a PDF (400.5 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Bollag R. J., Waldman A. S., Liskay R. M. Homologous recombination in mammalian cells. Annu Rev Genet. 1989;23:199–225. doi: 10.1146/annurev.ge.23.120189.001215. [DOI] [PubMed] [Google Scholar]
- Cai H., Levine M. Modulation of enhancer-promoter interactions by insulators in the Drosophila embryo. Nature. 1995 Aug 10;376(6540):533–536. doi: 10.1038/376533a0. [DOI] [PubMed] [Google Scholar]
- Capecchi M. R. Altering the genome by homologous recombination. Science. 1989 Jun 16;244(4910):1288–1292. doi: 10.1126/science.2660260. [DOI] [PubMed] [Google Scholar]
- Corces V. G. Chromatin insulators. Keeping enhancers under control. Nature. 1995 Aug 10;376(6540):462–463. doi: 10.1038/376462a0. [DOI] [PubMed] [Google Scholar]
- Corces V. G., Geyer P. K. Interactions of retrotransposons with the host genome: the case of the gypsy element of Drosophila. Trends Genet. 1991 Mar;7(3):86–90. doi: 10.1016/0168-9525(91)90277-W. [DOI] [PubMed] [Google Scholar]
- Engels W. R., Benz W. K., Preston C. R., Graham P. L., Phillis R. W., Robertson H. M. Somatic effects of P element activity in Drosophila melanogaster: pupal lethality. Genetics. 1987 Dec;117(4):745–757. doi: 10.1093/genetics/117.4.745. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Engels W. R., Johnson-Schlitz D. M., Eggleston W. B., Sved J. High-frequency P element loss in Drosophila is homolog dependent. Cell. 1990 Aug 10;62(3):515–525. doi: 10.1016/0092-8674(90)90016-8. [DOI] [PubMed] [Google Scholar]
- Engels W. R., Preston C. R., Johnson-Schlitz D. M. Long-range cis preference in DNA homology search over the length of a Drosophila chromosome. Science. 1994 Mar 18;263(5153):1623–1625. doi: 10.1126/science.8128250. [DOI] [PubMed] [Google Scholar]
- Gerasimova T. I., Gdula D. A., Gerasimov D. V., Simonova O., Corces V. G. A Drosophila protein that imparts directionality on a chromatin insulator is an enhancer of position-effect variegation. Cell. 1995 Aug 25;82(4):587–597. doi: 10.1016/0092-8674(95)90031-4. [DOI] [PubMed] [Google Scholar]
- Gloor G. B., Nassif N. A., Johnson-Schlitz D. M., Preston C. R., Engels W. R. Targeted gene replacement in Drosophila via P element-induced gap repair. Science. 1991 Sep 6;253(5024):1110–1117. doi: 10.1126/science.1653452. [DOI] [PubMed] [Google Scholar]
- Gloor G. B., Preston C. R., Johnson-Schlitz D. M., Nassif N. A., Phillis R. W., Benz W. K., Robertson H. M., Engels W. R. Type I repressors of P element mobility. Genetics. 1993 Sep;135(1):81–95. doi: 10.1093/genetics/135.1.81. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gonzy-Tréboul G., Lepesant J. A., Deutsch J. Enhancer-trap targeting at the Broad-Complex locus of Drosophila melanogaster. Genes Dev. 1995 May 1;9(9):1137–1148. doi: 10.1101/gad.9.9.1137. [DOI] [PubMed] [Google Scholar]
- Harrison D. A., Gdula D. A., Coyne R. S., Corces V. G. A leucine zipper domain of the suppressor of Hairy-wing protein mediates its repressive effect on enhancer function. Genes Dev. 1993 Oct;7(10):1966–1978. doi: 10.1101/gad.7.10.1966. [DOI] [PubMed] [Google Scholar]
- Heslip T. R., Hodgetts R. B. Targeted transposition at the vestigial locus of Drosophila melanogaster. Genetics. 1994 Dec;138(4):1127–1135. doi: 10.1093/genetics/138.4.1127. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hinnen A., Hicks J. B., Fink G. R. Transformation of yeast. Proc Natl Acad Sci U S A. 1978 Apr;75(4):1929–1933. doi: 10.1073/pnas.75.4.1929. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hoover K. K., Chien A. J., Corces V. G. Effects of transposable elements on the expression of the forked gene of Drosophila melanogaster. Genetics. 1993 Oct;135(2):507–526. doi: 10.1093/genetics/135.2.507. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Johnson-Schlitz D. M., Engels W. R. P-element-induced interallelic gene conversion of insertions and deletions in Drosophila melanogaster. Mol Cell Biol. 1993 Nov;13(11):7006–7018. doi: 10.1128/mcb.13.11.7006. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Karess R. E., Rubin G. M. Analysis of P transposable element functions in Drosophila. Cell. 1984 Aug;38(1):135–146. doi: 10.1016/0092-8674(84)90534-8. [DOI] [PubMed] [Google Scholar]
- Keeler K. J., Dray T., Penney J. E., Gloor G. B. Gene targeting of a plasmid-borne sequence to a double-strand DNA break in Drosophila melanogaster. Mol Cell Biol. 1996 Feb;16(2):522–528. doi: 10.1128/mcb.16.2.522. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Koller B. H., Smithies O. Altering genes in animals by gene targeting. Annu Rev Immunol. 1992;10:705–730. doi: 10.1146/annurev.iy.10.040192.003421. [DOI] [PubMed] [Google Scholar]
- Lankenau D. H. Genetics of genetics in Drosophila: P elements serving the study of homologous recombination, gene conversion and targeting. Chromosoma. 1995 Jul;103(10):659–668. doi: 10.1007/BF00344226. [DOI] [PubMed] [Google Scholar]
- Lankenau D. H., Huijser P., Jansen E., Miedema K., Hennig W. Micropia: a retrotransposon of Drosophila combining structural features of DNA viruses, retroviruses and non-viral transposable elements. J Mol Biol. 1988 Nov 20;204(2):233–246. doi: 10.1016/0022-2836(88)90572-4. [DOI] [PubMed] [Google Scholar]
- Marchuk D., Drumm M., Saulino A., Collins F. S. Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR products. Nucleic Acids Res. 1991 Mar 11;19(5):1154–1154. doi: 10.1093/nar/19.5.1154. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Marlor R. L., Parkhurst S. M., Corces V. G. The Drosophila melanogaster gypsy transposable element encodes putative gene products homologous to retroviral proteins. Mol Cell Biol. 1986 Apr;6(4):1129–1134. doi: 10.1128/mcb.6.4.1129. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McCall K., Bender W. Probes of chromatin accessibility in the Drosophila bithorax complex respond differently to Polycomb-mediated repression. EMBO J. 1996 Feb 1;15(3):569–580. [PMC free article] [PubMed] [Google Scholar]
- Nassif N., Penney J., Pal S., Engels W. R., Gloor G. B. Efficient copying of nonhomologous sequences from ectopic sites via P-element-induced gap repair. Mol Cell Biol. 1994 Mar;14(3):1613–1625. doi: 10.1128/mcb.14.3.1613. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Orr-Weaver T. L., Szostak J. W., Rothstein R. J. Yeast transformation: a model system for the study of recombination. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6354–6358. doi: 10.1073/pnas.78.10.6354. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Petersen N. S., Lankenau D. H., Mitchell H. K., Young P., Corces V. G. forked proteins are components of fiber bundles present in developing bristles of Drosophila melanogaster. Genetics. 1994 Jan;136(1):173–182. doi: 10.1093/genetics/136.1.173. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Rubin G. M., Spradling A. C. Genetic transformation of Drosophila with transposable element vectors. Science. 1982 Oct 22;218(4570):348–353. doi: 10.1126/science.6289436. [DOI] [PubMed] [Google Scholar]
- 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]
- Spana C., Harrison D. A., Corces V. G. The Drosophila melanogaster suppressor of Hairy-wing protein binds to specific sequences of the gypsy retrotransposon. Genes Dev. 1988 Nov;2(11):1414–1423. doi: 10.1101/gad.2.11.1414. [DOI] [PubMed] [Google Scholar]
- Spradling A. C., Rubin G. M. Transposition of cloned P elements into Drosophila germ line chromosomes. Science. 1982 Oct 22;218(4570):341–347. doi: 10.1126/science.6289435. [DOI] [PubMed] [Google Scholar]
- Taylor J. W., Ott J., Eckstein F. The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res. 1985 Dec 20;13(24):8765–8785. doi: 10.1093/nar/13.24.8765. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Taylor J. W., Schmidt W., Cosstick R., Okruszek A., Eckstein F. The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA. Nucleic Acids Res. 1985 Dec 20;13(24):8749–8764. doi: 10.1093/nar/13.24.8749. [DOI] [PMC free article] [PubMed] [Google Scholar]