Skip to main content
PMC home page
Genetics logoLink to Genetics
. 2003 Feb;163(2):611–623. doi: 10.1093/genetics/163.2.611

Knockout targeting of the Drosophila nap1 gene and examination of DNA repair tracts in the recombination products.

Susanne Lankenau 1, Thorsten Barnickel 1, Joachim Marhold 1, Frank Lyko 1, Bernard M Mechler 1, Dirk-Henner Lankenau 1
PMCID: PMC1462439  PMID: 12618400

Abstract

We used ends-in gene targeting to generate knockout mutations of the nucleosome assembly protein 1 (Nap1) gene in Drosophila melanogaster. Three independent targeted null-knockout mutations were produced. No wild-type NAP1 protein could be detected in protein extracts. Homozygous Nap1(KO) knockout flies were either embryonic lethal or poorly viable adult escapers. Three additional targeted recombination products were viable. To gain insight into the underlying molecular processes we examined conversion tracts in the recombination products. In nearly all cases the I-SceI endonuclease site of the donor vector was replaced by the wild-type Nap1 sequence. This indicated exonuclease processing at the site of the double-strand break (DSB), followed by replicative repair at donor-target junctions. The targeting products are best interpreted either by the classical DSB repair model or by the break-induced recombination (BIR) model. Synthesis-dependent strand annealing (SDSA), which is another important recombinational repair pathway in the germline, does not explain ends-in targeting products. We conclude that this example of gene targeting at the Nap1 locus provides added support for the efficiency of this method and its usefulness in targeting any arbitrary locus in the Drosophila genome.

Full Text

The Full Text of this article is available as a PDF (461.9 KB).

Selected References

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

  1. Adams M. D., Celniker S. E., Holt R. A., Evans C. A., Gocayne J. D., Amanatides P. G., Scherer S. E., Li P. W., Hoskins R. A., Galle R. F. The genome sequence of Drosophila melanogaster. Science. 2000 Mar 24;287(5461):2185–2195. doi: 10.1126/science.287.5461.2185. [DOI] [PubMed] [Google Scholar]
  2. Adams Melissa D., Sekelsky Jeff J. From sequence to phenotype: reverse genetics in Drosophila melanogaster. Nat Rev Genet. 2002 Mar;3(3):189–198. doi: 10.1038/nrg752. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. 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]
  6. Capecchi M. R. The new mouse genetics: altering the genome by gene targeting. Trends Genet. 1989 Mar;5(3):70–76. doi: 10.1016/0168-9525(89)90029-2. [DOI] [PubMed] [Google Scholar]
  7. Carthew R. W. Gene silencing by double-stranded RNA. Curr Opin Cell Biol. 2001 Apr;13(2):244–248. doi: 10.1016/s0955-0674(00)00204-0. [DOI] [PubMed] [Google Scholar]
  8. Curtis D., Clark S. H., Chovnick A., Bender W. Molecular analysis of recombination events in Drosophila. Genetics. 1989 Jul;122(3):653–661. doi: 10.1093/genetics/122.3.653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Deuring R., Fanti L., Armstrong J. A., Sarte M., Papoulas O., Prestel M., Daubresse G., Verardo M., Moseley S. L., Berloco M. The ISWI chromatin-remodeling protein is required for gene expression and the maintenance of higher order chromatin structure in vivo. Mol Cell. 2000 Feb;5(2):355–365. doi: 10.1016/s1097-2765(00)80430-x. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Engels W. R. Reversal of fortune for Drosophila geneticists? Science. 2000 Jun 16;288(5473):1973–1975. doi: 10.1126/science.288.5473.1973. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Ishimi Y., Hirosumi J., Sato W., Sugasawa K., Yokota S., Hanaoka F., Yamada M. Purification and initial characterization of a protein which facilitates assembly of nucleosome-like structure from mammalian cells. Eur J Biochem. 1984 Aug 1;142(3):431–439. doi: 10.1111/j.1432-1033.1984.tb08305.x. [DOI] [PubMed] [Google Scholar]
  14. Ito T., Bulger M., Kobayashi R., Kadonaga J. T. Drosophila NAP-1 is a core histone chaperone that functions in ATP-facilitated assembly of regularly spaced nucleosomal arrays. Mol Cell Biol. 1996 Jun;16(6):3112–3124. doi: 10.1128/mcb.16.6.3112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Kellogg D. R., Murray A. W. NAP1 acts with Clb1 to perform mitotic functions and to suppress polar bud growth in budding yeast. J Cell Biol. 1995 Aug;130(3):675–685. doi: 10.1083/jcb.130.3.675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kraus E., Leung W. Y., Haber J. E. Break-induced replication: a review and an example in budding yeast. Proc Natl Acad Sci U S A. 2001 Jul 17;98(15):8255–8262. doi: 10.1073/pnas.151008198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lankenau D. H., Corces V. G., Engels W. R. Comparison of targeted-gene replacement frequencies in Drosophila melanogaster at the forked and white loci. Mol Cell Biol. 1996 Jul;16(7):3535–3544. doi: 10.1128/mcb.16.7.3535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Lankenau D. H., Gloor G. B. In vivo gap repair in Drosophila: a one-way street with many destinations. Bioessays. 1998 Apr;20(4):317–327. doi: 10.1002/(SICI)1521-1878(199804)20:4<317::AID-BIES8>3.0.CO;2-M. [DOI] [PubMed] [Google Scholar]
  21. Li M., Strand D., Krehan A., Pyerin W., Heid H., Neumann B., Mechler B. M. Casein kinase 2 binds and phosphorylates the nucleosome assembly protein-1 (NAP1) in Drosophila melanogaster. J Mol Biol. 1999 Nov 12;293(5):1067–1084. doi: 10.1006/jmbi.1999.3207. [DOI] [PubMed] [Google Scholar]
  22. Malkova A., Ivanov E. L., Haber J. E. Double-strand break repair in the absence of RAD51 in yeast: a possible role for break-induced DNA replication. Proc Natl Acad Sci U S A. 1996 Jul 9;93(14):7131–7136. doi: 10.1073/pnas.93.14.7131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mansour S. L., Thomas K. R., Capecchi M. R. Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature. 1988 Nov 24;336(6197):348–352. doi: 10.1038/336348a0. [DOI] [PubMed] [Google Scholar]
  24. Myers E. W., Sutton G. G., Delcher A. L., Dew I. M., Fasulo D. P., Flanigan M. J., Kravitz S. A., Mobarry C. M., Reinert K. H., Remington K. A. A whole-genome assembly of Drosophila. Science. 2000 Mar 24;287(5461):2196–2204. doi: 10.1126/science.287.5461.2196. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. O'Neill S. L., Giordano R., Colbert A. M., Karr T. L., Robertson H. M. 16S rRNA phylogenetic analysis of the bacterial endosymbionts associated with cytoplasmic incompatibility in insects. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):2699–2702. doi: 10.1073/pnas.89.7.2699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pâques F., Haber J. E. Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae. Microbiol Mol Biol Rev. 1999 Jun;63(2):349–404. doi: 10.1128/mmbr.63.2.349-404.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Pâques F., Leung W. Y., Haber J. E. Expansions and contractions in a tandem repeat induced by double-strand break repair. Mol Cell Biol. 1998 Apr;18(4):2045–2054. doi: 10.1128/mcb.18.4.2045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rong Y. S., Golic K. G. A targeted gene knockout in Drosophila. Genetics. 2001 Mar;157(3):1307–1312. doi: 10.1093/genetics/157.3.1307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rong Y. S., Golic K. G. Gene targeting by homologous recombination in Drosophila. Science. 2000 Jun 16;288(5473):2013–2018. doi: 10.1126/science.288.5473.2013. [DOI] [PubMed] [Google Scholar]
  31. Rong Yikang S., Titen Simon W., Xie Heng B., Golic Mary M., Bastiani Michael, Bandyopadhyay Pradip, Olivera Baldomero M., Brodsky Michael, Rubin Gerald M., Golic Kent G. Targeted mutagenesis by homologous recombination in D. melanogaster. Genes Dev. 2002 Jun 15;16(12):1568–1581. doi: 10.1101/gad.986602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Seum Carole, Pauli Daniel, Delattre Marion, Jaquet Yannis, Spierer Anne, Spierer Pierre. Isolation of Su(var)3-7 mutations by homologous recombination in Drosophila melanogaster. Genetics. 2002 Jul;161(3):1125–1136. doi: 10.1093/genetics/161.3.1125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Silberman R., Kupiec M. Plasmid-mediated induction of recombination in yeast. Genetics. 1994 May;137(1):41–48. doi: 10.1093/genetics/137.1.41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Spradling A. C., Stern D., Beaton A., Rhem E. J., Laverty T., Mozden N., Misra S., Rubin G. M. The Berkeley Drosophila Genome Project gene disruption project: Single P-element insertions mutating 25% of vital Drosophila genes. Genetics. 1999 Sep;153(1):135–177. doi: 10.1093/genetics/153.1.135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Thomas K. R., Capecchi M. R. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell. 1987 Nov 6;51(3):503–512. doi: 10.1016/0092-8674(87)90646-5. [DOI] [PubMed] [Google Scholar]
  36. Török I., Herrmann-Horle D., Kiss I., Tick G., Speer G., Schmitt R., Mechler B. M. Down-regulation of RpS21, a putative translation initiation factor interacting with P40, produces viable minute imagos and larval lethality with overgrown hematopoietic organs and imaginal discs. Mol Cell Biol. 1999 Mar;19(3):2308–2321. doi: 10.1128/mcb.19.3.2308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Varga-Weisz P. D., Becker P. B. Chromatin-remodeling factors: machines that regulate? Curr Opin Cell Biol. 1998 Jun;10(3):346–353. doi: 10.1016/s0955-0674(98)80010-0. [DOI] [PubMed] [Google Scholar]
  38. White C. I., Haber J. E. Intermediates of recombination during mating type switching in Saccharomyces cerevisiae. EMBO J. 1990 Mar;9(3):663–673. doi: 10.1002/j.1460-2075.1990.tb08158.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Zhang Pumin, Li Mamei Z., Elledge Stephen J. Towards genetic genome projects: genomic library screening and gene-targeting vector construction in a single step. Nat Genet. 2001 Dec 20;30(1):31–39. doi: 10.1038/ng797. [DOI] [PubMed] [Google Scholar]
  40. Zhang Y., Buchholz F., Muyrers J. P., Stewart A. F. A new logic for DNA engineering using recombination in Escherichia coli. Nat Genet. 1998 Oct;20(2):123–128. doi: 10.1038/2417. [DOI] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES