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. 1991 Mar 15;88(6):2118–2121. doi: 10.1073/pnas.88.6.2118

Stable integrative transformation of Trypanosoma brucei that occurs exclusively by homologous recombination.

J Eid 1, B Sollner-Webb 1
PMCID: PMC51180  PMID: 2006150

Abstract

A calmodulin-neomycin-resistance fusion gene was introduced into Trypanosoma brucei by electroporation, and stably transformed cell lines were obtained. In all of the transformants, the fusion gene had integrated into the host genome at the cognate locus, evidently by homologous recombination within flanking calmodulin DNA. This unusual observation distinguishes trypanosomes as the only eukaryote other than yeast known to undergo gene targeting in essentially 100% of the stable transformants. It should now be possible to systematically manipulate the trypanosome genome, directing predetermined mutations to virtually any chromosomal locus.

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Selected References

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

  1. Auffray C., Rougeon F. Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA. Eur J Biochem. 1980 Jun;107(2):303–314. doi: 10.1111/j.1432-1033.1980.tb06030.x. [DOI] [PubMed] [Google Scholar]
  2. Beck E., Ludwig G., Auerswald E. A., Reiss B., Schaller H. Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5. Gene. 1982 Oct;19(3):327–336. doi: 10.1016/0378-1119(82)90023-3. [DOI] [PubMed] [Google Scholar]
  3. Borst P. Discontinuous transcription and antigenic variation in trypanosomes. Annu Rev Biochem. 1986;55:701–732. doi: 10.1146/annurev.bi.55.070186.003413. [DOI] [PubMed] [Google Scholar]
  4. Brun R., Schönenberger Cultivation and in vitro cloning or procyclic culture forms of Trypanosoma brucei in a semi-defined medium. Short communication. Acta Trop. 1979 Sep;36(3):289–292. [PubMed] [Google Scholar]
  5. Case M. E., Schweizer M., Kushner S. R., Giles N. H. Efficient transformation of Neurospora crassa by utilizing hybrid plasmid DNA. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5259–5263. doi: 10.1073/pnas.76.10.5259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cruz A., Beverley S. M. Gene replacement in parasitic protozoa. Nature. 1990 Nov 8;348(6297):171–173. doi: 10.1038/348171a0. [DOI] [PubMed] [Google Scholar]
  7. Cunningham I. New culture medium for maintenance of tsetse tissues and growth of trypanosomatids. J Protozool. 1977 May;24(2):325–329. doi: 10.1111/j.1550-7408.1977.tb00987.x. [DOI] [PubMed] [Google Scholar]
  8. De Lozanne A., Spudich J. A. Disruption of the Dictyostelium myosin heavy chain gene by homologous recombination. Science. 1987 May 29;236(4805):1086–1091. doi: 10.1126/science.3576222. [DOI] [PubMed] [Google Scholar]
  9. Firtel R. A., Cockburn A., Frankel G., Hershfield V. Structural organization of the genome of Dictyostelium discoideum: analysis by EcoR1 restriction endonuclease. J Mol Biol. 1976 Apr 25;102(4):831–852. doi: 10.1016/0022-2836(76)90294-1. [DOI] [PubMed] [Google Scholar]
  10. Gibson W. C., Osinga K. A., Michels P. A., Borst P. Trypanosomes of subgenus Trypanozoon are diploid for housekeeping genes. Mol Biochem Parasitol. 1985 Sep;16(3):231–242. doi: 10.1016/0166-6851(85)90066-0. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Imboden M. A., Laird P. W., Affolter M., Seebeck T. Transcription of the intergenic regions of the tubulin gene cluster of Trypanosoma brucei: evidence for a polycistronic transcription unit in a eukaryote. Nucleic Acids Res. 1987 Sep 25;15(18):7357–7368. doi: 10.1093/nar/15.18.7357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jasin M., Elledge S. J., Davis R. W., Berg P. Gene targeting at the human CD4 locus by epitope addition. Genes Dev. 1990 Feb;4(2):157–166. doi: 10.1101/gad.4.2.157. [DOI] [PubMed] [Google Scholar]
  14. Kapler G. M., Coburn C. M., Beverley S. M. Stable transfection of the human parasite Leishmania major delineates a 30-kilobase region sufficient for extrachromosomal replication and expression. Mol Cell Biol. 1990 Mar;10(3):1084–1094. doi: 10.1128/mcb.10.3.1084. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Laban A., Tobin J. F., Curotto de Lafaille M. A., Wirth D. F. Stable expression of the bacterial neor gene in Leishmania enriettii. Nature. 1990 Feb 8;343(6258):572–574. doi: 10.1038/343572a0. [DOI] [PubMed] [Google Scholar]
  16. Lee M. G., Van der Ploeg L. H. Homologous recombination and stable transfection in the parasitic protozoan Trypanosoma brucei. Science. 1990 Dec 14;250(4987):1583–1587. doi: 10.1126/science.2177225. [DOI] [PubMed] [Google Scholar]
  17. Murphy W. J., Watkins K. P., Agabian N. Identification of a novel Y branch structure as an intermediate in trypanosome mRNA processing: evidence for trans splicing. Cell. 1986 Nov 21;47(4):517–525. doi: 10.1016/0092-8674(86)90616-1. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Pays E., Steinert M. Control of antigen gene expression in African trypanosomes. Annu Rev Genet. 1988;22:107–126. doi: 10.1146/annurev.ge.22.120188.000543. [DOI] [PubMed] [Google Scholar]
  20. Rothstein R. J. One-step gene disruption in yeast. Methods Enzymol. 1983;101:202–211. doi: 10.1016/0076-6879(83)01015-0. [DOI] [PubMed] [Google Scholar]
  21. Schwartz D. C., Cantor C. R. Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell. 1984 May;37(1):67–75. doi: 10.1016/0092-8674(84)90301-5. [DOI] [PubMed] [Google Scholar]
  22. Sedivy J. M., Sharp P. A. Positive genetic selection for gene disruption in mammalian cells by homologous recombination. Proc Natl Acad Sci U S A. 1989 Jan;86(1):227–231. doi: 10.1073/pnas.86.1.227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Simpson L., Shaw J. RNA editing and the mitochondrial cryptogenes of kinetoplastid protozoa. Cell. 1989 May 5;57(3):355–366. doi: 10.1016/0092-8674(89)90911-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  25. Sutton R. E., Boothroyd J. C. Evidence for trans splicing in trypanosomes. Cell. 1986 Nov 21;47(4):527–535. doi: 10.1016/0092-8674(86)90617-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Tschudi C., Ullu E. Polygene transcripts are precursors to calmodulin mRNAs in trypanosomes. EMBO J. 1988 Feb;7(2):455–463. doi: 10.1002/j.1460-2075.1988.tb02833.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tschudi C., Young A. S., Ruben L., Patton C. L., Richards F. F. Calmodulin genes in trypanosomes are tandemly repeated and produce multiple mRNAs with a common 5' leader sequence. Proc Natl Acad Sci U S A. 1985 Jun;82(12):3998–4002. doi: 10.1073/pnas.82.12.3998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Vickerman K. Developmental cycles and biology of pathogenic trypanosomes. Br Med Bull. 1985 Apr;41(2):105–114. doi: 10.1093/oxfordjournals.bmb.a072036. [DOI] [PubMed] [Google Scholar]
  30. Weiner A. M., Maizels N. RNA editing: guided but not templated? Cell. 1990 Jun 15;61(6):917–920. doi: 10.1016/0092-8674(90)90053-h. [DOI] [PubMed] [Google Scholar]
  31. White T. C., Rudenko G., Borst P. Three small RNAs within the 10 kb trypanosome rRNA transcription unit are analogous to domain VII of other eukaryotic 28S rRNAs. Nucleic Acids Res. 1986 Dec 9;14(23):9471–9489. doi: 10.1093/nar/14.23.9471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. ten Asbroek A. L., Ouellette M., Borst P. Targeted insertion of the neomycin phosphotransferase gene into the tubulin gene cluster of Trypanosoma brucei. Nature. 1990 Nov 8;348(6297):174–175. doi: 10.1038/348174a0. [DOI] [PubMed] [Google Scholar]

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