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. 1987 Aug;6(8):2457–2461. doi: 10.1002/j.1460-2075.1987.tb02525.x

Stable introduction of exogenous DNA into Trypanosoma brucei.

W C Gibson 1, T C White 1, P W Laird 1, P Borst 1
PMCID: PMC553653  PMID: 3665883

Abstract

The lack of a homologous transformation system for trypanosomes is a serious handicap to the study of gene expression in these protozoans. Attempts to develop such a system have been complicated by the lack of suitable homologous vectors and ignorance of the requirements for mRNA synthesis which is discontinuous in trypanosomes. We have found that Trypanosoma congolense, a close relative of T. brucei, contains exceptionally small chromosomes, which can be isolated whole and distinguished from those of T. brucei by the presence of a unique satellite DNA. We show here that mini-chromosomes from T. congolense can be introduced into T. brucei by electroporation and detected by hybridisation with T. congolense satellite DNA. The introduced DNA can survive through several generations in the absence of any selective pressure. These results provide the basis for the development of a transformation system for trypanosomes.

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Selected References

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

  1. Boothroyd J. C. Antigenic variation in African trypanosomes. Annu Rev Microbiol. 1985;39:475–502. doi: 10.1146/annurev.mi.39.100185.002355. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Borst P., Fase-Fowler F., Weijers P. J., Barry J. D., Tetley L., Vickerman K. Kinetoplast DNA from Trypanosoma vivax and T. congolense. Mol Biochem Parasitol. 1985 May;15(2):129–142. doi: 10.1016/0166-6851(85)90114-8. [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. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  6. Fromm M. E., Taylor L. P., Walbot V. Stable transformation of maize after gene transfer by electroporation. 1986 Feb 27-Mar 5Nature. 319(6056):791–793. doi: 10.1038/319791a0. [DOI] [PubMed] [Google Scholar]
  7. Fromm M., Taylor L. P., Walbot V. Expression of genes transferred into monocot and dicot plant cells by electroporation. Proc Natl Acad Sci U S A. 1985 Sep;82(17):5824–5828. doi: 10.1073/pnas.82.17.5824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gibson W. C., Borst P. Size-fractionation of the small chromosomes of Trypanozoon and Nannomonas trypanosomes by pulsed field gradient gel electrophoresis. Mol Biochem Parasitol. 1986 Feb;18(2):127–140. doi: 10.1016/0166-6851(86)90033-2. [DOI] [PubMed] [Google Scholar]
  9. Hughes D. E., Simpson L. Introduction of plasmid DNA into the trypanosomatid protozoan Crithidia fasciculata. Proc Natl Acad Sci U S A. 1986 Aug;83(16):6058–6062. doi: 10.1073/pnas.83.16.6058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Johnson P. J., Borst P. Mapping of VSG genes on large expression-site chromosomes of Trypanosoma brucei separated by pulsed-field gradient electrophoresis. Gene. 1986;43(3):213–220. doi: 10.1016/0378-1119(86)90209-x. [DOI] [PubMed] [Google Scholar]
  11. Majiwa P. A., Hamers R., Van Meirvenne N., Matthyssens G. Evidence for genetic diversity in Trypanosoma (Nannomonas) congolense. Parasitology. 1986 Oct;93(Pt 2):291–304. doi: 10.1017/s0031182000051465. [DOI] [PubMed] [Google Scholar]
  12. Majiwa P. A., Masake R. A., Nantulya V. M., Hamers R., Matthyssens G. Trypanosoma (Nannomonas) congolense: identification of two karyotypic groups. EMBO J. 1985 Dec 1;4(12):3307–3313. doi: 10.1002/j.1460-2075.1985.tb04081.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Murray A. W., Szostak J. W. Construction of artificial chromosomes in yeast. Nature. 1983 Sep 15;305(5931):189–193. doi: 10.1038/305189a0. [DOI] [PubMed] [Google Scholar]
  16. Opperdoes F. R. Biochemical peculiarities of trypanosomes, African and South American. Br Med Bull. 1985 Apr;41(2):130–136. doi: 10.1093/oxfordjournals.bmb.a072039. [DOI] [PubMed] [Google Scholar]
  17. Potter H., Weir L., Leder P. Enhancer-dependent expression of human kappa immunoglobulin genes introduced into mouse pre-B lymphocytes by electroporation. Proc Natl Acad Sci U S A. 1984 Nov;81(22):7161–7165. doi: 10.1073/pnas.81.22.7161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Sloof P., Bos J. L., Konings A. F., Menke H. H., Borst P., Gutteridge W. E., Leon W. Characterization of satellite DNA in Trypanosoma brucei and Trypanosoma cruzi. J Mol Biol. 1983 Jun 15;167(1):1–21. doi: 10.1016/s0022-2836(83)80031-x. [DOI] [PubMed] [Google Scholar]
  21. Sloof P., Menke H. H., Caspers M. P., Borst P. Size fractionation of Trypanosoma brucei DNA: localization of the 177-bp repeat satellite DNA and a variant surface glycoprotein gene in a mini-chromosomal DNA fraction. Nucleic Acids Res. 1983 Jun 25;11(12):3889–3901. doi: 10.1093/nar/11.12.3889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Steinert M., Pays E. Genetic control of antigenic variation in trypanosomes. Br Med Bull. 1985 Apr;41(2):149–155. doi: 10.1093/oxfordjournals.bmb.a072042. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Van der Ploeg L. H., Bernards A., Rijsewijk F. A., Borst P. Characterization of the DNA duplication-transposition that controls the expression of two genes for variant surface glycoproteins in Trypanosoma brucei. Nucleic Acids Res. 1982 Jan 22;10(2):593–609. doi: 10.1093/nar/10.2.593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Van der Ploeg L. H., Schwartz D. C., Cantor C. R., Borst P. Antigenic variation in Trypanosoma brucei analyzed by electrophoretic separation of chromosome-sized DNA molecules. Cell. 1984 May;37(1):77–84. doi: 10.1016/0092-8674(84)90302-7. [DOI] [PubMed] [Google Scholar]
  27. Zimmermann U., Vienken J. Electric field-induced cell-to-cell fusion. J Membr Biol. 1982;67(3):165–182. doi: 10.1007/BF01868659. [DOI] [PubMed] [Google Scholar]

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