Skip to main content
The EMBO Journal logoLink to The EMBO Journal
. 1993 Jun;12(6):2529–2538. doi: 10.1002/j.1460-2075.1993.tb05908.x

Autonomously replicating single-copy episomes in Trypanosoma brucei show unusual stability.

P K Patnaik 1, S K Kulkarni 1, G A Cross 1
PMCID: PMC413491  PMID: 8508776

Abstract

We have obtained several autonomously replicating plasmids in procyclic Trypanosoma brucei. Two of these have been analyzed in detail. Both exist as monomeric single-copy episomes, which nevertheless demonstrate significant stability. These episomes transform procyclics at frequencies that are at least 60-fold higher than those achieved earlier with non-replicating molecules. A modified version of one of these episomes is able to replicate autonomously in bloodstream form T. brucei but it is unstable in this developmental stage of the organism. These episomes will prove useful as shuttle vectors, in functional complementation studies, in the construction of promoter-traps for the isolation of transcriptional promoters in T. brucei, and importantly, as models for the analysis of DNA replication in this ancient eukaryotic lineage.

Full text

PDF
2529

Images in this article

Selected References

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

  1. Agabian N. Trans splicing of nuclear pre-mRNAs. Cell. 1990 Jun 29;61(7):1157–1160. doi: 10.1016/0092-8674(90)90674-4. [DOI] [PubMed] [Google Scholar]
  2. Albertson D. G., Thomson J. N. The kinetochores of Caenorhabditis elegans. Chromosoma. 1982;86(3):409–428. doi: 10.1007/BF00292267. [DOI] [PubMed] [Google Scholar]
  3. Bellofatto V., Torres-Muñoz J. E., Cross G. A. Stable transformation of Leptomonas seymouri by circular extrachromosomal elements. Proc Natl Acad Sci U S A. 1991 Aug 1;88(15):6711–6715. doi: 10.1073/pnas.88.15.6711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Benne R. RNA editing in trypanosomes: is there a message? Trends Genet. 1990 Jun;6(6):177–181. doi: 10.1016/0168-9525(90)90173-4. [DOI] [PubMed] [Google Scholar]
  5. Bernards A., Van der Ploeg L. H., Frasch A. C., Borst P., Boothroyd J. C., Coleman S., Cross G. A. Activation of trypanosome surface glycoprotein genes involves a duplication-transposition leading to an altered 3' end. Cell. 1981 Dec;27(3 Pt 2):497–505. doi: 10.1016/0092-8674(81)90391-3. [DOI] [PubMed] [Google Scholar]
  6. Beverley S. M., Coburn C. M. Recurrent de novo appearance of small linear DNAs in Leishmania major and relationship to extra-chromosomal DNAs in other species. Mol Biochem Parasitol. 1990 Aug;42(1):133–141. doi: 10.1016/0166-6851(90)90121-2. [DOI] [PubMed] [Google Scholar]
  7. Bishop R. P., Miles M. A. Chromosome size polymorphisms of Leishmania donovani. Mol Biochem Parasitol. 1987 Jul;24(3):263–272. doi: 10.1016/0166-6851(87)90158-7. [DOI] [PubMed] [Google Scholar]
  8. Blackburn E. H., Challoner P. B. Identification of a telomeric DNA sequence in Trypanosoma brucei. Cell. 1984 Feb;36(2):447–457. doi: 10.1016/0092-8674(84)90238-1. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Carruthers V. B., Cross G. A. High-efficiency clonal growth of bloodstream- and insect-form Trypanosoma brucei on agarose plates. Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8818–8821. doi: 10.1073/pnas.89.18.8818. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Clark C. G., Lai E. Y., Fulton C., Cross G. A. Electrophoretic karyotype and linkage groups of the amoeboflagellate Naegleria gruberi. J Protozool. 1990 Sep-Oct;37(5):400–408. doi: 10.1111/j.1550-7408.1990.tb01164.x. [DOI] [PubMed] [Google Scholar]
  12. Clayton C. E., Fueri J. P., Itzhaki J. E., Bellofatto V., Sherman D. R., Wisdom G. S., Vijayasarathy S., Mowatt M. R. Transcription of the procyclic acidic repetitive protein genes of Trypanosoma brucei. Mol Cell Biol. 1990 Jun;10(6):3036–3047. doi: 10.1128/mcb.10.6.3036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Donelson J. E., Zeng W. A comparison of trans-RNA splicing in trypanosomes and nematodes. Parasitol Today. 1990 Oct;6(10):327–334. doi: 10.1016/0169-4758(90)90177-6. [DOI] [PubMed] [Google Scholar]
  14. Eid J., Sollner-Webb B. Stable integrative transformation of Trypanosoma brucei that occurs exclusively by homologous recombination. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2118–2121. doi: 10.1073/pnas.88.6.2118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Engman D. M., Reddy L. V., Donelson J. E., Kirchhoff L. V. Trypanosoma cruzi exhibits inter- and intra-strain heterogeneity in molecular karyotype and chromosomal gene location. Mol Biochem Parasitol. 1987 Jan 15;22(2-3):115–123. doi: 10.1016/0166-6851(87)90041-7. [DOI] [PubMed] [Google Scholar]
  16. Fangman W. L., Brewer B. J. Activation of replication origins within yeast chromosomes. Annu Rev Cell Biol. 1991;7:375–402. doi: 10.1146/annurev.cb.07.110191.002111. [DOI] [PubMed] [Google Scholar]
  17. Feagin J. E. RNA editing in kinetoplastid mitochondria. J Biol Chem. 1990 Nov 15;265(32):19373–19376. [PubMed] [Google Scholar]
  18. Felsenstein K. M., Emmons S. W. Nematode repetitive DNA with ARS and segregation function in Saccharomyces cerevisiae. Mol Cell Biol. 1988 Feb;8(2):875–883. doi: 10.1128/mcb.8.2.875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Gottesdiener K., Garciá-Anoveros J., Lee M. G., Van der Ploeg L. H. Chromosome organization of the protozoan Trypanosoma brucei. Mol Cell Biol. 1990 Nov;10(11):6079–6083. doi: 10.1128/mcb.10.11.6079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Herman R. K., Madl J. E., Kari C. K. Duplications in Caenorhabditis elegans. Genetics. 1979 Jun;92(2):419–435. doi: 10.1093/genetics/92.2.419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hill A., Bloom K. Genetic manipulation of centromere function. Mol Cell Biol. 1987 Jul;7(7):2397–2405. doi: 10.1128/mcb.7.7.2397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hirumi H., Hirumi K. Continuous cultivation of Trypanosoma brucei blood stream forms in a medium containing a low concentration of serum protein without feeder cell layers. J Parasitol. 1989 Dec;75(6):985–989. [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Kelly J. M., Ward H. M., Miles M. A., Kendall G. A shuttle vector which facilitates the expression of transfected genes in Trypanosoma cruzi and Leishmania. Nucleic Acids Res. 1992 Aug 11;20(15):3963–3969. doi: 10.1093/nar/20.15.3963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kimmerly W. J., Rine J. Replication and segregation of plasmids containing cis-acting regulatory sites of silent mating-type genes in Saccharomyces cerevisiae are controlled by the SIR genes. Mol Cell Biol. 1987 Dec;7(12):4225–4237. doi: 10.1128/mcb.7.12.4225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Lusky M., Botchan M. R. Genetic analysis of bovine papillomavirus type 1 trans-acting replication factors. J Virol. 1985 Mar;53(3):955–965. doi: 10.1128/jvi.53.3.955-965.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. McNally F. J., Rine J. A synthetic silencer mediates SIR-dependent functions in Saccharomyces cerevisiae. Mol Cell Biol. 1991 Nov;11(11):5648–5659. doi: 10.1128/mcb.11.11.5648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pagès M., Bastien P., Veas F., Rossi V., Bellis M., Wincker P., Rioux J. A., Roizès G. Chromosome size and number polymorphisms in Leishmania infantum suggest amplification/deletion and possible genetic exchange. Mol Biochem Parasitol. 1989 Sep;36(2):161–168. doi: 10.1016/0166-6851(89)90188-6. [DOI] [PubMed] [Google Scholar]
  32. Peden K. W., Pipas J. M., Pearson-White S., Nathans D. Isolation of mutants of an animal virus in bacteria. Science. 1980 Sep 19;209(4463):1392–1396. doi: 10.1126/science.6251547. [DOI] [PubMed] [Google Scholar]
  33. Pimpinelli S., Goday C. Unusual kinetochores and chromatin diminution in Parascaris. Trends Genet. 1989 Sep;5(9):310–315. doi: 10.1016/0168-9525(89)90114-5. [DOI] [PubMed] [Google Scholar]
  34. Rudenko G., Chung H. M., Pham V. P., Van der Ploeg L. H. RNA polymerase I can mediate expression of CAT and neo protein-coding genes in Trypanosoma brucei. EMBO J. 1991 Nov;10(11):3387–3397. doi: 10.1002/j.1460-2075.1991.tb04903.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rudenko G., Le Blancq S., Smith J., Lee M. G., Rattray A., Van der Ploeg L. H. Procyclic acidic repetitive protein (PARP) genes located in an unusually small alpha-amanitin-resistant transcription unit: PARP promoter activity assayed by transient DNA transfection of Trypanosoma brucei. Mol Cell Biol. 1990 Jul;10(7):3492–3504. doi: 10.1128/mcb.10.7.3492. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sherman D. R., Janz L., Hug M., Clayton C. Anatomy of the parp gene promoter of Trypanosoma brucei. EMBO J. 1991 Nov;10(11):3379–3386. doi: 10.1002/j.1460-2075.1991.tb04902.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sherman D. R., Janz L., Hug M., Clayton C. Anatomy of the parp gene promoter of Trypanosoma brucei. EMBO J. 1991 Nov;10(11):3379–3386. doi: 10.1002/j.1460-2075.1991.tb04902.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Snyder M., Sapolsky R. J., Davis R. W. Transcription interferes with elements important for chromosome maintenance in Saccharomyces cerevisiae. Mol Cell Biol. 1988 May;8(5):2184–2194. doi: 10.1128/mcb.8.5.2184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sogin M. L., Elwood H. J., Gunderson J. H. Evolutionary diversity of eukaryotic small-subunit rRNA genes. Proc Natl Acad Sci U S A. 1986 Mar;83(5):1383–1387. doi: 10.1073/pnas.83.5.1383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sogin M. L., Gunderson J. H., Elwood H. J., Alonso R. A., Peattie D. A. Phylogenetic meaning of the kingdom concept: an unusual ribosomal RNA from Giardia lamblia. Science. 1989 Jan 6;243(4887):75–77. doi: 10.1126/science.2911720. [DOI] [PubMed] [Google Scholar]
  42. Spithill T. W., Samaras N. The molecular karyotype of Leishmania major and mapping of alpha and beta tubulin gene families to multiple unlinked chromosomal loci. Nucleic Acids Res. 1985 Jun 11;13(11):4155–4169. doi: 10.1093/nar/13.11.4155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Stinchcomb D. T., Struhl K., Davis R. W. Isolation and characterisation of a yeast chromosomal replicator. Nature. 1979 Nov 1;282(5734):39–43. doi: 10.1038/282039a0. [DOI] [PubMed] [Google Scholar]
  44. Struhl K., Stinchcomb D. T., Scherer S., Davis R. W. High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1035–1039. doi: 10.1073/pnas.76.3.1035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Stuart K. RNA editing in trypanosomatid mitochondria. Annu Rev Microbiol. 1991;45:327–344. doi: 10.1146/annurev.mi.45.100191.001551. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. Van der Ploeg L. H., Liu A. Y., Borst P. Structure of the growing telomeres of Trypanosomes. Cell. 1984 Feb;36(2):459–468. doi: 10.1016/0092-8674(84)90239-3. [DOI] [PubMed] [Google Scholar]
  48. Wagner W., So M. Identification of a novel large extrachromosomal DNA (LED) in the Trypanosomatidae. Mol Microbiol. 1992 Aug;6(16):2299–2308. doi: 10.1111/j.1365-2958.1992.tb01405.x. [DOI] [PubMed] [Google Scholar]
  49. Zomerdijk J. C., Kieft R., Borst P. Efficient production of functional mRNA mediated by RNA polymerase I in Trypanosoma brucei. Nature. 1991 Oct 24;353(6346):772–775. doi: 10.1038/353772a0. [DOI] [PubMed] [Google Scholar]
  50. Zomerdijk J. C., Kieft R., Shiels P. G., Borst P. Alpha-amanitin-resistant transcription units in trypanosomes: a comparison of promoter sequences for a VSG gene expression site and for the ribosomal RNA genes. Nucleic Acids Res. 1991 Oct 11;19(19):5153–5158. doi: 10.1093/nar/19.19.5153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. 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]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES