Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1975 May 1;65(2):309–323. doi: 10.1083/jcb.65.2.309

Isolation of intracellular symbiotes by immune lysis of flagellate protozoa and characterization of their DNA

PMCID: PMC2109415  PMID: 805151

Abstract

A new method dependent on immune lysis is described for the isolation of intracellular symbiotes from two species of flagellate protozoa Blastocrithidia culicis and Crithidia oncopelti. The symbiote- containing flagellates are exposed to complement and antisera prepared in rabbits against symbiote-free organisms. The immune lysis seems to weaken the plasma membranes of the flagellates so that subsequent application of gentle shearing force liberates the intracellular entities in an undamaged condition. The symbiotes are then separated from other cellular components by DNAse digestion and differential centrifugation. The average recovery of symbiotes isolated by this method is 20%. Light and electron microscopy establishes the structural integrity and numerical abundance of isolated symbiotes in the final fractions. Integrity of symbiotes is further indicated by the high activity of a marker enzyme, uroporphyrinogen I synthetase. The DNA's of symbiote-containing and symbiote-free flagellates, and of isolated symbiotes were purified and compared after isopycnic centrifugation. The comparison establishes the presence of DNA's in symbiotes of both species. The guanine-cytosine (G-C) content of symbiote DNA differs from that of host DNA's in C. oncopelti, but resembles that of kinetoplast DNA in B. culicis. The latter observation was further shown by heat denaturation study. Renaturation kinetics indicate that the genome complexity of symbiote DNA in B. culicis is similar to that of bacteria.

Full Text

The Full Text of this article is available as a PDF (2.5 MB).

Selected References

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

  1. Bak A. L., Black F. T., Christiansen C., Freundt E. A. Genome size of mycoplasmal DNA. Nature. 1969 Dec 20;224(5225):1209–1210. doi: 10.1038/2241209a0. [DOI] [PubMed] [Google Scholar]
  2. Bak A. L., Christiansen C., Stenderup A. Bacterial genome sizes determined by DNA renaturation studies. J Gen Microbiol. 1970 Dec;64(3):377–380. doi: 10.1099/00221287-64-3-377. [DOI] [PubMed] [Google Scholar]
  3. Beale G. N., Jurand A., Preer J. R. The classes of endosymbiont of Paramecium aurelia. J Cell Sci. 1969 Jul;5(1):65–91. doi: 10.1242/jcs.5.1.65. [DOI] [PubMed] [Google Scholar]
  4. Borst P., Kroon A. M. Mitochondrial DNA: physicochemical properties, replication, and genetic function. Int Rev Cytol. 1969;26:107–190. doi: 10.1016/s0074-7696(08)61635-6. [DOI] [PubMed] [Google Scholar]
  5. Chang K. P., Trager W. Nutritional significance of symbiotic bacteria in two species of hemoflagellates. Science. 1974 Feb 8;183(4124):531–532. doi: 10.1126/science.183.4124.531. [DOI] [PubMed] [Google Scholar]
  6. Chang K. P. Ultrastructure of symbiotic bacteria in normal and antibiotic-treated Blastocrithidia culicis and Crithidia oncopelti. J Protozool. 1974 Nov;21(5):699–707. doi: 10.1111/j.1550-7408.1974.tb03733.x. [DOI] [PubMed] [Google Scholar]
  7. Fridovich I. Evidence for the symbiotic origin of mitochondria. Life Sci. 1974 Mar 1;14(5):819–826. doi: 10.1016/0024-3205(74)90071-x. [DOI] [PubMed] [Google Scholar]
  8. GRACE T. D. Establishment of four strains of cells from insect tissues grown in vitro. Nature. 1962 Aug 25;195:788–789. doi: 10.1038/195788a0. [DOI] [PubMed] [Google Scholar]
  9. GREEN H., BARROW P., GOLDBERG B. Effect of antibody and complement on permeability control in ascites tumor cells and erythrocytes. J Exp Med. 1959 Nov 1;110:699–713. doi: 10.1084/jem.110.5.699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gibson I., Chance M., Williams J. Extranuclear DNA and the endosymbionts of Paramecium aurelia. Nat New Biol. 1971 Nov 17;234(46):75–77. doi: 10.1038/newbio234075a0. [DOI] [PubMed] [Google Scholar]
  11. Kingsbury D. T. Estimate of the genome size of various microorganisms. J Bacteriol. 1969 Jun;98(3):1400–1401. doi: 10.1128/jb.98.3.1400-1401.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. LEDERBERG J. Cell genetics and hereditary symbiosis. Physiol Rev. 1952 Oct;32(4):403–430. doi: 10.1152/physrev.1952.32.4.403. [DOI] [PubMed] [Google Scholar]
  13. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  14. Lanham U. N. The Blochmann bodies: hereditary intracellular symbionts of insects. Biol Rev Camb Philos Soc. 1968 Aug;43(3):269–286. doi: 10.1111/j.1469-185x.1968.tb00961.x. [DOI] [PubMed] [Google Scholar]
  15. MARMUR J., DOTY P. Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J Mol Biol. 1962 Jul;5:109–118. doi: 10.1016/s0022-2836(62)80066-7. [DOI] [PubMed] [Google Scholar]
  16. Mandel M. New approaches to bacterial taxonomy: perspective and prospects. Annu Rev Microbiol. 1969;23:239–274. doi: 10.1146/annurev.mi.23.100169.001323. [DOI] [PubMed] [Google Scholar]
  17. NEWTON B. A., HORNE R. W. Intracellular structures in Strigomonas oncopelti. I. Cytoplasmic structures containing ribonucleoprotein. Exp Cell Res. 1957 Dec;13(3):563–574. doi: 10.1016/0014-4827(57)90086-1. [DOI] [PubMed] [Google Scholar]
  18. Preer L. B., Jurand A., Preer J. R., Jr, Rudman B. M. The classes of kappa in Paramecium aurelia. J Cell Sci. 1972 Sep;11(2):581–600. doi: 10.1242/jcs.11.2.581. [DOI] [PubMed] [Google Scholar]
  19. Raff R. A., Mahler H. R. The non symbiotic origin of mitochondria. Science. 1972 Aug 18;177(4049):575–582. doi: 10.1126/science.177.4049.575. [DOI] [PubMed] [Google Scholar]
  20. Rosse W. F., Dourmashkin R., Humphrey J. H. Immune lysis of normal human and paroxysmal nocturnal hemoglobinuria (PNH) red blood cells. 3. The membrane defects caused by complement lysis. J Exp Med. 1966 Jun 1;123(6):969–984. doi: 10.1084/jem.123.6.969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. SCHILDKRAUT C. L., MANDEL M., LEVISOHN S., SMITH-SONNEBORN J. E., MARMUR J. Deoxyribonucleic acid base composition and taxonomy of some protozoa. Nature. 1962 Nov 24;196:795–796. doi: 10.1038/196795a0. [DOI] [PubMed] [Google Scholar]
  22. SCHILDKRAUT C. L., MARMUR J., DOTY P. Determination of the base composition of deoxyribonucleic acid from its buoyant density in CsCl. J Mol Biol. 1962 Jun;4:430–443. doi: 10.1016/s0022-2836(62)80100-4. [DOI] [PubMed] [Google Scholar]
  23. SMITH-SONNEBORN J. E., VANWAGTENDONK W. J. PURIFICATION AND CHEMICAL CHARACTERIZATION OF KAPPA OF STOCK 51, PARAMECIUM AURELIA. Exp Cell Res. 1964 Jan;33:50–59. doi: 10.1016/s0014-4827(64)81011-9. [DOI] [PubMed] [Google Scholar]
  24. STUDIER F. W. SEDIMENTATION STUDIES OF THE SIZE AND SHAPE OF DNA. J Mol Biol. 1965 Feb;11:373–390. doi: 10.1016/s0022-2836(65)80064-x. [DOI] [PubMed] [Google Scholar]
  25. Sassa S., Granick S., Bickers D. R., Bradlow H. L., Kappas A. A microassay for uroporphyrinogen I synthase, one of three abnormal enzyme activities in acute intermittent porphyria, and its application to the study of the genetics of this disease. Proc Natl Acad Sci U S A. 1974 Mar;71(3):732–736. doi: 10.1073/pnas.71.3.732. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Simpson L. Behavior of the kinetoplast of Leishmania tarentolae upon cell rupture. J Protozool. 1968 Feb;15(1):132–136. doi: 10.1111/j.1550-7408.1968.tb02097.x. [DOI] [PubMed] [Google Scholar]
  27. Soldo A. T., Godoy G. A. Molecular complexity of Paramecium symbiont lambda deoxyribonucleic acid: evidence for the presence of a multicopy genome. J Mol Biol. 1973 Jan;73(1):93–108. doi: 10.1016/0022-2836(73)90161-7. [DOI] [PubMed] [Google Scholar]
  28. Soldo A. T., Van Wagtendonk W. J., Godoy G. A. Nucleic acid and protein content of purified endosymbiote particles of Paramecium aurelia. Biochim Biophys Acta. 1970 Apr 15;204(2):325–333. doi: 10.1016/0005-2787(70)90150-4. [DOI] [PubMed] [Google Scholar]
  29. Stevenson I. The biochemical status of mu particles in Paramecium aurelia. J Gen Microbiol. 1969 Jul;57(1):61–75. doi: 10.1099/00221287-57-1-61. [DOI] [PubMed] [Google Scholar]
  30. TRAGER W. The enhanced folic and folinic acid contents of erythrocytes infected with malaria parasites. Exp Parasitol. 1959 Jun;8(3):265–273. doi: 10.1016/0014-4894(59)90025-6. [DOI] [PubMed] [Google Scholar]
  31. Uzzell T., Spolsky C. Mitochondria and plastids as endosymbionts: a revival of special creation? Am Sci. 1974 May-Jun;62(3):334–343. [PubMed] [Google Scholar]
  32. VAN WAGTENDONKW, TANGUAY R. B. THE CHEMICAL COMPOSITION OF LAMBDA IN PARAMECIUM AURELIA, STOCK 299. J Gen Microbiol. 1963 Dec;33:395–400. doi: 10.1099/00221287-33-3-395. [DOI] [PubMed] [Google Scholar]
  33. VINOGRAD J., BRUNER R., KENT R., WEIGLE J. Band-centrifugation of macromolecules and viruses in self-generating density gradients. Proc Natl Acad Sci U S A. 1963 Jun;49:902–910. doi: 10.1073/pnas.49.6.902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wetmur J. G., Davidson N. Kinetics of renaturation of DNA. J Mol Biol. 1968 Feb 14;31(3):349–370. doi: 10.1016/0022-2836(68)90414-2. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES