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. 1975 Nov 1;67(2):378–399. doi: 10.1083/jcb.67.2.378

Physicochemical properties of kinetoplast DNA from Crithidia acanthocephali. Crithidia luciliae, and Trypanosoma lewisi

D L Fouts, J E Manning, D R Wolstenholme
PMCID: PMC2109601  PMID: 1104639

Abstract

The protozoa Crithidia and Trypanosoma contain within a mitochondrion a mass of DNA known as kinetoplast DNA (kDNA) which consists mainly of an association of thousands of small circular molecules of similar size held together by topological interlocking. Using kDNA from Crithidia acanthocephali, Crithidia luciliae, and Trypanosoma lewisi, physicochemical studies have been carried out with intact associations and with fractions of covalently closed single circular molecules, and of open single circular and unit length linear molecules obtained from kDNA associations by sonication, sucrose sedimentation, and cesium chloride-ethidium bromide equilibrium centrifugation. Buoyant density analyses failed to provide evidence for base composition heterogeneity among kDNA molecules within a species. The complementary nucleotide strands of kDNA molecules of all three species had distinct buoyant densities in both alkaline and neutral cesium chloride. For C. acanthocephali kDNA, these buoyant density differences were shown to be a reflection of differences in base composition between the complementary nucleotide strands. The molar ratios of adenine: thymine:guanine:cytosine, obtained from deoxyribonucleotide analyses were 16.8:41.0:28.1:14.1 for the heavy strand and 41.6:16.6:12.8:29.0 for the light strand. Covalently closed single circular molecules of C. acanthocephali (as well as intact kDNA associations of C. acanthocephali and T. lewisi) formed a single band in alkaline cesium chloride gradients, indicating their component nucleotide strands to be alkaline insensitive. Data from buoyant density, base composition, and thermal melting analyses suggested that minor bases are either rare or absent in Crithidia kDNA. The kinetics of renaturation of 32P labeled C. acanthocephali kDNA measured using hydroxyapatite chromatography were consistent with at least 70% of the circular molecules of this DNA having the same nucleotide sequence. Evidence for sequence homologies among the kDNAs of all three species was obtained from buoyant density analyses of DNA in annealed mixtures containing one component kDNA strand from each of two species.

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Selected References

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  1. Bauer W., Vinograd J. The interaction of closed circular DNA with intercalative dyes. I. The superhelix density of SV40 DNA in the presence and absence of dye. J Mol Biol. 1968 Apr 14;33(1):141–171. doi: 10.1016/0022-2836(68)90286-6. [DOI] [PubMed] [Google Scholar]
  2. Borst P., Aaij C. Identification of the heavy strand of rat-liver mitochondrial DNA as the messenger strand. Biochem Biophys Res Commun. 1969 Feb 7;34(3):358–364. doi: 10.1016/0006-291x(69)90841-9. [DOI] [PubMed] [Google Scholar]
  3. Borst P., Ruttenberg G. J. Renaturation of mitochondrial DNA. Biochim Biophys Acta. 1966 Mar 21;114(3):645–647. doi: 10.1016/0005-2787(66)90117-1. [DOI] [PubMed] [Google Scholar]
  4. Borst P., van Bruggen E. F., Ruttenberg G. J., Kroon A. M. Mitochondrial DNA. II. Sedimentation analysis and electron microscopy of mitochondrial DNA from chick liver. Biochim Biophys Acta. 1967 Nov 21;149(1):156–172. doi: 10.1016/0005-2787(67)90698-3. [DOI] [PubMed] [Google Scholar]
  5. Britten R. J., Kohne D. E. Repeated sequences in DNA. Hundreds of thousands of copies of DNA sequences have been incorporated into the genomes of higher organisms. Science. 1968 Aug 9;161(3841):529–540. doi: 10.1126/science.161.3841.529. [DOI] [PubMed] [Google Scholar]
  6. Corneo G., Ginelli E., Polli E. Isolation of the complementary strands of a human satellite DNA. J Mol Biol. 1968 Apr 14;33(1):331–335. doi: 10.1016/0022-2836(68)90301-x. [DOI] [PubMed] [Google Scholar]
  7. Corneo G., Moore C., Sanadi D. R., Grossman L. I., Marmur J. Mitochondrial DNA in yeast and some mammalian species. Science. 1966 Feb 11;151(3711):687–689. doi: 10.1126/science.151.3711.687. [DOI] [PubMed] [Google Scholar]
  8. Corneo G., Zardi L., Polli E. Human mitochondrial DNA. J Mol Biol. 1968 Sep 28;36(3):419–423. doi: 10.1016/0022-2836(68)90166-6. [DOI] [PubMed] [Google Scholar]
  9. Dawid I. B. Mitochondrial RNA In Xenopus laevis. I. The expression of the mitochondrial genome. J Mol Biol. 1972 Jan 28;63(2):201–216. doi: 10.1016/0022-2836(72)90370-1. [DOI] [PubMed] [Google Scholar]
  10. Dawid I. B., Wolstenholme D. R. Ultracentrifuge and electron microscope studies on the structure of mitochondrial DNA. J Mol Biol. 1967 Sep 14;28(2):233–245. doi: 10.1016/s0022-2836(67)80006-8. [DOI] [PubMed] [Google Scholar]
  11. Doerfler W., Hogness D. S. The strands of DNA from lambda and related bacteriophages: isolation and characterization. J Mol Biol. 1968 May 14;33(3):635–659. doi: 10.1016/0022-2836(68)90311-2. [DOI] [PubMed] [Google Scholar]
  12. Doty P., Boedtker H., Fresco J. R., Haselkorn R., Litt M. SECONDARY STRUCTURE IN RIBONUCLEIC ACIDS. Proc Natl Acad Sci U S A. 1959 Apr;45(4):482–499. doi: 10.1073/pnas.45.4.482. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dubnau D., Smith I., Morell P., Marmur J. Gene conservation in Bacillus species. I. Conserved genetic and nucleic acid base sequence homologies. Proc Natl Acad Sci U S A. 1965 Aug;54(2):491–498. doi: 10.1073/pnas.54.2.491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dusanic D. G. Growth and immunologic studies on the culture forms of Trypanosoma lewisi. J Protozool. 1968 May;15(2):328–333. doi: 10.1111/j.1550-7408.1968.tb02131.x. [DOI] [PubMed] [Google Scholar]
  15. Flamm W. G., McCallum M., Walker P. M. The isolation of complementary strands from a mouse DNA fraction. Proc Natl Acad Sci U S A. 1967 Jun;57(6):1729–1734. doi: 10.1073/pnas.57.6.1729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Follett E. A., Crawford L. V. Electron microscope study of the denaturation of human papilloma virus DNA. I. Loss and reversal of supercoiling turns. J Mol Biol. 1967 Sep 28;28(3):455–459. doi: 10.1016/s0022-2836(67)80095-0. [DOI] [PubMed] [Google Scholar]
  17. Freifelder D., Kleinschmidt A. K. Single-strand breaks in duplex DNA of coliphage T7 as demonstrated by electron microscopy. J Mol Biol. 1965 Nov;14(1):271–278. doi: 10.1016/s0022-2836(65)80246-7. [DOI] [PubMed] [Google Scholar]
  18. GUILD W. R., ROBINSON M. Evidence for message reading from a unique strand of pneumococcal DNA. Proc Natl Acad Sci U S A. 1963 Jul;50:106–112. doi: 10.1073/pnas.50.1.106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. GUTTMAN H. N., EISENMAN R. N. 'CURE' OF CRITHIDIA (STRIGOMONAS) ONCOPELTI OF ITS BACTERIAL ENDOSYMBIOTE. Nature. 1965 Apr 3;206:113–114. doi: 10.1038/206113a0. [DOI] [PubMed] [Google Scholar]
  20. Gillis M., De Ley J., De Cleene M. The determination of molecular weight of bacterial genome DNA from renaturation rates. Eur J Biochem. 1970 Jan;12(1):143–153. doi: 10.1111/j.1432-1033.1970.tb00831.x. [DOI] [PubMed] [Google Scholar]
  21. Grossman L. I., Watson R., Vinograd J. The presence of ribonucleotides in mature closed-circular mitochondrial DNA. Proc Natl Acad Sci U S A. 1973 Dec;70(12):3339–3343. doi: 10.1073/pnas.70.12.3339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. HONIGBERG B. M., BALAMUTH W., BOVEE E. C., CORLISS J. O., GOJDICS M., HALL R. P., KUDO R. R., LEVINE N. D., LOEBLICH A. R., Jr, WEISER J. A REVISED CLASSIFICATION OF THE PHYLUM PROTOZOA. J Protozool. 1964 Feb;11:7–20. doi: 10.1111/j.1550-7408.1964.tb01715.x. [DOI] [PubMed] [Google Scholar]
  23. HOTTA Y., BASSEL A. MOLECULAR SIZE AND CIRCULARITY OF DNA IN CELLS OF MAMMALS AND HIGHER PLANTS. Proc Natl Acad Sci U S A. 1965 Feb;53:356–362. doi: 10.1073/pnas.53.2.356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kram R., Botchan M., Hearst J. E. Arrangement of the highly reiterated DNA sequences in the centric heterochromatin of Drosophila melanogaster. Evidence for interspersed spacer DNA. J Mol Biol. 1972 Feb 28;64(1):103–117. doi: 10.1016/0022-2836(72)90323-3. [DOI] [PubMed] [Google Scholar]
  25. Laurent M., Steinert M. Electron microscopy of kinetoplastic DNA from Trypanosoma mega. Proc Natl Acad Sci U S A. 1970 Jun;66(2):419–424. doi: 10.1073/pnas.66.2.419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Laurent M., Van Assel S., Steinert M. Kinetoplast DNA. A unique macromolecular structure of considerable size and mechanical resistance. Biochem Biophys Res Commun. 1971 Apr 16;43(2):278–284. doi: 10.1016/0006-291x(71)90749-2. [DOI] [PubMed] [Google Scholar]
  27. Leffler A. T., 2nd, Creskoff E., Luborsky S. W., McFarland V., Mora P. T. Isolation and characterization of rat liver mitochondrial DNA. J Mol Biol. 1970 Mar;48(3):455–468. doi: 10.1016/0022-2836(70)90058-6. [DOI] [PubMed] [Google Scholar]
  28. Locker J., Rabinowitz M., Getz G. S. Tandem inverted repeats in mitochondrial DNA of petite mutants of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1366–1370. doi: 10.1073/pnas.71.4.1366. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Manning J. E., Schmid C. W., Davidson N. Interspersion of repetitive and nonrepetitive DNA sequences in the Drosophila melanogaster genome. Cell. 1975 Feb;4(2):141–155. doi: 10.1016/0092-8674(75)90121-x. [DOI] [PubMed] [Google Scholar]
  30. Meselson M., Stahl F. W., Vinograd J. EQUILIBRIUM SEDIMENTATION OF MACROMOLECULES IN DENSITY GRADIENTS. Proc Natl Acad Sci U S A. 1957 Jul 15;43(7):581–588. doi: 10.1073/pnas.43.7.581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Miyaki M., Koide K., Ono T. RNase and alkali sensitivity of closed circular mitochondrial DNA of rat ascites hepatoma cells. Biochem Biophys Res Commun. 1973 Jan 23;50(2):252–258. doi: 10.1016/0006-291x(73)90833-4. [DOI] [PubMed] [Google Scholar]
  32. Nass M. M., Buck C. A. Studies on mitochondrial tRNA from animal cells. II. Hybridization of aminoacyl-tRNA from rat liver mitochondria with heavy and light complementary strands of mitochondrial DNA. J Mol Biol. 1970 Dec 14;54(2):187–198. doi: 10.1016/0022-2836(70)90426-2. [DOI] [PubMed] [Google Scholar]
  33. Nass M. M. Mitochondrial DNA. II. Structure and physicochemical properties of isolated DNA. J Mol Biol. 1969 Jun 28;42(3):529–545. doi: 10.1016/0022-2836(69)90241-1. [DOI] [PubMed] [Google Scholar]
  34. Pikó L., Blair D. G., Tyler A., Vinograd J. Cytoplasmic DNA in the unfertilized sea urchin egg: physical properties of circular mitochondrial DNA and the occurrence of catenated forms. Proc Natl Acad Sci U S A. 1968 Mar;59(3):838–845. doi: 10.1073/pnas.59.3.838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Polan M. L., Friedman S., Gall J. G., Gehring W. Isolation and characterization of mitochondrial DNA from Drosophila melanogaster. J Cell Biol. 1973 Feb;56(2):580–589. doi: 10.1083/jcb.56.2.580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Radloff R., Bauer W., Vinograd J. A dye-buoyant-density method for the detection and isolation of closed circular duplex DNA: the closed circular DNA in HeLa cells. Proc Natl Acad Sci U S A. 1967 May;57(5):1514–1521. doi: 10.1073/pnas.57.5.1514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Renger H. C., Wolstenholme D. R. Kinetoplast and other satellite DNAs of kinetoplastic and dyskinetoplastic strains of Trypanosoma. J Cell Biol. 1971 Aug;50(2):533–540. doi: 10.1083/jcb.50.2.533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Renger H. C., Wolstenholme D. R. Kinetoplast deoxyribonucleic acid of the hemoflagellate Trypanosoma lewisi. J Cell Biol. 1970 Dec;47(3):689–702. doi: 10.1083/jcb.47.3.689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Renger H. C., Wolstenholme D. R. The form and structure of kinetoplast DNA of Crithidia. J Cell Biol. 1972 Aug;54(2):346–364. doi: 10.1083/jcb.54.2.346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Richards O. C., Ryan R. S., Manning J. E. Effects of cycloheximide and of chloramphenicol on DNA synthesis in Euglena gracilis. Biochim Biophys Acta. 1971 May 13;238(2):190–201. doi: 10.1016/0005-2787(71)90086-4. [DOI] [PubMed] [Google Scholar]
  41. Richardson C. C. The 5'-terminal nucleotides of T7 bacteriophage deoxyribonucleic acid. J Mol Biol. 1966 Jan;15(1):49–61. doi: 10.1016/s0022-2836(66)80208-5. [DOI] [PubMed] [Google Scholar]
  42. Riou G., Delain E. Electron microscopy of the circular kinetoplastic DNA from Trypanosoma cruzi: occurrence of catenated forms. Proc Natl Acad Sci U S A. 1969 Jan;62(1):210–217. doi: 10.1073/pnas.62.1.210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Riva S., Barrai I., Cavalli-Sforza L., Falaschi A. Dependence of the buoyant density of single-stranded DNA on base composition. J Mol Biol. 1969 Oct 28;45(2):367–374. doi: 10.1016/0022-2836(69)90111-9. [DOI] [PubMed] [Google Scholar]
  44. Riva S., Polsinelli M., Falaschi A. A new phage of Bacillus subtilis with infectious DNA having separable strands. J Mol Biol. 1968 Jul 28;35(2):347–356. doi: 10.1016/s0022-2836(68)80029-4. [DOI] [PubMed] [Google Scholar]
  45. Robberson D., Aloni Y., Attardi G. Electron microscopic visualization of mitochondrial RNA-DNA hybrids. J Mol Biol. 1971 Jan 28;55(2):267–270. doi: 10.1016/0022-2836(71)90196-3. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. 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]
  48. Schildkraut C. L., Maio J. J. Fractions of HeLa DNA differing in their content of guanine+cytosine. J Mol Biol. 1969 Dec 14;46(2):305–312. doi: 10.1016/0022-2836(69)90423-9. [DOI] [PubMed] [Google Scholar]
  49. Simpson L., Da Silva A. Isolation and characterization of kinetoplast DNA from Leishmania tarentolae. J Mol Biol. 1971 Mar 28;56(3):443–473. doi: 10.1016/0022-2836(71)90394-9. [DOI] [PubMed] [Google Scholar]
  50. Sinclair J. H., Stevens B. J., Sanghavi P., Rabinowitz M. Mitochondrial-satellite and circular DNA filaments in yeast. Science. 1967 Jun 2;156(3779):1234–1237. doi: 10.1126/science.156.3779.1234. [DOI] [PubMed] [Google Scholar]
  51. Stutz E., Rawson J. R. Separation and characterization of Euglena gracilis chloroplast single-strand DNA. Biochim Biophys Acta. 1970 May 21;209(1):16–23. doi: 10.1016/0005-2787(70)90656-8. [DOI] [PubMed] [Google Scholar]
  52. VINOGRAD J., MORRIS J., DAVIDSON N., DOVE W. F., Jr The bouyant behavior of viral and bacterial DNA in alkaline CsCl. Proc Natl Acad Sci U S A. 1963 Jan 15;49:12–17. doi: 10.1073/pnas.49.1.12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Vinograd J., Lebowitz J. Physical and topological properties of circular DNA. J Gen Physiol. 1966 Jul;49(6):103–125. doi: 10.1085/jgp.49.6.103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Welker N. E., Campbell L. L. Unrelatedness of Bacillus amyloliquefaciens and Bacillus subtilis. J Bacteriol. 1967 Oct;94(4):1124–1130. doi: 10.1128/jb.94.4.1124-1130.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Wesley R. D., Simpson L. Studies on kinetoplast DNA. 3. Kinetic complexity of kinetoplast and nuclear DNA from Leishmania tarentolae. Biochim Biophys Acta. 1973 Sep 7;319(3):267–276. doi: 10.1016/0005-2787(73)90165-2. [DOI] [PubMed] [Google Scholar]
  56. Wesley R. D., Simpson L. Studies on kinetoplast DNA. II. Biophysical properties of minicircular DNA from Leishmania tarentolae. Biochim Biophys Acta. 1973 Sep 7;319(3):254–266. doi: 10.1016/0005-2787(73)90164-0. [DOI] [PubMed] [Google Scholar]
  57. 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]
  58. Wolstenholme D. R., Gross N. J. The form and size of mitochondrial DNA of the red bean, Phaseolus vulgaris. Proc Natl Acad Sci U S A. 1968 Sep;61(1):245–252. doi: 10.1073/pnas.61.1.245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Wolstenholme D. R., Renger H. C., Manning J. E., Fouts D. L. Kinetoplast DNA of Crithidia. J Protozool. 1974 Nov;21(5):622–631. doi: 10.1111/j.1550-7408.1974.tb03716.x. [DOI] [PubMed] [Google Scholar]
  60. Wolstonholme D. R., Kirschner R. G., Gross N. J. Heart denaturation studies of rat liver mitrochondrial DNA. A denaturation map and changes in molecular configurations. J Cell Biol. 1972 May;53(2):393–406. doi: 10.1083/jcb.53.2.393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Wong-Staal F., Mendelsohn J., Goulian M. Ribonucleotides in closed circular mitochondrial DNA from HeLa cells. Biochem Biophys Res Commun. 1973 Jul 2;53(1):140–148. doi: 10.1016/0006-291x(73)91412-5. [DOI] [PubMed] [Google Scholar]

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