Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1969 Aug;63(4):1282–1289. doi: 10.1073/pnas.63.4.1282

PARTIAL PURIFICATION OF NATIVE RRNA AND TRNA CISTRONS FROM Mycoplasma SP. (KID)*

John L Ryan 1, Harold J Morowitz 1
PMCID: PMC223462  PMID: 5260931

Abstract

Precise optical melting profiles of purified DNA from Mycoplasma sp. (Kid) show a secondary hyperchromic rise, corresponding to 1.4 per cent of the total DNA, occurring at 88°C, while the bulk of the DNA melts at 79.5°C, indicating an average base composition of 24.9 per cent guanine-cytosine (G-C). A method is presented, using sonication followed by hydroxyapatite column chromatography, for the partial purification of regions of a genome which contain significantly higher G-C than the average value for the genome. The procedure does not involve denaturation and renaturation of the high G-C material so that purified DNA is in its native, double-stranded state and has a normal melting profile. When applied to Mycoplasma sp. (Kid), the method yielded a fraction of native DNA enriched 40 times with respect to those regions coding for rRNA and tRNA. This enriched DNA has a saturation hybridization value of 15.9 per cent with Kid rRNA plus tRNA. The saturation hybridization values of the bulk DNA with rRNA and tRNA are 0.26 per cent and 0.16 per cent, respectively. Based on a genome size of 6.84 × 108, obtained by electron microscopy, this indicates that Mycoplasma sp. (Kid) contains only enough ribosomal DNA to code for one set of 23S plus 16S rRNA and only enough DNA complementary to tRNA to code for 44 different tRNA molecules.

Full text

PDF
1282

Selected References

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

  1. Bode H. R., Morowitz H. J. Size and structure of the Mycoplasma hominis H39 chromosome. J Mol Biol. 1967 Jan 28;23(2):191–199. doi: 10.1016/s0022-2836(67)80026-3. [DOI] [PubMed] [Google Scholar]
  2. GEIDUSCHEK E. P., TOCCHINI-VALENTINI G. P., SARNAT M. T. ASYMMETRIC SYNTHESIS OF RNA IN VITRO: DEPENDENCE OF DNA CONTINUITY AND CONFORMATION. Proc Natl Acad Sci U S A. 1964 Aug;52:486–493. doi: 10.1073/pnas.52.2.486. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Gillespie D., Spiegelman S. A quantitative assay for DNA-RNA hybrids with DNA immobilized on a membrane. J Mol Biol. 1965 Jul;12(3):829–842. doi: 10.1016/s0022-2836(65)80331-x. [DOI] [PubMed] [Google Scholar]
  4. HAYASHI M., HAYASHI M. N., SPIEGELMAN S. RESTRICTION OF IN VIVO GENETIC TRANSCRIPTION TO ONE OF THE COMPLEMENTARY STRANDS OF DNA. Proc Natl Acad Sci U S A. 1963 Oct;50:664–672. doi: 10.1073/pnas.50.4.664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Kirk R. G., Morowitz H. J. Ribonucleic acids of Mycoplasma gallisepticum strain A5969. Am J Vet Res. 1969 Feb;30(2):287–293. [PubMed] [Google Scholar]
  6. Kohne D. E. Isolation and characterization of bacterial ribosomal RNA cistrons. Biophys J. 1968 Oct;8(10):1104–1118. doi: 10.1016/S0006-3495(68)86542-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kumar S., Bovre K., Guha A., Hradecna Z., Maher V. R., Szybalski W. Orientation and control of transcription in E. coli phage lambda. Nature. 1969 Mar 1;221(5183):823–825. doi: 10.1038/221823a0. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. MIURA K. I. The nucleotide composition of ribonucleic acids of soluble and particle fractions in several species of bacteria. Biochim Biophys Acta. 1962 Jan 22;55:62–70. doi: 10.1016/0006-3002(62)90931-9. [DOI] [PubMed] [Google Scholar]
  10. MIYAZAWA Y., THOMAS C. A., Jr NUCLEOTIDE COMPOSITION OF SHORT SEGMENTS OF DNA MOLECULES. J Mol Biol. 1965 Feb;11:223–237. doi: 10.1016/s0022-2836(65)80053-5. [DOI] [PubMed] [Google Scholar]
  11. McGee Z. A., Rogul M., Wittler R. G. Molecular genetic studies of relationships among mycoplasma, L-forms and bacteria. Ann N Y Acad Sci. 1967 Jul 28;143(1):21–30. doi: 10.1111/j.1749-6632.1967.tb27639.x. [DOI] [PubMed] [Google Scholar]
  12. Oishi M. The transcribing strands of bacillus subtilis DNA for ribosomal and transfer RNA. Proc Natl Acad Sci U S A. 1969 Jan;62(1):256–262. doi: 10.1073/pnas.62.1.256. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Warnaar S. O., Mulder G., Sigtenhorst-van der Slu, Cohen J. A. The transcription in vitro of various forms of phiX174 DNA. Biochim Biophys Acta. 1969 Jan 21;174(1):239–245. doi: 10.1016/0005-2787(69)90247-0. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES