Skip to main content
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1968 Nov 1;39(2):264–285. doi: 10.1083/jcb.39.2.264

CYTOPLASMIC LABEL FOLLOWING TRITIATED THYMIDINE TREATMENT OF ALLIUM CEPA L. ROOTS

Cytochemical and Electron Microscope Study

Catharine P Fussell 1
PMCID: PMC2107535  PMID: 5677966

Abstract

Tritiated thymidine routinely labels onion root cytoplasm during most of the cell cycle. One-third of this label could be cytochemically identified as DNA. The balance of the label was not RNA or a lipid, or attributable to labeled impurities in thymidine-3H. In electron microscope radioautographs one-third of the cytoplasmic silver grains was over organelles, presumably mitochondria and plastids. The other two-thirds of the silver grains in electron micrographs was distributed widely, 41% over ground cytoplasm and 10% over cell walls-cell membranes. Snake venom phosphodiesterase (SVDase) extracted a cytoplasmic fraction not degraded by DNase, and did not appear to extract nuclear DNA. The SVDase-extractable fraction may be DNA or a thymidine 5'-phosphoryl group in an ester linkage with another hydroxylic compound. The nature of the nonextractable fraction is considered. Possibilities discussed are: (1) technical problems such as the binding of an acid-labile nuclear DNA in the cytoplasm; (2) non-DNA, such as breakdown products, and thymine compounds other than DNA; (3) DNA, not extractable because of the nature of its binding to other compounds or because it is a "core" resistant to DNase. Until the chemical nature of this nonextractable fraction is known, cytoplasmic label following thymidine-3H treatment cannot necessarily be considered DNA, nor the assumption made that thymidine-3H exclusively labels DNA.

Full Text

The Full Text of this article is available as a PDF (2.2 MB).

Selected References

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

  1. APPLEBY C. A., MORTON R. K. Lactic dehydrogenase and cytochrome b2 of baker's yeast. The deoxyribose polynucleotide component and the physicochemical properties of the crystalline enzyme. Biochem J. 1960 May;75:258–269. doi: 10.1042/bj0750258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. CABIB E. CARBOHYDRATE METABOLISM. Annu Rev Biochem. 1963;32:321–354. doi: 10.1146/annurev.bi.32.070163.001541. [DOI] [PubMed] [Google Scholar]
  3. CARO L. G., VAN TUBERGEN R. P., KOLB J. A. High-resolution autoradiography. I. Methods. J Cell Biol. 1962 Nov;15:173–188. doi: 10.1083/jcb.15.2.173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. CHIBA Y., SUGAHARA K. The nucleic acid content of chloroplasts isolated from spinach and tobacco leaves. Arch Biochem Biophys. 1957 Oct;71(2):367–376. doi: 10.1016/0003-9861(57)90047-4. [DOI] [PubMed] [Google Scholar]
  5. CRONKITE E. P., BOND V. P., FLIEDNER T. M., RUBINI J. R. The use of tritiated thymidine in the study of DNS synthesis and cell turnover in hemopoietic tissues. Lab Invest. 1959 Jan-Feb;8(1):263–277. [PubMed] [Google Scholar]
  6. DAOUST R. IN VITRO BINDING OF NUCLEIC ACIDS TO TISSUE SECTIONS AFTER REMOVAL OF TISSUE NUCLEIC ACIDS. J Histochem Cytochem. 1964 Aug;12:640–645. doi: 10.1177/12.8.640. [DOI] [PubMed] [Google Scholar]
  7. Edelman M., Cowan C. A., Epstein H. T., Schiff J. A. STUDIES OF CHLOROPLAST DEVELOPMENT IN EUGLENA, VIII. CHLOROPLAST-ASSOCIATED DNA. Proc Natl Acad Sci U S A. 1964 Nov;52(5):1214–1219. doi: 10.1073/pnas.52.5.1214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. FRAENKEL-CONRAT H., SNELL N. S., DUCAY E. D. Avidin. I. Isolation and characterization of the protein and nucleic acid. Arch Biochem Biophys. 1952 Jul;39(1):80–96. doi: 10.1016/0003-9861(52)90263-4. [DOI] [PubMed] [Google Scholar]
  9. Fussell C. P. Methyl green-pyronin as a stain for autoradiographs of plant material. Stain Technol. 1966 Nov;41(6):315–316. doi: 10.3109/10520296609116331. [DOI] [PubMed] [Google Scholar]
  10. GIBOR A., GRANICK S. PLASTIDS AND MITOCHONDRIA: INHERITABLE SYSTEMS. Science. 1964 Aug 14;145(3633):890–897. doi: 10.1126/science.145.3635.890. [DOI] [PubMed] [Google Scholar]
  11. GIBOR A., IZAWA M. THE DNA CONTENT OF THE CHLOROPLASTS OF ACETABULARIA. Proc Natl Acad Sci U S A. 1963 Dec;50:1164–1169. doi: 10.1073/pnas.50.6.1164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. GINSBURG V. SUGAR NUCLEOTIDES AND THE SYNTHESIS OF CARBOHYDRATES. Adv Enzymol Relat Areas Mol Biol. 1964;26:35–88. doi: 10.1002/9780470122716.ch2. [DOI] [PubMed] [Google Scholar]
  13. GUTTES E., GUTTES S. THYMIDINE INCORPORATION BY MITOCHONDRIA IN PHYSARUM POLYCEPHALUM. Science. 1964 Sep 4;145(3636):1057–1058. doi: 10.1126/science.145.3636.1057. [DOI] [PubMed] [Google Scholar]
  14. HOLLEY R. W., APGAR J., EVERETT G. A., MADISON J. T., MARQUISEE M., MERRILL S. H., PENSWICK J. R., ZAMIR A. STRUCTURE OF A RIBONUCLEIC ACID. Science. 1965 Mar 19;147(3664):1462–1465. doi: 10.1126/science.147.3664.1462. [DOI] [PubMed] [Google Scholar]
  15. Hughes W. L., Bond V. P., Brecher G., Cronkite E. P., Painter R. B., Quastler H., Sherman F. G. CELLULAR PROLIFERATION IN THE MOUSE AS REVEALED BY AUTORADIOGRAPHY WITH TRITIATED THYMIDINE. Proc Natl Acad Sci U S A. 1958 May;44(5):476–483. doi: 10.1073/pnas.44.5.476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jagendorf A. T., Wildman S. G. The Proteins of Green Leaves. VI. Centrifugal Fractionation of Tobacco Leaf Homogenates and Some Properties of Isolated Chloroplasts. Plant Physiol. 1954 May;29(3):270–279. doi: 10.1104/pp.29.3.270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. KAUFMANN B. P., GAY H., McDONALD M. R. Localization of cellular proteins by enzymatic hydrolysis. Cold Spring Harb Symp Quant Biol. 1950;14:85–91. doi: 10.1101/sqb.1950.014.01.012. [DOI] [PubMed] [Google Scholar]
  18. LITTAU V. C. Cytological evidence that both RNA and DNA may form a complex with the same protein. J Biophys Biochem Cytol. 1959 Mar 25;5(2):231–234. doi: 10.1083/jcb.5.2.231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. LUCK D. J., REICH E. DNA IN MITOCHONDRIA OF NEUROSPORA CRASSA. Proc Natl Acad Sci U S A. 1964 Oct;52:931–938. doi: 10.1073/pnas.52.4.931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. LUFT J. H. Improvements in epoxy resin embedding methods. J Biophys Biochem Cytol. 1961 Feb;9:409–414. doi: 10.1083/jcb.9.2.409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. LUFT J. H. Permanganate; a new fixative for electron microscopy. J Biophys Biochem Cytol. 1956 Nov 25;2(6):799–802. doi: 10.1083/jcb.2.6.799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. MANTSAVINOS R., ZAMENHOF S. Pathways for the biosynthesis of thymidylic acid in bacterial mutants. J Biol Chem. 1961 Mar;236:876–882. [PubMed] [Google Scholar]
  23. MARTIN E. M., MORTON R. K. The chemical composition of microsomes and mitochondria from silver beet. Biochem J. 1956 Oct;64(2):221–235. doi: 10.1042/bj0640221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Muckenthaler F. A., Mahowald A. P. DNA synthesis in the ooplasm of Drosophila melanogaster. J Cell Biol. 1966 Feb;28(2):199–208. doi: 10.1083/jcb.28.2.199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. NASS S., NASS M. M., HENNIX U. DEOXYRIBONUCLEIC ACID IN ISOLATED RAT-LIVER MITOCHONDRIA. Biochim Biophys Acta. 1965 Mar 15;95:426–435. doi: 10.1016/0005-2787(65)90189-9. [DOI] [PubMed] [Google Scholar]
  26. NECAS O. Nuclear structure of growing naked protoplasts of yearts. Nature. 1959 Nov 21;184(Suppl 21):1664–1665. doi: 10.1038/1841664b0. [DOI] [PubMed] [Google Scholar]
  27. PAZUR J. H., ANDERSON J. S. ENZYMIC TRANSFER OF RHAMNOSYL UNITS FROM THYMIDINE DIPHOSPHATE RHAMNOSE TO BACTERIAL CELL-WALL FRAGMENTS. Biochim Biophys Acta. 1963 Sep 10;74:788–790. doi: 10.1016/0006-3002(63)91435-5. [DOI] [PubMed] [Google Scholar]
  28. PLAUT W., SAGAN L. A. Incorporation of thymidine in the cytoplasm of Amoeba proteus. J Biophys Biochem Cytol. 1958 Nov 25;4(6):843–846. doi: 10.1083/jcb.4.6.843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. POLLISTER A. W. AN AUTORADIOGRAPHIC STUDY OF RNA SYNTHESIS IN ISOLATED SALIVARY GLANDS OF DROSOPHILA HYDEI. II. INTERFEROMETRIC STUDIES. J Morphol. 1965 Jan;116:89–98. doi: 10.1002/jmor.1051160106. [DOI] [PubMed] [Google Scholar]
  30. PORTER K. R., MACHADO R. D. Studies on the endoplasmic reticulum. IV. Its form and distribution during mitosis in cells of onion root tip. J Biophys Biochem Cytol. 1960 Feb;7:167–180. doi: 10.1083/jcb.7.1.167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. PRIVAT DE GARILHE M., LASKOWSKI M. Studies of the phosphodiesterase from rattlesnake venom. Biochim Biophys Acta. 1955 Nov;18(3):370–378. doi: 10.1016/0006-3002(55)90100-1. [DOI] [PubMed] [Google Scholar]
  32. RABINOVITCH M., PLAUT W. Cytoplasmic DNA synthesis in Amoeba proteus. I. On the particulate nature of the DNA-containing elements. J Cell Biol. 1962 Dec;15:525–534. doi: 10.1083/jcb.15.3.525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. RICHTER G., SENGER H. ISOLATION OF A DNA-RNA COMPLEX FROM CHLORELLA CELLS. Biochim Biophys Acta. 1965 Feb 8;95:362–364. doi: 10.1016/0005-2787(65)90504-6. [DOI] [PubMed] [Google Scholar]
  34. RIS H., PLAUT W. Ultrastructure of DNA-containing areas in the chloroplast of Chlamydomonas. J Cell Biol. 1962 Jun;13:383–391. doi: 10.1083/jcb.13.3.383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rabinowitz M., Sinclair J., DeSalle L., Haselkorn R., Swift H. H. Isolation of deoxyribonucleic acid from mitochondria of chick embryo heart and liver. Proc Natl Acad Sci U S A. 1965 May;53(5):1126–1133. doi: 10.1073/pnas.53.5.1126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. SAGER R., ISHIDA M. R. CHLOROPLAST DNA IN CHLAMYDOMONAS. Proc Natl Acad Sci U S A. 1963 Oct;50:725–730. doi: 10.1073/pnas.50.4.725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. SCHERBAUM O. H. Possible sites of metabolic control during the induction of synchronous cell division. Ann N Y Acad Sci. 1960 Oct 7;90:565–579. doi: 10.1111/j.1749-6632.1960.tb23273.x. [DOI] [PubMed] [Google Scholar]
  38. STONE G. E., PRESCOTT D. M. CELL DIVISION AND DNA SYNTHESIS IN TETRAHYMENA PYRIFORMIS DEPRIVED OF ESSENTIAL AMINO ACIDS. J Cell Biol. 1964 May;21:275–281. doi: 10.1083/jcb.21.2.275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Steffensen D. M., Sheridan W. F. Incorporation of H3-thymidine into chloroplast DNA of marine algae. J Cell Biol. 1965 Jun;25(3):619–626. doi: 10.1083/jcb.25.3.619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Suyama Y., Bonner W. D. DNA from plant mitochondria. Plant Physiol. 1966 Mar;41(3):383–388. doi: 10.1104/pp.41.3.383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. TAYLOR J. H. Autoradiography with tritium-labeled substances. Adv Biol Med Phys. 1960;7:107–130. doi: 10.1016/b978-1-4832-3113-6.50006-3. [DOI] [PubMed] [Google Scholar]
  42. TAYLOR J. H., McMASTER R. D. Autoradiographic and microphotometric studies of desoxyribose nucleic acid during microgametogenesis in Lilium longiflorum. Chromosoma. 1954;6(6-7):489–521. doi: 10.1007/BF01259951. [DOI] [PubMed] [Google Scholar]
  43. Taylor J. H., Woods P. S., Hughes W. L. THE ORGANIZATION AND DUPLICATION OF CHROMOSOMES AS REVEALED BY AUTORADIOGRAPHIC STUDIES USING TRITIUM-LABELED THYMIDINEE. Proc Natl Acad Sci U S A. 1957 Jan 15;43(1):122–128. doi: 10.1073/pnas.43.1.122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. WOLSTENHOLME D. R., PLAUT W. CYTOPLASMIC DNA SYNTHESIS IN AMOEBA PROTEUS. 3. FURTHER STUDIES ON THE NATURE OF THE DNA--CONTAINING ELEMENTS. J Cell Biol. 1964 Sep;22:505–513. doi: 10.1083/jcb.22.3.505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. WOODARD J., RASCH E., SWIFT H. Nucleic acid and protein metabolism during the mitotic cycle in Vicia faba. J Biophys Biochem Cytol. 1961 Feb;9:445–462. doi: 10.1083/jcb.9.2.445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. WOODS P. S. A chromatographic study of hydrolysis in the Feulgen nucleal reaction. J Biophys Biochem Cytol. 1957 Jan 25;3(1):71–88. doi: 10.1083/jcb.3.1.71. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES