1887

Abstract

Methods are described that allow extraction of high molecular weight DNA from germinated conidia of By labelling DNA with ribonucleosides, early conidia were shown to be active in DNA synthesis. These cells when treated with the enzyme Zymolyase became fragile and could be readily lysed with ionic detergents to release high molecular weight DNA.

The DNA extracted from Zymolyase treated cells on to alkaline sucrose gradients sedimented as a heterogeneous species of up to 150 ×10 molecular weight. A minor DNA species (presumably mitochondrial) of 20×10 molecular weight comprised 2-7% of the total. The identity of the DNA was confirmed by sensitivity to DNAase, the diphenylamine assay and TLC. Sedimentation patterns were unaffected by protease digestions and no anomalous high speed rotor effects were evident. Isopycnic gradients suggested that the DNA released was uncomplexed with either protein or carbohydrates. Sepharose chromatography of extracted, RNAase-treated Zymolyase lysate resulted in clearly separate high molecular weight DNA and RNA-protein elution profiles.

UV light preferentially inhibited nuclear DNA synthesis and drastically reduced the size and amount of nascent DNA being synthesized in the excision defective mutant. Sites in parental DNA sensitive to UV endonuclease were measured in cells made permeable with Triton X-100.

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1983-02-01
2024-12-10
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References

  1. Agsteribbe E. 1979; Protoplasts from Neurospora crassa. . Neurospora Newsletter 26:19–20
    [Google Scholar]
  2. Boyd J. B., Setlow R. B. 1976; Characterization of postreplication repair in mutagen-sensitive strains of Drosophila melanogaster. . Genetics 84:507–526
    [Google Scholar]
  3. Carrier W. L., Setlow R. B. 1971; The excision of pyrimidine dimers (the detection of dimers in small amounts). Methods in Enzymology 21:230–237
    [Google Scholar]
  4. Cryer D. R., Eccleshall R., Marmur J. 1975; Isolation of yeast DNA. Methods in Cellular Biology 12:39–44
    [Google Scholar]
  5. Di Caprio L., Cox B. 1981; DNA synthesis in UV- irradiated yeast. Mutation Research 82:69–85
    [Google Scholar]
  6. Dingman C. W., Kakunaga T. 1976; DNA strand breaking and rejoining in response to ultraviolet light in normal human and xeroderma pigmentosum cells. International Journal of Radiation Biology 30:55–66
    [Google Scholar]
  7. Dutta S. K., Richman N., Woodward V. W., Mandel M. 1967; Relatedness among species of fungi and higher plants measured by DNA hybridization and base ratios. Genetics 57:719–727
    [Google Scholar]
  8. Elkind M. M., Kamper C. 1970; Two forms of repair of DNA in mammalian cells following irradiation. Biophysical Journal 10:237–245
    [Google Scholar]
  9. Emerson S. 1963; A plasmodiploid variant of Neuro- spora crassa. . Genetics 34:162–182
    [Google Scholar]
  10. Farkas V. 1979; Biosynthesis of cell walls of fungi. Microbiological Reviews 43:117–144
    [Google Scholar]
  11. Ganesan A. K. 1974; Persistance of pyrimidine dimers during postreplication repair in excision-deficient derivatives of Escherichia coli K-12 after ultraviolet irradiation. Journal of Molecular Biology 87:103–119
    [Google Scholar]
  12. Grivell A. R., Jackson J. F. 1968; Thymidine kinase: evidence for its absence from Neurospora crassa and some other micro-organisms, and the relevance of this to the specific labelling of DNA. Journal of General Microbiology 54:307–317
    [Google Scholar]
  13. Hatayama T., Goldberg I. H. 1979; DNA damage and repair in relation to cell killing in neoxarzinostatin-treated HeLa cells. Biochimica et biophysica acta 565:59–71
    [Google Scholar]
  14. Hill E. B., Wayne L. G., Gross W. M. 1972; Purification of mycobacterial deoxyribonucleic acid. Journal of Bacteriology 112:1033–1039
    [Google Scholar]
  15. Horowitz N. H., Macleod H. 1960; The DNA content of Neurospora nuclei. Microbial Genetics Bulletin 17:6
    [Google Scholar]
  16. Kielman J. K., Deering R. A. 1980; Ultraviolet light-induced inhibition of cell division and DNA synthesis in axenically grown repair mutants of Dictyostelium discoideum. . Photochemistry and Photobiology 32:149–156
    [Google Scholar]
  17. Luck D. J. L., Reich E. 1964; DNA in mitochondria of Neurospora crassa. . Proceedings of the National Academy of Sciences of the United States of America 64931–938
    [Google Scholar]
  18. Lurquin P., Mergeay M., Van Der Parren J. 1972; Banding of depolymerized DNA’s in CsCl density gradients studied by computer-aided simulations. In Uptake of Informative Molecules by Living Cells pp. 47–50 Edited by Ledoux L. Amsterdam: North-Holland Publishing Company;
    [Google Scholar]
  19. Lurquin P. F., Tshitenge G., Delaunoit G., Ledoux L. 1975; Isolation of DNA from plant cells by gel filtration on agarose. Analytical Biochemistry 65:1–10
    [Google Scholar]
  20. Mangold H. K. 1969; Nucleic acids and nucleotides. In Thin Layer Chromatography pp. 787–807 Edited by Stahl E. New York: Springer-Verlag;
    [Google Scholar]
  21. Martin R. G., Ames B. N. 1961; A method for determining the sedimentation behavior of enzymes: application to protein mixtures. Journal of Biological Chemistry 236:1372–1379
    [Google Scholar]
  22. Minagawa T., Wagner B., Strauss B. 1959; The nucleic acid content of Neurospora crassa. . Archives of Biochemistry and Biophysics 80:442–445
    [Google Scholar]
  23. Price P. A. 1975; The essential role of Ca2+ in the activity of bovine pancreatic deoxyribonuclease. Journal of Biological Chemistry 250:1981–1985
    [Google Scholar]
  24. Regan J. D., Setlow R. B. 1974; Two forms of repair in the DNA of human cells damaged by chemical carcinogens and mutagens. Cancer Research 34:3318–3325
    [Google Scholar]
  25. Resnick M. A., Boyce J., Cox B. 1981; Postreplication repair in Saccharomyces cerevisiae. . Journal of Bacteriology 146:285–290
    [Google Scholar]
  26. Reynolds R. J. 1978; Removal of pyrimidine dimers from Saccharomyces cerevisiae nuclear DNA under nongrowth conditions as detected by a sensitive, enzymatic assay. Mutation Research 50:43–56
    [Google Scholar]
  27. Richards G. M. 1974; Modification of the diphenyl- amine reaction giving increased sensitivity and simplicity in the estimation of DNA. Analytical Biochemistry 57:369–376
    [Google Scholar]
  28. Rupp W. D., Howard-Flanders P. 1968; Discontinuities in the DNA synthesized in an excision-defective strain of Escherichia coli following ultraviolet irradiation. Journal of Molecular Biology 31:291–304
    [Google Scholar]
  29. Sancar A., Hack A. M., Rupp W. D. 1979; Simple method for identification of plasmid-coded proteins. Journal of Bacteriology 137:692–693
    [Google Scholar]
  30. Schildkraut C. L., Marmur J., Doty P. 1962; Determination of the base composition of deoxyribonucleic acid from its buoyant density in CsCl. Journal of Molecular Biology 4:430–443
    [Google Scholar]
  31. Schroeder A. L. 1970a; Ultraviolet-sensitive mutants of Neurospora. I. Genetic basis and effect on recombination. Molecular and General Genetics 107:291–304
    [Google Scholar]
  32. Schroeder A. L. 1970b; Ultraviolet-sensitive mutants of Neurospora. II. Radiation studies. Molecular and General Genetics 107:305–320
    [Google Scholar]
  33. Schroeder A. L. 1975; Genetic control of radiation sensitivity and DNA repair in Neurospora. . In Molecular Mechanisms for the Repair of DNA pp. 567–576 Edited by Hanawalt P., Setlow R. New York: Plenum Publishing Corporation;
    [Google Scholar]
  34. Stadler D. R., Smith D. A. 1968; A new mutation in Neurospora for sensitivity to ultraviolet. Canadian Journal of Genetics and Cytology 10:916–919
    [Google Scholar]
  35. Studier F. W. 1965; Sedimentation studies of the size and shape of DNA. Journal of Molecular Biology 11:373–390
    [Google Scholar]
  36. Terpstra P., Holtrop M., Kroon A. M. 1976; Restriction fragment map of Neurospora crassa mito-chondrial DNA. In The Genetic Function of Mitochondrial DNA pp. 111–118 Edited by Saccone A., Kroon A. M. New York: Prentiss Hall;
    [Google Scholar]
  37. Villa V. D., Storck R. 1968; Nucleotide composition of nuclear and mitochondrial deoxyribonucleic acid of fungi. Journal of Bacteriology 96:741–746
    [Google Scholar]
  38. Vogel H. J. 1964; Distribution of lysine pathways among fungi: evolutionary implications. American Naturalist 98:435–446
    [Google Scholar]
  39. Wilkin R. J. 1973; DNA repair: a simple enzymatic assay for human cells. International Journal of Radiation Biology 24:609–613
    [Google Scholar]
  40. Worthy T. E., Epler J. L. 1972; Repair of ultraviolet light-induced damage to the deoxyribonucleic acid of Neurospora crassa. . Journal of Bacteriology 110:1010–1016
    [Google Scholar]
  41. Zeeland A. A., Smith C. A., Hanawalt P. C. 1981; Sensitive determination of pyrimidine dimers in DNA of UV-irradiated mammalian cells. Mutation Research 82:173–189
    [Google Scholar]
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