Skip to main content
PMC home page
The Journal of General Physiology logoLink to The Journal of General Physiology
. 1966 Jul 1;49(6):233–258. doi: 10.1085/jgp.49.6.233

Integration of Deoxyribonuclease-Treated DNA in Bacillus subtilis Transformation

Walter F Bodmer 1
PMCID: PMC2195534  PMID: 4961186

Abstract

Normal preparations of B. subtilis DNA have weight average native molecular weights of 10 to 30 x 106. For any given preparation the upper and lower 95% size limits may differ by a factor of ten or more. Single-stranded molecular weights indicate an average of 1 to 4 breaks per single strand of the native DNA. The reduction in transforming activity and viscosity following DNAase I digestion can be accounted for by a direct relationship between the transforming activity of a DNA and its single-stranded molecular weight. Uptake studies with DNAase I treated heavy (2H15N 3H) DNA show that single strand breaks inhibit integration less than transformation. A provisional estimate of the size of the integrated region based on correlating the single strand size of the donor-recipient complex with the donor-recipient density differences following alkali denaturation came to 1530 nucleotides. Using a competent, nonleaky thymine-requiring strain of B. subtilis grown in 5-BU medium before and after transformation, it was shown that (a) No detectable amount of DNA synthesis is necessary for the initial stages of integration, (b) Cells which have recently been replicating DNA are not competent. (c) Cells containing donor DNA show a lag in DNA replication following transformation, (d) When donor DNA is replicated it initially appears in a density region between light and hybrid. This indicates that it includes the transition point formed at the time of reinitiation of DNA synthesis in the presence of 5-BU following transformation. A model is proposed in which donor DNA is integrated at the stationary growing point of the competent cell, which is in a state of suspended DNA synthesis.

Full Text

The Full Text of this article is available as a PDF (1.5 MB).

Selected References

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

  1. BODMER W., SCHILDKRAUT C. PREPARATION AND CHARACTERIZATION OF 15N2H3H-LABELED DNA FROM BACILLUS SUBTILIS AND ESCHERICHIA COLI PHAGE T2. Anal Biochem. 1964 Jun;8:229–243. doi: 10.1016/0003-2697(64)90051-x. [DOI] [PubMed] [Google Scholar]
  2. BURGI E., HERSHEY A. D. Sedimentation rate as a measure of molecular weight of DNA. Biophys J. 1963 Jul;3:309–321. doi: 10.1016/s0006-3495(63)86823-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bodmer W. F. Recombination and integration in Bacillus subtilis transformation: involvement of DNA synthesis. J Mol Biol. 1965 Dec;14(2):534–557. doi: 10.1016/s0022-2836(65)80203-0. [DOI] [PubMed] [Google Scholar]
  4. CLARK A. J., MARGULIES A. D. ISOLATION AND CHARACTERIZATION OF RECOMBINATION-DEFICIENT MUTANTS OF ESCHERICHIA COLI K12. Proc Natl Acad Sci U S A. 1965 Feb;53:451–459. doi: 10.1073/pnas.53.2.451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. FARMER J. L., ROTHMAN F. TRANSFORMABLE THYMINE-REQUIRING MUTANT OF BACILLUS SUBTILS. J Bacteriol. 1965 Jan;89:262–263. doi: 10.1128/jb.89.1.262-263.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. FOX M. S., ALLEN M. K. ON THE MECHANISM OF DEOXYRIBONUCLEATE INTEGRATION IN PNEUMOCOCCAL TRANSFORMATION. Proc Natl Acad Sci U S A. 1964 Aug;52:412–419. doi: 10.1073/pnas.52.2.412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. FOX M. S., HOTCHKISS R. D. Fate of transforming deoxyribonucleate following fixation by transformable bacteria. Nature. 1960 Sep 17;187:1002–1006. doi: 10.1038/1871002a0. [DOI] [PubMed] [Google Scholar]
  8. GANESAN A. T., LEDERBERG J. PHYSICAL AND BIOLOGICAL STUDIES ON TRANSFORMING DNA. J Mol Biol. 1964 Sep;9:683–695. doi: 10.1016/s0022-2836(64)80175-3. [DOI] [PubMed] [Google Scholar]
  9. HANAWALT P. C., RAY D. S. ISOLATION OF THE GROWING POINT IN THE BACTERIAL CHROMOSOME. Proc Natl Acad Sci U S A. 1964 Jul;52:125–132. doi: 10.1073/pnas.52.1.125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. KAISER A. D., HOGNESS D. S. The transformation of Escherichia coli with deoxyribonucleic acid isolated from bacteriophage lambda-dg. J Mol Biol. 1960 Dec;2:392–415. doi: 10.1016/s0022-2836(60)80050-2. [DOI] [PubMed] [Google Scholar]
  11. KENT J. L., ROGER M., HOTCHKISS R. D. ON THE ROLE OF INTEGRITY OF DNA PARTICLES IN GENETIC RECOMBINATION DURING PNEUMOCOCCAL TRANSFORMATION. Proc Natl Acad Sci U S A. 1963 Oct;50:717–725. doi: 10.1073/pnas.50.4.717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. LEHMAN I. R., RICHARDSON C. C. THE DEOXYRIBONUCLEASES OF ESCHERICHIA COLI. IV. AN EXONUCLEASE ACTIVITY PRESENT IN PURIFIED PREPARATIONS OF DEOXYRIBONUCLEIC ACID POLYMERASE. J Biol Chem. 1964 Jan;239:233–241. [PubMed] [Google Scholar]
  13. LERMAN L. S., TOLMACH L. J. Genetic transformation. II. The significance of damage to the DNA molecule. Biochim Biophys Acta. 1959 Jun;33(2):371–387. doi: 10.1016/0006-3002(59)90127-1. [DOI] [PubMed] [Google Scholar]
  14. MANDELL J. D., HERSHEY A. D. A fractionating column for analysis of nucleic acids. Anal Biochem. 1960 Jun;1:66–77. doi: 10.1016/0003-2697(60)90020-8. [DOI] [PubMed] [Google Scholar]
  15. MARMUR J., DOTY P. Thermal renaturation of deoxyribonucleic acids. J Mol Biol. 1961 Oct;3:585–594. doi: 10.1016/s0022-2836(61)80023-5. [DOI] [PubMed] [Google Scholar]
  16. MESELSON M. ON THE MECHANISM OF GENETIC RECOMBINATION BETWEEN DNA MOLECULES. J Mol Biol. 1964 Sep;9:734–745. doi: 10.1016/s0022-2836(64)80178-9. [DOI] [PubMed] [Google Scholar]
  17. NESTER E. W. PENICILLIN RESISTANCE OF COMPETENT CELLS IN DEOXYRIBONUCLEIC ACID TRANSFORMATION OF BACILLUS SUBTILIS. J Bacteriol. 1964 Apr;87:867–875. doi: 10.1128/jb.87.4.867-875.1964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. NESTER E. W., STOCKER B. A. BIOSYNTHETIC LATENCY IN EARLY STAGES OF DEOXYRIBONUCLEIC ACIDTRANSFORMATION IN BACILLUS SUBTILIS. J Bacteriol. 1963 Oct;86:785–796. doi: 10.1128/jb.86.4.785-796.1963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Nester E W, Schafer M, Lederberg J. Gene Linkage in DNA Transfer: A Cluster of Genes Concerned with Aromatic Biosynthesis in Bacillus Subtilis. Genetics. 1963 Apr;48(4):529–551. doi: 10.1093/genetics/48.4.529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. OISHI M., YOSHIKAWA H., SUEOKA N. SYNCHRONOUS AND DICHOTOMOUS REPLICATIONS OF THE BACILLUS SUBTILIS CHROMOSOME DURING SPORE GERMINATION. Nature. 1964 Dec 12;204:1069–1073. doi: 10.1038/2041069a0. [DOI] [PubMed] [Google Scholar]
  21. OTH A., FREDERICQ E., HACHA R. Enzymic degradation of deoxyribonucleic acid. II. Enzymic properties of thymus acid deoxyribonuclease. Biochim Biophys Acta. 1958 Aug;29(2):287–296. doi: 10.1016/0006-3002(58)90187-2. [DOI] [PubMed] [Google Scholar]
  22. PENE J. J., ROMIG W. R. ON THE MECHANISM OF GENETIC RECOMBINATION IN TRANSFORMING BACILLUS SUBTILIS. J Mol Biol. 1964 Jul;9:236–245. doi: 10.1016/s0022-2836(64)80103-0. [DOI] [PubMed] [Google Scholar]
  23. PETTIJOHN D., HANAWALT P. EVIDENCE FOR REPAIR-REPLICATION OF ULTRAVIOLET DAMAGED DNA IN BACTERIA. J Mol Biol. 1964 Aug;9:395–410. doi: 10.1016/s0022-2836(64)80216-3. [DOI] [PubMed] [Google Scholar]
  24. PRITCHARD R. H., LARK K. G. INDUCTION OF REPLICATION BY THYMINE STARVATION AT THE CHROMOSOME ORIGIN IN ESCHERICHIA COLI. J Mol Biol. 1964 Aug;9:288–307. doi: 10.1016/s0022-2836(64)80208-4. [DOI] [PubMed] [Google Scholar]
  25. RICHARDSON C. C., INMAN R. B., KORNBERG A. ENZYMIC SYNTHESIS OF DEOXYRIBONUCLEIC ACID. 18. THE REPAIR OF PARTIALLY SINGLE-STRANDED DNA TEMPLATES BY DNA POLYMERASE. J Mol Biol. 1964 Jul;9:46–69. doi: 10.1016/s0022-2836(64)80090-5. [DOI] [PubMed] [Google Scholar]
  26. RYTER A., LANDMAN O. E. ELECTRON MICROSCOPE STUDY OF THE RELATIONSHIP BETWEEN MESOSOME LOSS AND THE STABLE L STATE (OR PROTOPLAST STATE) IN BACILLUS SUBTILIS. J Bacteriol. 1964 Aug;88:457–467. doi: 10.1128/jb.88.2.457-467.1964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. STRAUSS B. S. Recovery of deoxyribonucleic acid from the effects of alkylation. J Gen Microbiol. 1963 Jan;30:89–103. doi: 10.1099/00221287-30-1-89. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. WHITEHOUSE H. L. A THEORY OF CROSSING-OVER BY MEANS OF HYBRID DEOXYRIBONUCLEIC ACID. Nature. 1963 Sep 14;199:1034–1040. doi: 10.1038/1991034a0. [DOI] [PubMed] [Google Scholar]
  30. YOUNG E. T., 2nd, SINSHEIMER R. L. A COMPARISON OF THE INITIAL ACTIONS OF SPLEEN DEOXYRIBONUCLEASE AND PANCREATIC DEOXYRIBONUCLEASE. J Biol Chem. 1965 Mar;240:1274–1280. [PubMed] [Google Scholar]
  31. ZAMENHOF S., ALEXANDER H. E., LEIDY G. Studies on the chemistry of the transforming activity. I. Resistance to physical and chemical agents. J Exp Med. 1953 Oct;98(4):373–397. doi: 10.1084/jem.98.4.373. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of General Physiology are provided here courtesy of The Rockefeller University Press

RESOURCES