Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1976 May;18(2):672–684. doi: 10.1128/jvi.18.2.672-684.1976

Multiple structures of adeno-associated virus DNA: analysis of terminally labeled molecules with endonuclease R-Hae III.

D T Denhardt, S Eisenberg, K Bartok, B J Carter
PMCID: PMC515595  PMID: 1271522

Abstract

The double-stranded form of adeno-associated virus (AAV) DNA has about 20 sites sensitive to endonuclease R.Hae III from Haemophilus aegypitus; the fragments produced fall into about 13 size classes, 8 of which contain single fragments. The location of the Hae III-produced AAV fragments relative to the three EcoR1 fragments was determined. Using revised figures for the molecular weights of the Hae III cleavage products of phiX174 replicative form DNA, we calculated that AAV DNA contains about 4,000 nucleotides. After Hae III digestiion of duplex DNA terminally labeled with 32P using polynucleotide kinase, the majority of fragments containing a 5' 32P label were about 40 nucleotides in length, and fragments of similar size were generated from each end, suggesting that the Hae site closest to the end is within the terminal repetition. Two more-slowly-migrating cleavage products also bore 5' 32P end label. These three terminally labeled species were also generated from single-stranded AAV DNA by digestion with Hae III, and evidence that one may have a nonlinear ("rabbit-ear") structure is presented. The predominant 5' terminal base was identified as thymine for both the plus and minus strands of AAV. Single-stranded AAV molecules could not be efficiently covalently circularized by incubation with polynucleotide ligase or ligase plus T4 DNA polymerase.

Full text

PDF
684

Images in this article

682Image
on p.682

Selected References

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

  1. Bartok K., Harbers B., Denhardt D. T. Isolation and characterization of self-complementary sequences from phiX174 viral DNA. J Mol Biol. 1975 Nov 25;99(1):93–105. doi: 10.1016/s0022-2836(75)80161-6. [DOI] [PubMed] [Google Scholar]
  2. Berkowitz S. A., Day L. A. Molecular weight of single-stranded fd bacteriophage DNA. High speed equilibrium sedimentation and light scattering measurements. Biochemistry. 1974 Nov 5;13(23):4825–4831. doi: 10.1021/bi00720a022. [DOI] [PubMed] [Google Scholar]
  3. Berns K. I., Kelly T. J., Jr Letter: Visualization of the inverted terminal repetition in adeno-associated virus DNA. J Mol Biol. 1974 Jan 15;82(2):267–271. doi: 10.1016/0022-2836(74)90344-1. [DOI] [PubMed] [Google Scholar]
  4. Berns K. I., Kort J., Fife K. H., Grogan E. W., Spear I. Study of the fine structure of adeno-associated virus DNA with bacterial restriction endonucleases. J Virol. 1975 Sep;16(3):712–719. doi: 10.1128/jvi.16.3.712-719.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blakesley R. W., Wells R. D. 'Single-stranded' DNA from phiX174 and M13 is cleaved by certain restriction endonucleases. Nature. 1975 Oct 2;257(5525):421–422. doi: 10.1038/257421a0. [DOI] [PubMed] [Google Scholar]
  6. Carter B. J., Khoury G., Denhardt D. T. Physical map and strand polarity of specific fragments of adenovirus-associated virus DNA produced by endonuclease R-EcoRI. J Virol. 1975 Sep;16(3):559–568. doi: 10.1128/jvi.16.3.559-568.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Carter B. J., Khoury G., Rose J. A. Adenovirus-associated virus multiplication. IX. Extent of transcription of the viral genome in vivo. J Virol. 1972 Dec;10(6):1118–1125. doi: 10.1128/jvi.10.6.1118-1125.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Carter B. J., Khoury G. Specific cleavage of adenovirus-associated virus DNA by restriction endonuclease R-EcoRI--characterization of cleavage products. Virology. 1975 Feb;63(2):523–538. doi: 10.1016/0042-6822(75)90325-6. [DOI] [PubMed] [Google Scholar]
  9. Carter B. J., Koczot F. J., Garrison J., Rose J. A., Dolin R. Separate helper functions provided by adenovirus for adenovirus-associated virus multiplication. Nat New Biol. 1973 Jul 18;244(133):71–73. doi: 10.1038/newbio244071a0. [DOI] [PubMed] [Google Scholar]
  10. Carter B. J., Rose J. A. Transcription in vivo of a defective parvovirus: sedimentation and electrophoretic analysis of RNA synthesized by adenovirus-associated virus and its helper adenovirus. Virology. 1974 Sep;61(1):182–199. doi: 10.1016/0042-6822(74)90253-0. [DOI] [PubMed] [Google Scholar]
  11. Cavalier-Smith T. Palindromic base sequences and replication of eukaryote chromosome ends. Nature. 1974 Aug 9;250(5466):467–470. doi: 10.1038/250467a0. [DOI] [PubMed] [Google Scholar]
  12. Denhardt D. T. A theory of DNA replication. J Theor Biol. 1972 Mar;34(3):487–508. doi: 10.1016/0022-5193(72)90137-3. [DOI] [PubMed] [Google Scholar]
  13. Dingman C. W., Fisher M. P., Kakefuda T. Role of molecular conformation in determining the electrophoretic properties of polynucleotides in agarose-acrylamide gels. II. Biochemistry. 1972 Mar 28;11(7):1242–1250. doi: 10.1021/bi00757a020. [DOI] [PubMed] [Google Scholar]
  14. Edgell M. H., Hutchison C. A., 3rd, Sclair M. Specific endonuclease R fragments of bacteriophage phiX174 deoxyribonucleic acid. J Virol. 1972 Apr;9(4):574–582. doi: 10.1128/jvi.9.4.574-582.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Eisenberg S., Harbers B., Hours C., Denhardt D. T. The mechanism of replication of phiX174 DNA. XII. Non-random location of gaps in nascent phiX174 RF II DNA. J Mol Biol. 1975 Nov 25;99(1):107–123. doi: 10.1016/s0022-2836(75)80162-8. [DOI] [PubMed] [Google Scholar]
  16. FIERS W., SINSHEIMER R. L. The structure of the DNA of bacteriophage phi-X174. III. Ultracentrifugal evidence for a ring structure. J Mol Biol. 1962 Oct;5:424–434. doi: 10.1016/s0022-2836(62)80031-x. [DOI] [PubMed] [Google Scholar]
  17. Gerry H. W., Kelly T. J., Jr, Berns K. I. Arrangement of nucleotide sequences in adeno-associated virus DNA. J Mol Biol. 1973 Sep 15;79(2):207–225. doi: 10.1016/0022-2836(73)90001-6. [DOI] [PubMed] [Google Scholar]
  18. Glynn I. M., Chappell J. B. A simple method for the preparation of 32-P-labelled adenosine triphosphate of high specific activity. Biochem J. 1964 Jan;90(1):147–149. doi: 10.1042/bj0900147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hedgpeth J., Goodman H. M., Boyer H. W. DNA nucleotide sequence restricted by the RI endonuclease. Proc Natl Acad Sci U S A. 1972 Nov;69(11):3448–3452. doi: 10.1073/pnas.69.11.3448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Horiuchi K., Zinder N. D. Site-specific cleavage of single-stranded DNA by a Hemophilus restriction endonuclease. Proc Natl Acad Sci U S A. 1975 Jul;72(7):2555–2558. doi: 10.1073/pnas.72.7.2555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Johnson F. B., Ozer H. L., Hoggan M. D. Structural proteins of adenovirus-associated virus type 3. J Virol. 1971 Dec;8(6):860–863. doi: 10.1128/jvi.8.6.860-863.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Johnson P. H., Lee A. S., Sinsheimer R. L. Production of specific fragments of phi X174 replicative form DNA by a restriction enzyme from Haemophilus parainfluenzae, endonuclease HP. J Virol. 1973 Apr;11(4):596–599. doi: 10.1128/jvi.11.4.596-599.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Koczot F. J., Carter B. J., Garon C. F., Rose J. A. Self-complementarity of terminal sequences within plus or minus strands of adenovirus-associated virus DNA. Proc Natl Acad Sci U S A. 1973 Jan;70(1):215–219. doi: 10.1073/pnas.70.1.215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lee A. S., Sinsheimer R. L. A cleavage map of bacteriophage phiX174 genome. Proc Natl Acad Sci U S A. 1974 Jul;71(7):2882–2886. doi: 10.1073/pnas.71.7.2882. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lillehaug J. R., Kleppe K. Kinetics and specificity of T4 polynucleotide kinase. Biochemistry. 1975 Mar 25;14(6):1221–1225. doi: 10.1021/bi00677a020. [DOI] [PubMed] [Google Scholar]
  26. Mayor H. D., Torikai K., Melnick J. L., Mandel M. Plus and minus single-stranded DNA separately encapsidated in adeno-associated satellite virions. Science. 1969 Dec 5;166(3910):1280–1282. doi: 10.1126/science.166.3910.1280. [DOI] [PubMed] [Google Scholar]
  27. Middleton J. H., Edgell M. H., Hutchison C. A., 3rd Specific fragments of phi X174 deoxyribonucleic acid produced by a restriction enzyme from Haemophilus aegyptius, endonuclease Z. J Virol. 1972 Jul;10(1):42–50. doi: 10.1128/jvi.10.1.42-50.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Roberts R. J., Breitmeyer J. B., Tabachnik N. F., Myers P. A. A second specific endonuclease from Haemophilus aegyptius. J Mol Biol. 1975 Jan 5;91(1):121–123. doi: 10.1016/0022-2836(75)90375-7. [DOI] [PubMed] [Google Scholar]
  29. Rose J. A., Berns K. I., Hoggan M. D., Koczot F. J. Evidence for a single-stranded adenovirus-associated virus genome: formation of a DNA density hybrid on release of viral DNA. Proc Natl Acad Sci U S A. 1969 Nov;64(3):863–869. doi: 10.1073/pnas.64.3.863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rose J. A., Maizel J. V., Jr, Inman J. K., Shatkin A. J. Structural proteins of adenovirus-associated viruses. J Virol. 1971 Nov;8(5):766–770. doi: 10.1128/jvi.8.5.766-770.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Schekman R. W., Iwaya M., Bromstrup K., Denhardt D. T. The mechanism of replication of phi X174 single-stranded DNA. 3. An enzymic study of the structure of the replicative form II DNA. J Mol Biol. 1971 Apr 28;57(2):177–199. doi: 10.1016/0022-2836(71)90340-8. [DOI] [PubMed] [Google Scholar]
  32. Sinsheimer R. L. Bacteriophage phi-X174 and related viruses. Prog Nucleic Acid Res Mol Biol. 1968;8:115–169. [PubMed] [Google Scholar]
  33. Smith H. O., Nathans D. Letter: A suggested nomenclature for bacterial host modification and restriction systems and their enzymes. J Mol Biol. 1973 Dec 15;81(3):419–423. doi: 10.1016/0022-2836(73)90152-6. [DOI] [PubMed] [Google Scholar]
  34. Thomas M., Davis R. W. Studies on the cleavage of bacteriophage lambda DNA with EcoRI Restriction endonuclease. J Mol Biol. 1975 Jan 25;91(3):315–328. doi: 10.1016/0022-2836(75)90383-6. [DOI] [PubMed] [Google Scholar]
  35. Williamson R. Properties of rapidly labelled deoxyribonucleic acid fragments isolated from the cytoplasm of primary cultures of embryonic mouse liver cells. J Mol Biol. 1970 Jul 14;51(1):157–168. doi: 10.1016/0022-2836(70)90277-9. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES