Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1972 Feb;9(2):297–308. doi: 10.1128/jvi.9.2.297-308.1972

Intracellular Forms of Adenovirus Deoxyribonucleic Acid I. Evidence for a Deoxyribonucleic Acid-Protein Complex in Baby Hamster Kidney Cells Infected with Adenovirus Type 12

Walter Doerfler 1,1, Ulla Lundholm 1,1, Monica Hirsch-Kauffmann 1
PMCID: PMC356297  PMID: 5062681

Abstract

The total intracellular deoxyribonucleic acid (DNA) from baby hamster kidney cells abortively infected with 3H-adenovirus type 12 was analyzed in dye-buoyant density gradients. Between 10 and 20% of the cell-associated radioactivity derived from viral DNA bands in a density position which is 0.043 to 0.085 g/cm3 higher than that of viral DNA extracted from purified virions. The DNA in the high-density region (HP-fraction) is almost completely absent when DNA, ribonucleic acid (RNA) or protein synthesis is chemically inhibited in separate experiments. The HP-fraction is not found when the virus does not adsorb to and enter the cell. The DNA in the HP-fraction appears as early as 2 hr after inoculation. At 2 hr after infection, the HP-fraction is present both in the nucleus and the cytoplasm. This DNA hybridizes exclusively with viral DNA and sediments at approximately the same rate in both neutral and alkaline sucrose density gradients. Electron microscopy has revealed no circular DNA molecules in this fraction. Evidence indicates that the viral DNA in the HP-fraction exists in a complex with protein and possibly RNA. The protein component of the complex is resistant to enzymatic digestion, whereas the complex is susceptible to ribonuclease treatment. Digestion with deoxyribonuclease reduces the amount of DNA found in the HP-fraction. The structure and biological function of this complex are currently being investigated.

Full text

PDF
297

Selected References

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

  1. BABLANIAN R., EGGERS H. J., TAMM I. STUDIES ON THE MECHANISM OF POLIOVIRUS-INDUCED CELL DAMAGE. I. THE RELATION BETWEEN POLIOVIRUS,-INDUCED METABOLIC AND MORPHOLOGICAL ALTERATIONS IN CULTURED CELLS. Virology. 1965 May;26:100–113. doi: 10.1016/0042-6822(65)90030-9. [DOI] [PubMed] [Google Scholar]
  2. Bode V. C., Kaiser A. D. Changes in the structure and activity of lambda DNA in a superinfected immune bacterium. J Mol Biol. 1965 Dec;14(2):399–417. doi: 10.1016/s0022-2836(65)80190-5. [DOI] [PubMed] [Google Scholar]
  3. Bourgaux P., Bourgaux-Ramoisy D., Seiler P. The replication of the ring-shaped DNA of polyoma virus. II. Identification of molecules at various stages of replication. J Mol Biol. 1971 Jul 14;59(1):195–206. doi: 10.1016/0022-2836(71)90421-9. [DOI] [PubMed] [Google Scholar]
  4. Burlingham B. T., Doerfler W., Pettersson U., Philipson L. Adenovirus endonuclease: association with the penton of adenovirus type 2. J Mol Biol. 1971 Aug 28;60(1):45–64. doi: 10.1016/0022-2836(71)90446-3. [DOI] [PubMed] [Google Scholar]
  5. Burlingham B. T., Doerfler W. Three size-classes of intracellular adenovirus deoxyribonucleic acid. J Virol. 1971 Jun;7(6):707–719. doi: 10.1128/jvi.7.6.707-719.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Clewell D. B., Helinski D. R. Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an opern circular DNA form. Proc Natl Acad Sci U S A. 1969 Apr;62(4):1159–1166. doi: 10.1073/pnas.62.4.1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. DULBECCO R., VOGT M. Plaque formation and isolation of pure lines with poliomyelitis viruses. J Exp Med. 1954 Feb;99(2):167–182. doi: 10.1084/jem.99.2.167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Denhardt D. T. A membrane-filter technique for the detection of complementary DNA. Biochem Biophys Res Commun. 1966 Jun 13;23(5):641–646. doi: 10.1016/0006-291x(66)90447-5. [DOI] [PubMed] [Google Scholar]
  9. Doerfler W., Hogness D. S. The strands of DNA from lambda and related bacteriophages: isolation and characterization. J Mol Biol. 1968 May 14;33(3):635–659. doi: 10.1016/0022-2836(68)90311-2. [DOI] [PubMed] [Google Scholar]
  10. Doerfler W. Integration of the deoxyribonucleic acid of adenovirus type 12 into the deoxyribonucleic acid of baby hamster kidney cells. J Virol. 1970 Nov;6(5):652–666. doi: 10.1128/jvi.6.5.652-666.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Doerfler W., Kleinschmidt A. K. Denaturation pattern of the DNA of adenovirus type 2 as determined by electron microscopy. J Mol Biol. 1970 Jun 28;50(3):579–593. doi: 10.1016/0022-2836(70)90086-0. [DOI] [PubMed] [Google Scholar]
  12. Doerfler W., Lundholm U. Absence of replication of the DNA of adenovirus type 12 in BHK21 cells. Virology. 1970 Mar;40(3):754–757. doi: 10.1016/0042-6822(70)90222-9. [DOI] [PubMed] [Google Scholar]
  13. Doerfler W. Nonproductive infection of baby hamster kidney cells (BHK21) with adenovirus type 12. Virology. 1969 Aug;38(4):587–606. doi: 10.1016/0042-6822(69)90179-2. [DOI] [PubMed] [Google Scholar]
  14. Doerfler W. The fate of the DNA of adenovirus type 12 in baby hamster kidney cells. Proc Natl Acad Sci U S A. 1968 Jun;60(2):636–643. doi: 10.1073/pnas.60.2.636. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. EAGLE H. Amino acid metabolism in mammalian cell cultures. Science. 1959 Aug 21;130(3373):432–437. doi: 10.1126/science.130.3373.432. [DOI] [PubMed] [Google Scholar]
  16. EAGLE H. Propagation in a fluid medium of a human epidermoid carcinoma, strain KB. Proc Soc Exp Biol Med. 1955 Jul;89(3):362–364. doi: 10.3181/00379727-89-21811. [DOI] [PubMed] [Google Scholar]
  17. Gellert M. Formation of covalent circles of lambda DNA by E. coli extracts. Proc Natl Acad Sci U S A. 1967 Jan;57(1):148–155. doi: 10.1073/pnas.57.1.148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gilbert W., Dressler D. DNA replication: the rolling circle model. Cold Spring Harb Symp Quant Biol. 1968;33:473–484. doi: 10.1101/sqb.1968.033.01.055. [DOI] [PubMed] [Google Scholar]
  19. Goldberg A. R., Howe M. New mutations in the S cistron of bacteriophage lambda affecting host cell lysis. Virology. 1969 May;38(1):200–202. doi: 10.1016/0042-6822(69)90148-2. [DOI] [PubMed] [Google Scholar]
  20. Hirai K., Lehman J., Defendi V. Integration of simian virus 40 deoxyribonucleic acid into the deoxyribonucleic acid of primary infected Chinese hamster cells. J Virol. 1971 Nov;8(5):708–715. doi: 10.1128/jvi.8.5.708-715.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hudson B., Upholt W. B., Devinny J., Vinograd J. The use of an ethidium analogue in the dye-buoyant density procedure for the isolation of closed circular DNA: the variation of the superhelix density of mitochondrial DNA. Proc Natl Acad Sci U S A. 1969 Mar;62(3):813–820. doi: 10.1073/pnas.62.3.813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Klenk H. D., Caliguiri L. A., Choppin P. W. The proteins of the parainfluenza virus SV5. II. The carbohydrate content and glycoproteins of the virion. Virology. 1970 Oct;42(2):473–481. doi: 10.1016/0042-6822(70)90290-4. [DOI] [PubMed] [Google Scholar]
  23. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  24. Levine A. J., Kang H. S., Billheimer F. E. DNA replication in SV40 infected cells. I. Analysis of replicating SV40 DNA. J Mol Biol. 1970 Jun 14;50(2):549–568. doi: 10.1016/0022-2836(70)90211-1. [DOI] [PubMed] [Google Scholar]
  25. Lindberg U., Darnell J. E. SV40-specific RNA in the nucleus and polyribosomes of transformed cells. Proc Natl Acad Sci U S A. 1970 Apr;65(4):1089–1096. doi: 10.1073/pnas.65.4.1089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Maizel J. V., Jr, White D. O., Scharff M. D. The polypeptides of adenovirus. II. Soluble proteins, cores, top components and the structure of the virion. Virology. 1968 Sep;36(1):126–136. doi: 10.1016/0042-6822(68)90122-0. [DOI] [PubMed] [Google Scholar]
  27. Pearson G. D., Hanawalt P. C. Isolation of DNA replication complexes from uninfected and adenovirus-infected HeLa cells. J Mol Biol. 1971 Nov 28;62(1):65–80. doi: 10.1016/0022-2836(71)90131-8. [DOI] [PubMed] [Google Scholar]
  28. Price P. A., Stein W. H., Moore S. Effect of divalent cations on the reduction and re-formation of the disulfide bonds of deoxyribonuclease. J Biol Chem. 1969 Feb 10;244(3):929–932. [PubMed] [Google Scholar]
  29. STOKER M., MACPHERSON I. SYRIAN HAMSTER FIBROBLAST CELL LINE BHK21 AND ITS DERIVATIVES. Nature. 1964 Sep 26;203:1355–1357. doi: 10.1038/2031355a0. [DOI] [PubMed] [Google Scholar]
  30. Strauss J. H., Jr, Burge B. W., Darnell J. E. Carbohydrate content of the membrane protein of Sindbis virus. J Mol Biol. 1970 Feb 14;47(3):437–448. doi: 10.1016/0022-2836(70)90313-x. [DOI] [PubMed] [Google Scholar]
  31. VINOGRAD J., HEARST J. E. Equilibrium sedimentation of macromolecules and viruses in a density gradient. Fortschr Chem Org Naturst. 1962;20:373–422. [PubMed] [Google Scholar]
  32. Vinograd J., Lebowitz J., Radloff R., Watson R., Laipis P. The twisted circular form of polyoma viral DNA. Proc Natl Acad Sci U S A. 1965 May;53(5):1104–1111. doi: 10.1073/pnas.53.5.1104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Zur Hausen H., Sokol F. Fate of adenovirus type 12 genomes in nonpermissive cells. J Virol. 1969 Sep;4(3):256–263. doi: 10.1128/jvi.4.3.256-263.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES