Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1974 Sep;71(9):3545–3549. doi: 10.1073/pnas.71.9.3545

Packaging and Maturation of DNA of Bacteriophage T7 In Vitro

Carol Kerr 1,2, Paul D Sadowski 1,2
PMCID: PMC433811  PMID: 4530321

Abstract

We have developed an in vitro complementation assay to demonstrate packaging and maturation of DNA of phage T7. Cells of Escherichia coli B infected with an appropriate T7 amber mutant are concentrated 200-fold and lysed by freezing and thawing. Two extracts from cells infected with different amber mutants are mixed and incubated at 30°. Positive complementation results in a 100-fold increase in phage titer.

Using this assay we have demonstrated the packaging of phage DNA from an extract that contains no phage heads (gene 9-, 10-), within head structures present in an extract that contains no phage DNA (gene 5-). We have also demonstrated an activity in extracts that contain no phage DNA or heads (gene 5-, 9-, 10-), which complements gene 19--infected cells. We have proven that this activity is due to the gene-19 product by showing that the activity is temperature-sensitive if the extract is made from cells infected with a mutant having a temperature-sensitive mutation in gene 19. This assay should be useful in elucidating the mechanism of packaging and maturation of DNA of phage T7.

Keywords: morphogenesis, in vitro complementation

Full text

PDF

Selected References

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

  1. Botstein D., Waddell C. H., King J. Mechanism of head assembly and DNA encapsulation in Salmonella phage p22. I. Genes, proteins, structures and DNA maturation. J Mol Biol. 1973 Nov 15;80(4):669–695. doi: 10.1016/0022-2836(73)90204-0. [DOI] [PubMed] [Google Scholar]
  2. Edgar R. S., Lielausis I. Some steps in the assembly of bacteriophage T4. J Mol Biol. 1968 Mar 14;32(2):263–276. doi: 10.1016/0022-2836(68)90008-9. [DOI] [PubMed] [Google Scholar]
  3. Edgar R. S., Wood W. B. Morphogenesis of bacteriophage T4 in extracts of mutant-infected cells. Proc Natl Acad Sci U S A. 1966 Mar;55(3):498–505. doi: 10.1073/pnas.55.3.498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Enquist L. W., Skalka A. Replication of bacteriophage lambda DNA dependent on the function of host and viral genes. I. Interaction of red, gam and rec. J Mol Biol. 1973 Apr 5;75(2):185–212. doi: 10.1016/0022-2836(73)90016-8. [DOI] [PubMed] [Google Scholar]
  5. Frankel F. R. DNA replication after T4 infection. Cold Spring Harb Symp Quant Biol. 1968;33:485–493. doi: 10.1101/sqb.1968.033.01.056. [DOI] [PubMed] [Google Scholar]
  6. Frankel F. R. Evidence for long DNA strands in the replicating pool after T4 infection. Proc Natl Acad Sci U S A. 1968 Jan;59(1):131–138. doi: 10.1073/pnas.59.1.131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hausmann R., LaRue K. Variations in sedimentation patterns among deoxyribonucleic acids synthesized after infection of Escherichia coli by different amber mutants of bacteriophage T7. J Virol. 1969 Feb;3(2):278–281. doi: 10.1128/jvi.3.2.278-281.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hohn B., Hohn T. Activity of empty, headlike particles for packaging of DNA of bacteriophage lambda in vitro. Proc Natl Acad Sci U S A. 1974 Jun;71(6):2372–2376. doi: 10.1073/pnas.71.6.2372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kaiser D., Masuda T. In vitro assembly of bacteriophage Lambda heads. Proc Natl Acad Sci U S A. 1973 Jan;70(1):260–264. doi: 10.1073/pnas.70.1.260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kelly T. J., Jr, Thomas C. A., Jr An intermediate in the replication of bacteriophage T7 DNA molecules. J Mol Biol. 1969 Sep 28;44(3):459–475. doi: 10.1016/0022-2836(69)90373-8. [DOI] [PubMed] [Google Scholar]
  11. King J. Assembly of the tail of bacteriophage T4. J Mol Biol. 1968 Mar 14;32(2):231–262. doi: 10.1016/0022-2836(68)90007-7. [DOI] [PubMed] [Google Scholar]
  12. King J. Bacteriophage T4 tail assembly: four steps in core formation. J Mol Biol. 1971 Jun 28;58(3):693–709. doi: 10.1016/0022-2836(71)90034-9. [DOI] [PubMed] [Google Scholar]
  13. King J., Laemmli U. K. Bacteriophage T4 tail assembly: structural proteins and their genetic identification. J Mol Biol. 1973 Apr 5;75(2):315–337. doi: 10.1016/0022-2836(73)90024-7. [DOI] [PubMed] [Google Scholar]
  14. King J., Laemmli U. K. Polypeptides of the tail fibres of bacteriophage T4. J Mol Biol. 1971 Dec 28;62(3):465–477. doi: 10.1016/0022-2836(71)90148-3. [DOI] [PubMed] [Google Scholar]
  15. King J., Lenk E. V., Botstein D. Mechanism of head assembly and DNA encapsulation in Salmonella phage P22. II. Morphogenetic pathway. J Mol Biol. 1973 Nov 15;80(4):697–731. doi: 10.1016/0022-2836(73)90205-2. [DOI] [PubMed] [Google Scholar]
  16. King J., Mykolajewycz N. Bacteriophage T4 tail assembly: proteins of the sheath, core and baseplate. J Mol Biol. 1973 Apr 5;75(2):339–358. doi: 10.1016/0022-2836(73)90025-9. [DOI] [PubMed] [Google Scholar]
  17. King J., Wood W. B. Assembly of bacteriophage T4 tail fibers: the sequence of gene product interaction. J Mol Biol. 1969 Feb 14;39(3):583–601. doi: 10.1016/0022-2836(69)90147-8. [DOI] [PubMed] [Google Scholar]
  18. Laemmli U. K., Favre M. Maturation of the head of bacteriophage T4. I. DNA packaging events. J Mol Biol. 1973 Nov 15;80(4):575–599. doi: 10.1016/0022-2836(73)90198-8. [DOI] [PubMed] [Google Scholar]
  19. Mackinlay A. G., Kaiser A. D. DNA replication in head mutants of bacteriophage lambda. J Mol Biol. 1969 Feb 14;39(3):679–683. doi: 10.1016/0022-2836(69)90155-7. [DOI] [PubMed] [Google Scholar]
  20. McClure S. C., MacHattie L., Gold M. A sedimentation analysis of DNA found in Escherichia coli infected with phage lambda mutants. Virology. 1973 Jul;54(1):1–18. doi: 10.1016/0042-6822(73)90109-8. [DOI] [PubMed] [Google Scholar]
  21. Meezan E., Wood W. B. The sequence of gene product interaction in bacteriophage T4 tail core assembly. J Mol Biol. 1971 Jun 28;58(3):685–692. doi: 10.1016/0022-2836(71)90033-7. [DOI] [PubMed] [Google Scholar]
  22. Pruss G., Goldstein R. N., Calendar R. In vitro packaging of satellite phage P4 DNA. Proc Natl Acad Sci U S A. 1974 Jun;71(6):2367–2371. doi: 10.1073/pnas.71.6.2367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sadowski P., McGeer A., Becker A. Terminal cross-linking of DNA catalyzed by an enzyme system containing DNA ligase, DNA polymerase, and exonuclease of bacteriophage T7. Can J Biochem. 1974 Jun;52(6):525–535. doi: 10.1139/o74-077. [DOI] [PubMed] [Google Scholar]
  24. Schlegel R. A., Thomas C. A., Jr Some special structural features of intracellular bacteriophage T7 concatemers. J Mol Biol. 1972 Jul 21;68(2):319–345. doi: 10.1016/0022-2836(72)90216-1. [DOI] [PubMed] [Google Scholar]
  25. Strätling W., Krause E., Knippers R. Fast sedimenting deoxyribonucleic acid in bacteriophage T7-infected cells. Virology. 1973 Jan;51(1):109–119. doi: 10.1016/0042-6822(73)90371-1. [DOI] [PubMed] [Google Scholar]
  26. Studier F. W. Bacteriophage T7. Science. 1972 Apr 28;176(4033):367–376. doi: 10.1126/science.176.4033.367. [DOI] [PubMed] [Google Scholar]
  27. Studier F. W. The genetics and physiology of bacteriophage T7. Virology. 1969 Nov;39(3):562–574. doi: 10.1016/0042-6822(69)90104-4. [DOI] [PubMed] [Google Scholar]
  28. Ward S., Dickson R. C. Assembly of bacteriophage T4 tail fibers. 3. Genetic control of the major tail fiber polypeptides. J Mol Biol. 1971 Dec 28;62(3):479–492. doi: 10.1016/0022-2836(71)90149-5. [DOI] [PubMed] [Google Scholar]
  29. Ward S., Luftig R. B., Wilson J. H., Eddleman H., Lyle H., Wood W. B. Assembly of bacteriophage T4 tail fibers. II. Isolation and characterization of tail fiber precursors. J Mol Biol. 1970 Nov 28;54(1):15–31. doi: 10.1016/0022-2836(70)90443-2. [DOI] [PubMed] [Google Scholar]
  30. Yamamoto K. R., Alberts B. M., Benzinger R., Lawhorne L., Treiber G. Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to large-scale virus purification. Virology. 1970 Mar;40(3):734–744. doi: 10.1016/0042-6822(70)90218-7. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES