Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1978 Jul;27(1):149–163. doi: 10.1128/jvi.27.1.149-163.1978

In vitro packaging of bacteriophate T7 DNA synthesized in vitro.

W E Masker, N B Kuemmerle, D P Allison
PMCID: PMC354148  PMID: 691109

Abstract

An in vitro DNA packaging system was used to encapsulate T7 DNA that had been synthesized by extracts prepared from gently lysed Escherchia coli infected with bacteriophage T7 carrying amber mutations in gene 3 or in both genes 3 and 6. Isopycnic centrifugation of density-labeled wild-type DNA was employed in an effort to separate product from template; suppressor-free indicator bacteria were used to eliminate contributions from endogenous DNA or contaminating phage. Additional controls indicated that fragmented DNA is packaged in vitro only with very low efficiency and that the frequency of recombination during packaging is too low to affect interpretation of these experiments. T7 DNA replicated by extracts prepared using T7 mutants deficient in both genes 3 and 6 could be packaged in vitro with an efficiency comparable to that found when highly purified virion T7 DNA was used. When T7 deficient in the gene 3 endonuclease but with normal levels of the gene 6 exonuclease was used, fast-sedimentingconcatemer-like DNA structures were formed during in vitro DNA synthesis. Electron microscopy revealed many branched and highly complex DNA structures formed during this reaction. This concatemer-like DNA was encapsulated in vitro with an efficiency significantly greater than that found for DNA the length of a single T7 genome.

Full text

PDF
149

Images in this article

159Image
on p.159
160Image
on p.160

Selected References

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

  1. 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]
  2. Broker T. R., Lehman I. R. Branched DNA molecules: intermediates in T4 recombination. J Mol Biol. 1971 Aug 28;60(1):131–149. doi: 10.1016/0022-2836(71)90453-0. [DOI] [PubMed] [Google Scholar]
  3. Center M. S., Studier F. W., Richardson C. C. The structural gene for a T7 endonuclease essential for phage DNA synthesis. Proc Natl Acad Sci U S A. 1970 Jan;65(1):242–248. doi: 10.1073/pnas.65.1.242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Davis R. W., Davidson N. Electron-microscopic visualization of deletion mutations. Proc Natl Acad Sci U S A. 1968 May;60(1):243–250. doi: 10.1073/pnas.60.1.243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dressler D., Wolfson J., Magazin M. Initiation and reinitiation of DNA synthesis during replication of bacteriophage T7. Proc Natl Acad Sci U S A. 1972 Apr;69(4):998–1002. doi: 10.1073/pnas.69.4.998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Freifelder D. Molecular weights of coliphages and coliphage DNA. IV. Molecular weights of DNA from bacteriophages T4, T5 and T7 and the general problem of determination of M. J Mol Biol. 1970 Dec 28;54(3):567–577. doi: 10.1016/0022-2836(70)90127-0. [DOI] [PubMed] [Google Scholar]
  7. Fröhlich B., Powling A., Knippers R. Formation of concatemeric DNA in bacteriophage T7-infected bacteria. Virology. 1975 Jun;65(2):455–468. doi: 10.1016/0042-6822(75)90051-3. [DOI] [PubMed] [Google Scholar]
  8. Grippo P., Richardson C. C. Deoxyribonucleic acid polymerase of bacteriophage T7. J Biol Chem. 1971 Nov 25;246(22):6867–6873. [PubMed] [Google Scholar]
  9. Hausmann R. Bacteriophage T7 genetics. Curr Top Microbiol Immunol. 1976;75:77–110. doi: 10.1007/978-3-642-66530-1_3. [DOI] [PubMed] [Google Scholar]
  10. Hausmann R. The genetics of T-odd phages. Annu Rev Microbiol. 1973;27:51–67. doi: 10.1146/annurev.mi.27.100173.000411. [DOI] [PubMed] [Google Scholar]
  11. Henner W. D., Kleber I., Benzinger R. Transfection of Escherichia coli spheroplasts. 3. Facilitation of transfection and stabilization of spheroplasts by different basic polymers. J Virol. 1973 Oct;12(4):741–747. doi: 10.1128/jvi.12.4.741-747.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hinkle D. C., Richardson C. C. Bacteriophage T7 deoxyribonucleic acid replication in vitro. Purification and properties of the gene 4 protein of bacteriophage T7. J Biol Chem. 1975 Jul 25;250(14):5523–5529. [PubMed] [Google Scholar]
  13. Hinkle D. C., Richardson C. C. Bacteriophage T7 deoxyribonucleic acid replication in vitro. Requirements for deoxyribonucleic acid synthesis and characterization of the product. J Biol Chem. 1974 May 10;249(9):2974–2980. [PubMed] [Google Scholar]
  14. Inman R. B., Schnös M. Partial denaturation of thymine- and 5-bromouracil-containing lambda DNA in alkali. J Mol Biol. 1970 Apr 14;49(1):93–98. doi: 10.1016/0022-2836(70)90378-5. [DOI] [PubMed] [Google Scholar]
  15. KLEINSCHMIDT A. K., LANG D., JACHERTS D., ZAHN R. K. [Preparation and length measurements of the total desoxyribonucleic acid content of T2 bacteriophages]. Biochim Biophys Acta. 1962 Dec 31;61:857–864. [PubMed] [Google Scholar]
  16. 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]
  17. Kerr C., Sadowski P. D. Gene 6 exonuclease of bacteriophage T7. I. Purification and properties of the enzyme. J Biol Chem. 1972 Jan 10;247(1):305–310. [PubMed] [Google Scholar]
  18. Kerr C., Sadowski P. D. Gene 6 exonuclease of bacteriophage T7. II. Mechanism of the reaction. J Biol Chem. 1972 Jan 10;247(1):311–318. [PubMed] [Google Scholar]
  19. Kerr C., Sadowski P. D. Packaging and maturation of DNA of bacteriophage T7 in vitro. Proc Natl Acad Sci U S A. 1974 Sep;71(9):3545–3549. doi: 10.1073/pnas.71.9.3545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kerr C., Sadowski P. D. The involvement of genes 3,4,5 and 6 in genetic recombination in bacteriophage T7. Virology. 1975 May;65(1):281–285. doi: 10.1016/0042-6822(75)90031-8. [DOI] [PubMed] [Google Scholar]
  21. Kolodner R., Richardson C. C. Replication of duplex DNA by bacteriophage T7 DNA polymerase and gene 4 protein is accompanied by hydrolysis of nucleoside 5'-triphosphates. Proc Natl Acad Sci U S A. 1977 Apr;74(4):1525–1529. doi: 10.1073/pnas.74.4.1525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kuemmerle N. B., Masker W. E. In vitro packaging of UV radiation-damaged DNA from bacteriophage T7. J Virol. 1977 Sep;23(3):509–516. doi: 10.1128/jvi.23.3.509-516.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lee M., Miller R. C., Jr, Scraba D., Paetkau V. The essential role of bacteriophage T7 endonuclease (gene 3) in molecular recombination. J Mol Biol. 1976 Jul 15;104(4):883–888. doi: 10.1016/0022-2836(76)90189-3. [DOI] [PubMed] [Google Scholar]
  24. Lee M., Miller R. C., Jr T7 exonuclease (gene 6) is necessary for molecular recombination of bacteriophage T7. J Virol. 1974 Nov;14(5):1040–1048. doi: 10.1128/jvi.14.5.1040-1048.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Masamune Y., Frenkel G. D., Richardson C. C. A mutant of bacteriophage T7 deficient in polynucleotide ligase. J Biol Chem. 1971 Nov 25;246(22):6874–6879. [PubMed] [Google Scholar]
  26. Masker W. E. Deoxyribonucleic acid repair in vitro by extracts of Escherichia coli. J Bacteriol. 1977 Mar;129(3):1415–1423. doi: 10.1128/jb.129.3.1415-1423.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Masker W. E., Richardson C. C. Bacteriophage T7 deoxyribonucleic acid replication in vitro VI. Synthesis of biologically active T7 DNA. J Mol Biol. 1976 Feb 5;100(4):557–567. doi: 10.1016/s0022-2836(76)80045-9. [DOI] [PubMed] [Google Scholar]
  28. Masker W. E., Richardson C. C. Bacteriophage T7 deoxyribonucleic acid replication in vitro. V. Synthesis of intact chromosomes of bacteriophage T7. J Mol Biol. 1976 Feb 5;100(4):543–556. doi: 10.1016/s0022-2836(76)80044-7. [DOI] [PubMed] [Google Scholar]
  29. Miller R. C., Jr, Lee M. The role of bacteriophage T7 exonuclease (gene 6) in genetic recombination and production of concatemers. J Mol Biol. 1976 Feb 25;101(2):223–234. doi: 10.1016/0022-2836(76)90374-0. [DOI] [PubMed] [Google Scholar]
  30. Modrich P., Richardson C. C. Bacteriophage T7 deoxyribonucleic acid replication invitro. Bacteriophage T7 DNA polymerase: an an emzyme composed of phage- and host-specific subunits. J Biol Chem. 1975 Jul 25;250(14):5515–5522. [PubMed] [Google Scholar]
  31. Paetkau V., Langman L., Bradley R., Scraba D., Miller R. C., Jr Folded, concatenated genomes as replication intermediates of bacteriophage T7 DNA. J Virol. 1977 Apr;22(1):130–141. doi: 10.1128/jvi.22.1.130-141.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Richardson C. C. The 5'-terminal nucleotides of T7 bacteriophage deoxyribonucleic acid. J Mol Biol. 1966 Jan;15(1):49–61. doi: 10.1016/s0022-2836(66)80208-5. [DOI] [PubMed] [Google Scholar]
  33. Roeder G. S., Sadowski P. D. Bacteriophage T7 morphogenesis: phage-related particles in cells infected with wild-type and mutant T7 phage. Virology. 1977 Jan;76(1):263–285. doi: 10.1016/0042-6822(77)90302-6. [DOI] [PubMed] [Google Scholar]
  34. Sadowski P. D. Bacteriophage T7 endonuclease. I. Properties of the enzyme purified from T7 phage-infected Escherichia coli B. J Biol Chem. 1971 Jan 10;246(1):209–216. [PubMed] [Google Scholar]
  35. Sadowski P. D. Genetic recombination of bacteriophage T7 DNA in vitro. II. Further properties of the in vitro recombination-packaging reaction. Virology. 1977 May 1;78(1):192–202. doi: 10.1016/0042-6822(77)90091-5. [DOI] [PubMed] [Google Scholar]
  36. Sadowski P. D., Vetter D. Genetic recombination of bacteriophage T7 DNA in vitro. Proc Natl Acad Sci U S A. 1976 Mar;73(3):692–696. doi: 10.1073/pnas.73.3.692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Scherzinger E., Litfin F. In vitro studies on the role of phage T7 gene 4 product in DNA replication. Mol Gen Genet. 1974;135(1):73–86. doi: 10.1007/BF00433903. [DOI] [PubMed] [Google Scholar]
  38. Scherzinger E., Seiffert D. Studies on bacteriophage T7 DNA synthesis in vitro. I. Resolution of the T7 replication system into its components. Mol Gen Genet. 1975 Dec 1;141(3):213–232. doi: 10.1007/BF00341801. [DOI] [PubMed] [Google Scholar]
  39. Serwer P. Fast sedimenting bacteriophage T7 DNA from T7-infected Escherichia coli. Virology. 1974 May;59(1):70–88. doi: 10.1016/0042-6822(74)90207-4. [DOI] [PubMed] [Google Scholar]
  40. Strätling W., Ferdinand F. J., Krause E., Knippers R. Bacteriophage T7-DNA replication in vitro: an experimental system. Eur J Biochem. 1973 Sep 21;38(1):160–169. doi: 10.1111/j.1432-1033.1973.tb03046.x. [DOI] [PubMed] [Google Scholar]
  41. Strätling W., Knippers R. Function and purification of gene 4 protein of phage T7. Nature. 1973 Sep 28;245(5422):195–197. doi: 10.1038/245195a0. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. Studier F. W. Bacteriophage T7. Science. 1972 Apr 28;176(4033):367–376. doi: 10.1126/science.176.4033.367. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. Transfection of restrictionless Escherichia coli by bacteriophage T7 dna: effect of in vitro erosion of DNA by gamma exonuclease. J Mol Biol. 1976 Aug 25;105(4):603–609. doi: 10.1016/0022-2836(76)90238-2. [DOI] [PubMed] [Google Scholar]
  46. Vlachopoulou P. J., Sadowksi P. D. Genetic recombination of bacteriophage T7 DNA in vitro III. A physical assay for recombinant DNA. Virology. 1977 May 1;78(1):203–215. doi: 10.1016/0042-6822(77)90092-7. [DOI] [PubMed] [Google Scholar]
  47. Watson J. D. Origin of concatemeric T7 DNA. Nat New Biol. 1972 Oct 18;239(94):197–201. doi: 10.1038/newbio239197a0. [DOI] [PubMed] [Google Scholar]
  48. Wickner W., Brutlag D., Schekman R., Kornberg A. RNA synthesis initiates in vitro conversion of M13 DNA to its replicative form. Proc Natl Acad Sci U S A. 1972 Apr;69(4):965–969. doi: 10.1073/pnas.69.4.965. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES