Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1983 Jul;47(1):96–105. doi: 10.1128/jvi.47.1.96-105.1983

Bacteriophage P22 in vitro DNA packaging monitored by agarose gel electrophoresis: rate of DNA entry into capsids.

R Gope, P Serwer
PMCID: PMC255206  PMID: 6191043

Abstract

Bacteriophage P22, like other double-stranded DNA bacteriophages, packages DNA in a preassembled, DNA-free procapsid. The P22 procapsid and P22 bacteriophage have been electrophoretically characterized; the procapsid has a negative average electrical surface charge density (sigma) higher in magnitude than the negative sigma of the mature bacteriophage. Dextrans, sucrose, and maltose were shown to have a dramatic stimulatory effect on the in vitro packaging of DNA by the P22 procapsid. However, sedoheptulose, smaller sugars, and smaller polyols did not stimulate in vitro P22 DNA packaging. These and other data suggest that an osmotic pressure difference across some particle, probably a capsid, stimulates P22 DNA packaging. After in vitro packaging was optimized by including dextran 40 in extracts, the entry kinetics of DNA into P22 capsids were measured. Packaged DNA was detected by: (i) DNA-specific staining of intact capsids after fractionation by agarose gel electrophoresis and (ii) agarose gel electrophoresis of DNase-resistant DNA after release of DNase-resistant DNA from capsids. It was found that the first DNA was packaged by 1.5 min after the start of incubation. The data further suggest that either P22 capsids with DNA partially packaged in vitro are too unstable to be detected by the above procedures or entry of DNA into the capsid occurs in less than 0.25 min.

Full text

PDF
96

Images in this article

99Image
on p.99
101Image
on p.101

Selected References

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

  1. Benchimol S., Lucko H., Becker A. Bacteriophage lambda DNA packaging in vitro. The involvement of the lambda FI gene product, single-strand DNA, and a novel lambda-directed protein in the packaging reaction. J Biol Chem. 1982 May 10;257(9):5201–5210. [PubMed] [Google Scholar]
  2. Bjornsti M. A., Reilly B. E., Anderson D. L. In vitro assembly of the Bacillus subtilis bacteriophage phi 29. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5861–5865. doi: 10.1073/pnas.78.9.5861. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bjornsti M. A., Reilly B. E., Anderson D. L. Morphogenesis of bacteriophage phi 29 of Bacillus subtilis: DNA-gp3 intermediate in in vivo and in vitro assembly. J Virol. 1982 Feb;41(2):508–517. doi: 10.1128/jvi.41.2.508-517.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bjornsti M. A., Reilly B. E., Anderson D. L. Morphogenesis of bacteriophage phi 29 of Bacillus subtilis: oriented and quantized in vitro packaging of DNA protein gp3. J Virol. 1983 Jan;45(1):383–396. doi: 10.1128/jvi.45.1.383-396.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Black L. W. In vitro packaging of bacteriophage T4 DNA. Virology. 1981 Aug;113(1):336–344. doi: 10.1016/0042-6822(81)90160-4. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Casjens S., King J. P22 morphogenesis. I: Catalytic scaffolding protein in capsid assembly. J Supramol Struct. 1974;2(2-4):202–224. doi: 10.1002/jss.400020215. [DOI] [PubMed] [Google Scholar]
  8. Casjens S., King J. Virus assembly. Annu Rev Biochem. 1975;44:555–611. doi: 10.1146/annurev.bi.44.070175.003011. [DOI] [PubMed] [Google Scholar]
  9. Earnshaw W. C., Casjens S. R. DNA packaging by the double-stranded DNA bacteriophages. Cell. 1980 Sep;21(2):319–331. doi: 10.1016/0092-8674(80)90468-7. [DOI] [PubMed] [Google Scholar]
  10. Earnshaw W., Casjens S., Harrison S. C. Assembly of the head of bacteriophage P22: x-ray diffraction from heads, proheads and related structures. J Mol Biol. 1976 Jun 25;104(2):387–410. doi: 10.1016/0022-2836(76)90278-3. [DOI] [PubMed] [Google Scholar]
  11. Fujisawa H., Yamagishi M. Studies on factors involved in in vitro packaging of phage T3 dna. Prog Clin Biol Res. 1981;64:239–252. [PubMed] [Google Scholar]
  12. Hohn T., Katsura I. Structure and assembly of bacteriophage lambda. Curr Top Microbiol Immunol. 1977;78:69–110. doi: 10.1007/978-3-642-66800-5_3. [DOI] [PubMed] [Google Scholar]
  13. Hsiao C. L., Black L. W. DNA packaging and the pathway of bacteriophage T4 head assembly. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3652–3656. doi: 10.1073/pnas.74.9.3652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. King J., Casjens S. Catalytic head assembling protein in virus morphogenesis. Nature. 1974 Sep 13;251(5471):112–119. doi: 10.1038/251112a0. [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. 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]
  17. Masker W. E. In vitro packaging of bacteriophage T7 DNA requires ATP. J Virol. 1982 Jul;43(1):365–367. doi: 10.1128/jvi.43.1.365-367.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Masker W. E., Serwer P. DNA packaging in vitro by an isolated bacteriophage T7 procapsid. J Virol. 1982 Sep;43(3):1138–1142. doi: 10.1128/jvi.43.3.1138-1142.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. McDonell M. W., Simon M. N., Studier F. W. Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J Mol Biol. 1977 Feb 15;110(1):119–146. doi: 10.1016/s0022-2836(77)80102-2. [DOI] [PubMed] [Google Scholar]
  20. Miyazaki J. I., Fujisawa H., Minagawa T. Biological activity of purified bacteriophage T3 prohead and proheadlike structures as precursors for in vitro head assembly. Virology. 1978 Dec;91(2):283–290. doi: 10.1016/0042-6822(78)90376-8. [DOI] [PubMed] [Google Scholar]
  21. Murialdo H., Becker A. Head morphogenesis of complex double-stranded deoxyribonucleic acid bacteriophages. Microbiol Rev. 1978 Sep;42(3):529–576. doi: 10.1128/mr.42.3.529-576.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Poteete A. R., Botstein D. Purification and properties of proteins essential to DNA encapsulation by phage P22. Virology. 1979 Jun;95(2):565–573. doi: 10.1016/0042-6822(79)90509-9. [DOI] [PubMed] [Google Scholar]
  23. Poteete A. R., Jarvik V., Botstein D. Encapsulation of phage P22 DNA in vitro. Virology. 1979 Jun;95(2):550–564. doi: 10.1016/0042-6822(79)90508-7. [DOI] [PubMed] [Google Scholar]
  24. Pruss G. J., Wang J. C., Calendar R. In vitro packaging of covalently closed circular monomers of bacteriophage DNA. J Mol Biol. 1975 Nov 5;98(3):465–478. doi: 10.1016/s0022-2836(75)80080-5. [DOI] [PubMed] [Google Scholar]
  25. Rutila J. E., Jackson E. N. Physical map of the bacteriophage P22 genome. Virology. 1981 Sep;113(2):769–775. doi: 10.1016/0042-6822(81)90206-3. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Serwer P. A metrizamide-impermeable capsid in the DNA packaging pathway of bacteriophage T7. J Mol Biol. 1980 Mar 25;138(1):65–91. doi: 10.1016/s0022-2836(80)80005-2. [DOI] [PubMed] [Google Scholar]
  29. Serwer P. A technique for electrophoresis in multiple-concentration agarose gels. Anal Biochem. 1980 Jan 1;101(1):154–159. doi: 10.1016/0003-2697(80)90054-8. [DOI] [PubMed] [Google Scholar]
  30. Serwer P. Electrophoresis of duplex deoxyribonucleic acid in multiple-concentration agarose gels: fractionation of molecules with molecular weights between 2 X 10(6) and 110 X 10(6). Biochemistry. 1980 Jun 24;19(13):3001–3004. doi: 10.1021/bi00554a026. [DOI] [PubMed] [Google Scholar]
  31. Serwer P. Internal proteins of bacteriophage T7. J Mol Biol. 1976 Nov 5;107(3):271–291. doi: 10.1016/s0022-2836(76)80005-8. [DOI] [PubMed] [Google Scholar]
  32. Serwer P., Masker W. E., Allen J. L. Stability and in vitro DNA packaging of bacteriophages: effects of dextrans, sugars, and polyols. J Virol. 1983 Feb;45(2):665–671. doi: 10.1128/jvi.45.2.665-671.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Serwer P., Pichler M. E. Electrophoresis of bacteriophage T7 and T7 capsids in agarose gels. J Virol. 1978 Dec;28(3):917–928. doi: 10.1128/jvi.28.3.917-928.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Serwer P., Watson R. H. Electrophoresis in density gradients of metrizamide. Anal Biochem. 1981 Jul 1;114(2):342–348. doi: 10.1016/0003-2697(81)90491-7. [DOI] [PubMed] [Google Scholar]
  35. Steven A. C. Visualization of virus structure in three dimensions. Methods Cell Biol. 1981;22:297–323. doi: 10.1016/s0091-679x(08)61881-6. [DOI] [PubMed] [Google Scholar]
  36. Studier F. W. Analysis of bacteriophage T7 early RNAs and proteins on slab gels. J Mol Biol. 1973 Sep 15;79(2):237–248. doi: 10.1016/0022-2836(73)90003-x. [DOI] [PubMed] [Google Scholar]
  37. Studier F. W. Bacteriophage T7. Science. 1972 Apr 28;176(4033):367–376. doi: 10.1126/science.176.4033.367. [DOI] [PubMed] [Google Scholar]
  38. Tye B. K., Botstein D. P22 morphogenesis. II: Mechanism of DNA encapsulation. J Supramol Struct. 1974;2(2-4):225–238. doi: 10.1002/jss.400020216. [DOI] [PubMed] [Google Scholar]
  39. Wellauer P. K., Reeder R. H., Carroll D., Brown D. D., Deutch A., Higashinakagawa T., Dawid I. B. Amplified ribosomal DNA from Xenopus laevis has heterogeneous spacer lengths. Proc Natl Acad Sci U S A. 1974 Jul;71(7):2823–2827. doi: 10.1073/pnas.71.7.2823. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES