Skip to main content
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
. 1972 Sep;69(9):2717–2721. doi: 10.1073/pnas.69.9.2717

In Vitro Complementation as an Assay for New Proteins Required for Bacteriophage T4 DNA Replication: Purification of the Complex Specified by T4 Genes 44 and 62

Jack Barry 1, Bruce Alberts 1
PMCID: PMC427024  PMID: 4560698

Abstract

We have developed an in vitro complementation assay for six T4 bacteriophage gene products believed to be components of the T4 DNA replication apparatus. This assay is based upon the fact that DNA synthesis in an infected cell lysate that lacks a given gene product is specifically stimulated by addition of the missing product. By the use of such an assay, two proteins that appear to be the products of T4 genes 44 and 62 have been purified to electrophoretic homogeneity as a single complex of the two polypeptide chains.

Keywords: gene products, mutants, cell lysates

Full text

PDF
2717

Selected References

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

  1. APOSHIAN H. V., KORNBERG A. Enzymatic synthesis of deoxyribonucleic acid. IX. The polymerase formed after T2 bacteriophage infection of Escherichia coli: a new enzyme. J Biol Chem. 1962 Feb;237:519–525. [PubMed] [Google Scholar]
  2. Alberts B. M., Amodio F. J., Jenkins M., Gutmann E. D., Ferris F. L. Studies with DNA-cellulose chromatography. I. DNA-binding proteins from Escherichia coli. Cold Spring Harb Symp Quant Biol. 1968;33:289–305. doi: 10.1101/sqb.1968.033.01.033. [DOI] [PubMed] [Google Scholar]
  3. Alberts B. M., Frey L. T4 bacteriophage gene 32: a structural protein in the replication and recombination of DNA. Nature. 1970 Sep 26;227(5265):1313–1318. doi: 10.1038/2271313a0. [DOI] [PubMed] [Google Scholar]
  4. Alberts B. M. Function of gene 32-protein, a new protein essential for the genetic recombination and replication of T4 bacteriophage DNA. Fed Proc. 1970 May-Jun;29(3):1154–1163. [PubMed] [Google Scholar]
  5. De Waard A., Paul A. V., Lehman I. R. The structural gene for deoxyribonucleic acid polymerase in bacteriophages T4 and T5. Proc Natl Acad Sci U S A. 1965 Oct;54(4):1241–1248. doi: 10.1073/pnas.54.4.1241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Delius H., Mantell N. J., Alberts B. Characterization by electron microscopy of the complex formed between T4 bacteriophage gene 32-protein and DNA. J Mol Biol. 1972 Jun 28;67(3):341–350. doi: 10.1016/0022-2836(72)90454-8. [DOI] [PubMed] [Google Scholar]
  7. Godson G. N. A simple apparatus for rapid isoelectrofocusing of multiple samples on a micro scale. Anal Biochem. 1970 May;35(1):66–76. doi: 10.1016/0003-2697(70)90010-2. [DOI] [PubMed] [Google Scholar]
  8. Godson G. N., Sinsheimer R. L. Lysis of Escherichia coli with a neutral detergent. Biochim Biophys Acta. 1967 Dec 19;149(2):476–488. doi: 10.1016/0005-2787(67)90175-x. [DOI] [PubMed] [Google Scholar]
  9. Goulian M., Lucas Z. J., Kornberg A. Enzymatic synthesis of deoxyribonucleic acid. XXV. Purification and properties of deoxyribonucleic acid polymerase induced by infection with phage T4. J Biol Chem. 1968 Feb 10;243(3):627–638. [PubMed] [Google Scholar]
  10. Huberman J. A., Kornberg A., Alberts B. M. Stimulation of T4 bacteriophage DNA polymerase by the protein product of T4 gene 32. J Mol Biol. 1971 Nov 28;62(1):39–52. doi: 10.1016/0022-2836(71)90129-x. [DOI] [PubMed] [Google Scholar]
  11. Johns E. W. The electrophoresis of histones in polyacrylamide gel and their quantitative determination. Biochem J. 1967 Jul;104(1):78–82. doi: 10.1042/bj1040078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Moses R. E., Richardson C. C. Replication and repair of DNA in cells of Escherichia coli treated with toluene. Proc Natl Acad Sci U S A. 1970 Oct;67(2):674–681. doi: 10.1073/pnas.67.2.674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Nomura M. Bacterial ribosome. Bacteriol Rev. 1970 Sep;34(3):228–277. doi: 10.1128/br.34.3.228-277.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Okazaki R., Sugimoto K., Okazaki T., Imae Y., Sugino A. DNA chain growth: in vivo and in vitro synthesis in a DNA polymerase-negative mutant of E. coli. Nature. 1970 Oct 17;228(5268):223–226. doi: 10.1038/228223a0. [DOI] [PubMed] [Google Scholar]
  15. Riva S., Cascino A., Geiduschek E. P. Coupling of late transcription to viral replication in bacteriophage T4 development. J Mol Biol. 1970 Nov 28;54(1):85–102. doi: 10.1016/0022-2836(70)90447-x. [DOI] [PubMed] [Google Scholar]
  16. Schaller H., Otto B., Nüsslein V., Huf J., Herrmann R., Bonhoeffer F. Deoxyribonucleic acid replication in vitro. J Mol Biol. 1972 Jan 28;63(2):183–200. doi: 10.1016/0022-2836(72)90369-5. [DOI] [PubMed] [Google Scholar]
  17. Shapiro A. L., Viñuela E., Maizel J. V., Jr Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem Biophys Res Commun. 1967 Sep 7;28(5):815–820. doi: 10.1016/0006-291x(67)90391-9. [DOI] [PubMed] [Google Scholar]
  18. Siegel L. M., Monty K. J. Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductases. Biochim Biophys Acta. 1966 Feb 7;112(2):346–362. doi: 10.1016/0926-6585(66)90333-5. [DOI] [PubMed] [Google Scholar]
  19. Smith D. W., Schaller H. E., Bonhoeffer F. J. DNA synthesis in vitro. Nature. 1970 May 23;226(5247):711–713. doi: 10.1038/226711a0. [DOI] [PubMed] [Google Scholar]
  20. Warner H. R., Barnes J. E. Deoxyribonucleic acid synthesis in Escherichia coli infected with some deoxyribonucleic acid polymerase-less mutants of bacteriophage T4. Virology. 1966 Jan;28(1):100–107. doi: 10.1016/0042-6822(66)90310-2. [DOI] [PubMed] [Google Scholar]
  21. Warner H. R., Hobbs M. D. Incorporation of uracil-14C into nucleic acids in Escherichia coli infected with bacteriophage T4 and T4 amber mutants. Virology. 1967 Nov;33(3):376–384. doi: 10.1016/0042-6822(67)90113-4. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Yegian C. D., Mueller M., Selzer G., Russo V., Stahl F. W. Properties of the DNA-delay mutants of bacteriophage T4. Virology. 1971 Dec;46(3):900–919. doi: 10.1016/0042-6822(71)90090-0. [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