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. 1982 Aug;79(16):5033–5037. doi: 10.1073/pnas.79.16.5033

Cloning and mapping of a gene for translational initiation factor IF2 in Escherichia coli.

J A Plumbridge, J G Howe, M Springer, D Touati-Schwartz, J W Hershey, M Grunberg-Manago
PMCID: PMC346821  PMID: 6214789

Abstract

A novel method, not relying on genetic complementation of a mutation, was used to clone a gene for translational initiation factor IF2. Two clones from a cosmid library of total Escherichia coli DNA were isolated for their ability to overproduce IF2 in vivo as determined by quantitative immunoblotting. "Maxicell" analysis of cosmid-encoded proteins and specific immune precipitation of the labeled proteins showed that the structural gene for IF2 (inf B) had been cloned. Subcloning fragments from the original cosmids located the inf B gene to a 4.8-kilobase pair HindIII/BamHI fragment. This fragment has been inserted into an integration-deficient recombinant lambda phage that lysogenizes by homology. By mapping the point of lysogenization on the E. coli chromosome, inf B has been located at 68 min, very close to argG, nusA, rpsO, and pnp. Because the gene for initiation factor IF3 is located at 38 min on the chromosome, the genes for translational initiation factors are not grouped together.

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Selected References

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

  1. Bachmann B. J., Low K. B. Linkage map of Escherichia coli K-12, edition 6. Microbiol Rev. 1980 Mar;44(1):1–56. doi: 10.1128/mr.44.1.1-56.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Borck K., Beggs J. D., Brammar W. J., Hopkins A. S., Murray N. E. The construction in vitro of transducing derivatives of phage lambda. Mol Gen Genet. 1976 Jul 23;146(2):199–207. doi: 10.1007/BF00268089. [DOI] [PubMed] [Google Scholar]
  3. Chamberlain J. P. Fluorographic detection of radioactivity in polyacrylamide gels with the water-soluble fluor, sodium salicylate. Anal Biochem. 1979 Sep 15;98(1):132–135. doi: 10.1016/0003-2697(79)90716-4. [DOI] [PubMed] [Google Scholar]
  4. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Enquist L., Tiemeier D., Leder P., Weisberg R., Sternberg N. Safer derivatives of bacteriophage lambdagt-lambdaC for use in cloning of recombinant DNA molecules. Nature. 1976 Feb 19;259(5544):596–598. doi: 10.1038/259596a0. [DOI] [PubMed] [Google Scholar]
  6. Friedman D. I., Baron L. S. Genetic characterization of a bacterial locus involved in the activity of the N function of phage lambda. Virology. 1974 Mar;58(1):141–148. doi: 10.1016/0042-6822(74)90149-4. [DOI] [PubMed] [Google Scholar]
  7. Friedman D. I., Baumann M., Baron L. S. Cooperative effects of bacterial mutations affecting lambda N gene expression. I. Isolation and characterization of a nusB mutant. Virology. 1976 Aug;73(1):119–127. doi: 10.1016/0042-6822(76)90066-0. [DOI] [PubMed] [Google Scholar]
  8. Greenblatt J., Li J. The nusA gene protein of Escherichia coli. Its identification and a demonstration that it interacts with the gene N transcription anti-termination protein of bacteriophage lambda. J Mol Biol. 1981 Mar 25;147(1):11–23. doi: 10.1016/0022-2836(81)90076-0. [DOI] [PubMed] [Google Scholar]
  9. Greenblatt J., McLimont M., Hanly S. Termination of transcription by nusA gene protein of Escherichia coli. Nature. 1981 Jul 16;292(5820):215–220. doi: 10.1038/292215a0. [DOI] [PubMed] [Google Scholar]
  10. Hohn B., Collins J. A small cosmid for efficient cloning of large DNA fragments. Gene. 1980 Nov;11(3-4):291–298. doi: 10.1016/0378-1119(80)90069-4. [DOI] [PubMed] [Google Scholar]
  11. Hohn B. In vitro packaging of lambda and cosmid DNA. Methods Enzymol. 1979;68:299–309. doi: 10.1016/0076-6879(79)68021-7. [DOI] [PubMed] [Google Scholar]
  12. Howe J. G., Hershey J. W. A sensitive immunoblotting method for measuring protein synthesis initiation factor levels in lysates of Escherichia coli. J Biol Chem. 1981 Dec 25;256(24):12836–12839. [PubMed] [Google Scholar]
  13. Howe J. G., Hershey J. W. Immunochemical analysis of molecular forms of protein synthesis initiation factors in crude cell lysates of Escherichia coli. Arch Biochem Biophys. 1982 Apr 1;214(2):446–451. doi: 10.1016/0003-9861(82)90048-0. [DOI] [PubMed] [Google Scholar]
  14. Howe J. G., Yanov J., Meyer L., Johnston K., Hershey J. W. Determination of protein synthesis initiation factor levels in crude lysates of Escherichia coli by a sensitive radioimmune assay. Arch Biochem Biophys. 1978 Dec;191(2):813–820. doi: 10.1016/0003-9861(78)90424-1. [DOI] [PubMed] [Google Scholar]
  15. Kenny J. W., Lambert J. M., Traut R. R. Cross-linking of ribosomes using 2-iminothiolane (methyl 4-mercaptobutyrimidate) and identification of cross-linked proteins by diagonal polyacrylamide/sodium dodecyl sulfate gel electrophoresis. Methods Enzymol. 1979;59:534–550. doi: 10.1016/0076-6879(79)59112-5. [DOI] [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Lestienne P., Dondon J., Plumbridge J. A., Howe J. G., Mayaux J. F., Springer M., Blanquet S., Hershey J. W., Grunberg-Manago M. Expression of the gene for Escherichia coli initiation factor IE-3 in vivo and in vitro. Eur J Biochem. 1982 Apr;123(3):483–488. doi: 10.1111/j.1432-1033.1982.tb06556.x. [DOI] [PubMed] [Google Scholar]
  18. Low B. Rapid mapping of conditional and auxotrophic mutations in Escherichia coli K-12. J Bacteriol. 1973 Feb;113(2):798–812. doi: 10.1128/jb.113.2.798-812.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Plumbridge J. A., Springer M., Graffe M., Goursot R., Grunberg-Manago M. Physical localisation and cloning of the structural gene for E. coli initiation factor IF3 from a group of genes concerned with translation. Gene. 1980 Oct;11(1-2):33–42. doi: 10.1016/0378-1119(80)90084-0. [DOI] [PubMed] [Google Scholar]
  20. Portier C. Isolation of a polynucleotide phosphorylase mutant using a kanamycin resistant determinant. Mol Gen Genet. 1980;178(2):343–349. doi: 10.1007/BF00270482. [DOI] [PubMed] [Google Scholar]
  21. Portier C., Migot C., Grumberg-Manago M. Cloning of E. coli pnp gene from an episome. Mol Gen Genet. 1981;183(2):298–305. doi: 10.1007/BF00270632. [DOI] [PubMed] [Google Scholar]
  22. Sancar A., Hack A. M., Rupp W. D. Simple method for identification of plasmid-coded proteins. J Bacteriol. 1979 Jan;137(1):692–693. doi: 10.1128/jb.137.1.692-693.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Shimada K., Weisberg R. A., Gottesman M. E. Prophage lambda at unusual chromosomal locations. I. Location of the secondary attachment sites and the properties of the lysogens. J Mol Biol. 1972 Feb 14;63(3):483–503. doi: 10.1016/0022-2836(72)90443-3. [DOI] [PubMed] [Google Scholar]
  24. Springer M., Graffe M., Grunberg-Manago M. Characterization of an E. coli mutant with a thermolabile initiation factor IF3 activity. Mol Gen Genet. 1977 Feb 28;151(1):17–26. doi: 10.1007/BF00446908. [DOI] [PubMed] [Google Scholar]
  25. Springer M., Graffe M., Hennecke H. Specialized transducing phage for the initiation factor 3 gene in Escherichia coli. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3970–3974. doi: 10.1073/pnas.74.9.3970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Szybalski E. H., Szybalski W. A comprehensive molecular map of bacteriophage lambda. Gene. 1979 Nov;7(3-4):217–270. doi: 10.1016/0378-1119(79)90047-7. [DOI] [PubMed] [Google Scholar]

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