1887

Abstract

SUMMARY: A series of plasmids has been constructed that can be used to fuse the β-galactosidase gene () of to chromosomal genes of . Insertion of the gene is facilitated by the use of a selectable chloramphenicol acetyl-transferase () gene. The latter is included, along with the gene, in a single DNA fragment or “cartridge” that can be removed from the plasmid with a variety of different restriction endonucleases. Methods applicable to any cloned gene are described that enable the cartridge to be inserted at specific sites, or at random, directly into the chromosome in a single step. These single-copy chromosomal fusions can be readily transferred, by selection for chloramphenicol resistance, to a temperate phage such as ϕ105, to permit a more extensive genetic analysis of the expression of the target gene. Alternatively, the cartridge and flanking DNA sequences can be transferred into different genetic backgrounds by transformation. These techniques have been used to construct, in a single step, fusions to genes in the sporulation operons and .

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1986-11-01
2024-04-18
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References

  1. Biggin M. D., Gibson T. J., Hong G. F. 1983; Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. Proceedings of the National Academy of Sciences of the United States of America volume80:3963–3965
    [Google Scholar]
  2. Brickman E., Silhavy T. J., Bassford P. J., Jr, Shuman H. A., Beckwith J. R. 1979; Sites within gene lacZ of Escherichia coli for formation of active hybrid β-galactosidase molecules. Journal of Bacteriology 139:13–18
    [Google Scholar]
  3. Bugaichuk U. D., Deadman M., Errington J., Savva D. 1984; Restriction enzyme analysis of Bacillus subtilis bacteriophage 0105 DNA. Journal of General Microbiology 130:2165–2167
    [Google Scholar]
  4. Casadaban M. J., Martinez-Arias A., Shapira S. K., Chou J. 1983; β-Galactosidase gene fusions for analyzing gene expression in Escherichia coli and yeast. Methods in Enzymology 100:293–308
    [Google Scholar]
  5. Clarke S., Lopez-Diaz I., Mandelstam J. 1986; Use of lacZ gene fusions to determine the dependence pattern of the sporulation gene spoIID in spo mutants of Bacillus subtilis. Journal of General Microbiology 132:2987–2944
    [Google Scholar]
  6. Donnelly C. E., Sonenshein A. L. 1984; Promoter-probe plasmid for Bacillus subtilis. Journal of Bacteriology 157:965–967
    [Google Scholar]
  7. Duncan C. H., Wilson G. A., Young F. E. 1978; Mechanism of integrating foreign DNA during transformation of Bacillus subtilis. Proceedings of the National Academy of Sciences of the United States of America 75:3664–3668
    [Google Scholar]
  8. Errington J. 1984; Efficient Bacillus subtilis cloning system using bacteriophage vector ϕ105J9. Journal of General Microbiology 130:2615–2628
    [Google Scholar]
  9. Errington J., Mandelstam J. 1983; Variety of sporulation phenotypes resulting from mutations in a single regulatory locus, spoIIA, in Bacillus subtilis. Journal of General Microbiology 129:2091–2101
    [Google Scholar]
  10. Errington J., Mandelstam J. 1986a; Use of a lacZ gene fusion to determine the dependence pattern of sporulation operon spoIIA in spo mutants of Bacillus subtilis. Journal of General Microbiology 132:2967–2976
    [Google Scholar]
  11. Errington J., Mandelstam J. 1986b; Use of a lacZ gene fusion to determine the dependence pattern and the spore compartment expression of sporulation operon spo VA in spo mutants of Bacillus subtilis. Journal of General Microbiology 132:2977–2985
    [Google Scholar]
  12. Ferrari E., Hoch J. A. 1983; A single copy, transducible system for complementation and domi¬nance analyses in Bacillus subtilis. Molecular and General Genetics 189:321–325
    [Google Scholar]
  13. Flock J.-I. 1977; Deletion mutants of temperate Bacillus subtilis bacteriophage 105. Molecular and General Genetics 155:241–247
    [Google Scholar]
  14. Fort,P. Errington J. 1985; Nucleotide sequence and complementation analysis of a polycistronic sporulation operon, spoVA, in Bacillus subtilis. Journal of General Microbiology 131:1091–1105
    [Google Scholar]
  15. Fort P., Piggot P. J. 1984; Nucleotide sequence of sporulation locus spoil A in Bacillus subtilis. Journal of General Microbiology 130:2147–2153
    [Google Scholar]
  16. Grunstein M., Hogness D. S. 1975; Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proceedings of the National Academy of Sciences of the United States of America 72:3961–3965
    [Google Scholar]
  17. Gryczan T., Contente S., Dubnau D. 1980; Molecular cloning of heterologous chromosomal DNA by recombination between a plasmid vector and a homologous resident plasmid in Bacillus subtilis. Molecular and General Genetics 177:459–467
    [Google Scholar]
  18. Guo L.-H., Yang R. C. A., Wu R. 1983; An improved strategy for rapid direct sequencing of both strands of long DNA molecules cloned in a plasmid. Nucleic Acids Research 11:5521–5540
    [Google Scholar]
  19. Haldenwang W. G., Banner C. D. B., Ollington J. F., Losick R., Hoch J. A., O’Connor M. B., Sonenshein A. L. 1980; Mapping a cloned gene under sporulation control by insertion of a drug resistance marker into the Bacillus subtilis chromosome. Journal of Bacteriology 142:90–98
    [Google Scholar]
  20. Hirota Y. 1960; The effect of acridine dyes on mating type factors in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America 46:57–64
    [Google Scholar]
  21. Ionesco H., Schaeffer P. 1968; Localisation chromosomique de certain mutants asporogenes de Bacillus subtilis Marburg. Annales de I’Institut Pasteur 114:1–9
    [Google Scholar]
  22. Iordanescu S., Surdeanu M., Della Latta P., Novick R. 1978; Incompatibility and molecular relationships between small staphylococcal plasmids carrying the same resistance marker. Plasmid 1:468–479
    [Google Scholar]
  23. Jenkinson H. F., Mandelstam J. 1983; Cloning of the Bacillus subtilis lys and spoIIIB genes in phage ϕ105. Journal of General Microbiology 129:2229–2240
    [Google Scholar]
  24. Kawamura F., Saito H., Ikeda Y. 1979; A method for construction of specialized transducing phage pll of Bacillus subtilis. Gene 5:87–91
    [Google Scholar]
  25. Liu H.-M., Chak K. F., Piggot P. J. 1982; Isolation and characterization of a recombinant plasmid carrying a functional part of the Bacillus subtilis spoil A locus. Journal of General Microbiology 128:2805–2812
    [Google Scholar]
  26. Messing J. 1983; New M13 vectors for cloning. Methods in Enzymology 101:20–78
    [Google Scholar]
  27. Messing J., Crea R., Seeburg P. H. 1981; A system for shotgun DNA sequencing. Nucleic Acids Research 9:309–321
    [Google Scholar]
  28. Miller J. 1972 Experiments in Molecular Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  29. Moran C. P., Lang N. Jr, Le Grice S. F. J., Lee G., Stephens M., Sonenshein A. L., Pero J., Losick R. 1982; Nucleotide sequences that signal the initiation of transcription and translation in Bacillus subtilis. Molecular and General Genetics 186:339–346
    [Google Scholar]
  30. Niaudet B., Goze A., Ehrlich S. D. 1982; Insertional mutagenesis in Bacillus subtilis: mech¬anism and use in gene cloning. Gene 19:277–284
    [Google Scholar]
  31. Norrander J., Kempe T., Messing J. 1983; Construction of improved M13 vectors using oligo-deoxynucleotide-directed mutagenesis. Gene 26:101–106
    [Google Scholar]
  32. Piggot P. J., Curtis C. A. M., Lencastre H. 1984; Use of integrational plasmid vectors to demonstrate the polycistronic nature of a transcriptional unit (spoIIA) required for sporulation of Bacillus subtilis. Journal of General Microbiology 130:2123–2136
    [Google Scholar]
  33. Piggot P. J., Chapman J. W., Curtis C. A. M. 1985; Analysis of the control of spo gene expression in Bacillus subtilis. In Molecular Biology of Microbial Differentiation pp. 15–21 Edited by Setlow P., Hoch J. Washington, DC: American Society for Microbiology.;
    [Google Scholar]
  34. Rigby P. W. J., Dieckmann M., Rhodes C., Berg P. 1977; Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. Journal of Molecular Biology 113:237–251
    [Google Scholar]
  35. Sanger F., Nicklen S., Coulson A. R. 1977; DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences of the United States of America 74:5463–5467
    [Google Scholar]
  36. Sanger F., Coulson A. R., Barrell B. G., Smith A. J. H., Roe B. A. 1980; Cloning in single¬stranded bacteriophage as an aid to rapid DNA sequencing. Journal of Molecular Biology 143:161–178
    [Google Scholar]
  37. Savva D., Mandelstam J. 1984; Cloning of the Bacillus subtilis spoIIA and spoVA loci in phage ϕ105DI:lt. Journal of General Microbiology 130:2137–2145
    [Google Scholar]
  38. Savva D., Mandelstam J. 1985; Use of cloned spoIIA and spoVA probes to study synthesis of mRNA in wild-type and asporogenous mutants of Bacillus subtilis. In Molecular Biology of Microbial Differentiation pp. 55–59 Edited by Setlow P., Hoch J. Washington, DC: American Society for Microbiology;
    [Google Scholar]
  39. Southern E. M. 1975; Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology 98:503–517
    [Google Scholar]
  40. Stephens M. A., Lang N., Sandman K., Losick R. 1984; A promoter whose utilization is temporally regulated during sporulation in Bacillus subtilis. Journal of Molecular Biology 176:333–348
    [Google Scholar]
  41. Turner S. M., Errington J., Mandelstam J. 1986; Use of a lacZ gene fusion to determine the dependence pattern of sporulation operon spoIIIC in spo mutants of Bacillus subtilis: a branched pathway of expression of sporulation operons. Journal of General Microbiology 132:2995–3003
    [Google Scholar]
  42. Youngman P., Zuber P., Perkins J. B., Sandman K., Igo M., Losick R. 1985; New ways to study developmental genes in spore-forming bacteria. Science 228:285–291
    [Google Scholar]
  43. Zahler S. A. 1982; Specialized transduction in Bacillus subtilis. In The Molecular Biology of the Bacilli vol. 1269–305 Edited by Dubnau D. A. New York London: Academic Press;
    [Google Scholar]
  44. Zuber P., Losick R. 1983; Use of a lacZ fusion to study the role of spoO genes of Bacillus subtilis in developmental regulation. Cell 35:275–283
    [Google Scholar]
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