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Analyzing the Developmental Expression of Sigma Factors with S1-Nuclease Mapping

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Nuclease Methods and Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 160))

Abstract

Streptomycetes are mycelial, Gram-positive soil bacteria which produce a variety of biologically active secondary metabolites, including the majority of known antibiotics. In addition to this physiological differentiation, they undergo a complex cycle of morphological differentiation. Spores after germination on solid-growth media form a network of vegetative “substrate mycelium” that grows into the medium. After unknown signal(s), the substrate mycelium differentiates, and white aerial mycelium is formed that grows into the air. Later it undergoes septation and, in the last step of the process, forms chains of spores. The differentiation of Streptomyces is controlled on several levels, of which heterogeneity of σ factors of RNA polymerase seems to play an important role (1). Therefore, we examined the Streptomyces differentiation through the study of these key proteins in this process.

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References

  1. Chater, K. F. (1993) Genetics of differentiation in Streptomyces. Annu. Rev. Microbiol. 47, 685–713.

    Article  PubMed  CAS  Google Scholar 

  2. Lonetto, M., Gribskov, M., and Gross, C. A. (1992) The σ70 family: sequence conservation and evolutionary relationships. J. Bacteriol. 174, 3843–3849.

    PubMed  CAS  Google Scholar 

  3. Kormanec, J., Farkasovsky, M., and Potuckova, L. (1992) Four genes in Streptomyces aureofaciens containing a domain characteristic of principal sigma factors. Gene 122, 63–70.

    Article  PubMed  CAS  Google Scholar 

  4. Kormanec, J., Potuckova, L., and Rezuchova, B. (1994) The Streptomyces aureofaciens homologue of the whiG encoding a putative sigma factor essential for sporulation. Gene 143, 101–103.

    Article  PubMed  CAS  Google Scholar 

  5. Potuckova, L., Kelemen, G. H., Findlay, K. C, Lonetto, M. A., Buttner, M. J., and Kormanec, J. (1995) A new RNA polymerase sigma factor, σF, is required for the late stages of morphological differentiation in Streptomyces spp. Mol. Microbiol. 17, 37–48.

    Article  PubMed  CAS  Google Scholar 

  6. Rezuchova, B., Barak, I., and Kormanec, J. (1997) Disruption of a sigma factor gene, sigF, affects an intermediate stage of spore pigment production in Streptomyces aureofaciens. FEMS Microbiol. Let. 153, 371–377.

    Article  CAS  Google Scholar 

  7. Kormanec, J. and Farkasovsky, M. (1993) Differential expression of principal sigma factor homologues of Streptomyces aureofaciens correlates with developmental stage. Nucleic Acids Res. 21, 3647–3652.

    Article  PubMed  CAS  Google Scholar 

  8. Kormanec, J., Homerovà, D., Potuckova, L., Novakova, R., and Rezuchova, B. (1996) Differential expression of two sporulation specific σ factors of Streptomyces aureofaciens correlates with the developmental stage. Gene 181, 19–27.

    Article  PubMed  CAS  Google Scholar 

  9. Berk, A. J. and Sharp, P. A. (1977) Sizing and mapping of early adenovirus mRNA by gel electrophoresis of S1 endonuclease hybrids. Cell 12, 721–732.

    Article  PubMed  CAS  Google Scholar 

  10. Burke, J. F. (1984) High-sensitivity S1 mapping with single-stranded [32P]DNA probes synthesized from bacteriophage M13mp templates. Gene 30, 63–68.

    Article  PubMed  CAS  Google Scholar 

  11. Murray, M. G. (1986) Use of sodium trichloracetate and mung bean nuclease to increase sensistivity and precision during transcript mapping. Anal. Biochem. 158, 165–170.

    Article  PubMed  CAS  Google Scholar 

  12. Aldea, M., Claverie-Martin, F., Diaz-Torres, M. R., and Kushner, S. R. (1988) Transcript mapping using [35S]DNA probes, trichloracetate solvent and dideoxy sequencing ladders: a rapid method for identification of transcriptional start points. Gene 65, 101–110.

    Article  PubMed  CAS  Google Scholar 

  13. Weaver, R. F. and Weissmann, C. (1979) Mapping of RNA by a modification of the Berk-Sharp procedure: the 5′ termini of 15 S beta-globin mRNA precursor and mature 10 S beta-globin mRNA have identical map coordinates. Nucleic Acids Res 7, 1175–1193.

    Article  PubMed  CAS  Google Scholar 

  14. Maxam, A. M. and Gilbert, W. (1980) Sequencing end-labelled DNA with base specific chemical cleavages. Methods Enzymol 65, 449–560.

    Article  Google Scholar 

  15. Kormanec, J. and Farkasovsky, M. (1994) Isolation of total RNA from yeast and bacteria and detection of rRNA in Northern blots. BioTechniques 17, 838–842.

    PubMed  CAS  Google Scholar 

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Kormanec, J. (2001). Analyzing the Developmental Expression of Sigma Factors with S1-Nuclease Mapping. In: Schein, C.H. (eds) Nuclease Methods and Protocols. Methods in Molecular Biology™, vol 160. Humana Press. https://doi.org/10.1385/1-59259-233-3:481

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  • DOI: https://doi.org/10.1385/1-59259-233-3:481

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-679-6

  • Online ISBN: 978-1-59259-233-3

  • eBook Packages: Springer Protocols

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