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Selection analyses of insertional mutants using subgenic-resolution arrays

Nature Biotechnology volume 19, pages 1060–1065 (2001)Cite this article

Abstract

We describe a method of genome-wide analysis of quantitative growth phenotypes using insertional mutagenesis and DNA microarrays. We applied the method to assess the fitness contributions of Escherichia coli gene domains under specific growth conditions. A transposon library was subjected to competitive growth selection in Luria–Bertani (LB) and in glucose minimal media. Transposon-containing genomic DNA fragments from the selected libraries were compared with the initial unselected transposon insertion library on DNA microarrays to identify insertions that affect fitness. Genes involved in the biosynthesis of nutrients not provided in the growth medium were found to be significantly enriched in the set of genes containing negatively selected insertions. The data also identify fitness contributions of several uncharacterized genes, including putative transcriptional regulators and enzymes. The applicability of this high-resolution array selection in other species is discussed.

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References

  1. Riley, M. Genes and proteins of Escherichia coli K-12. Nucleic Acids Res. 26, 54 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Smith, V., Chou, K.N., Lashkari, D., Botstein, D. & Brown, P.O. Functional analysis of the genes of yeast chromosome V by genetic footprinting. Science 274, 2069–2074 (1996).

    Article  CAS  PubMed  Google Scholar 

  3. Winzeler, E.A. et al. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285, 901–906 (1999).

    Article  CAS  PubMed  Google Scholar 

  4. Akerley, B.J. et al. Systematic identification of essential genes by in vitro mariner mutagenesis. Proc. Natl. Acad. Sci. USA 95, 8927–8932 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Wong, S.M. & Mekalanos, J.J. Genetic footprinting with mariner-based transposition in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 97, 10191–10196 (2000).

    CAS  Google Scholar 

  6. Hutchison, C.A. et al. Global transposon mutagenesis and a minimal Mycoplasma genome. Science 286, 2165–2169 (1999).

    Article  CAS  PubMed  Google Scholar 

  7. Hare, R.S. et al. Genetic footprinting in bacteria. J. Bacteriol. 183, 1694–1706 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Schena, M., Shalon, D., Davis, R.W. & Brown, P.O. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270, 467–470 (1995).

    Article  CAS  PubMed  Google Scholar 

  9. Iyer, V.R. et al. Genomic binding sites of the yeast cell-cycle transcription factors SBF and MBF. Nature 409, 533–538 (2001).

    Article  CAS  PubMed  Google Scholar 

  10. Ren, B. et al. Genome-wide location and function of DNA binding proteins. Science 290, 2306–2309 (2000).

    Article  CAS  PubMed  Google Scholar 

  11. Pollack, J.R. et al. Genome-wide analysis of DNA copy-number changes using cDNA microarrays. Nat. Genet. 23, 41–46 (1999).

    Article  CAS  PubMed  Google Scholar 

  12. Alexeyev, M.F. & Shokolenko, I.N. Mini-Tn10 transposon derivatives for insertion mutagenesis and gene delivery into the chromosome of gram-negative bacteria. Gene 160, 59–62 (1995).

    Article  CAS  PubMed  Google Scholar 

  13. Bender, J. & Kleckner, N. IS10 transposase mutations that specifically alter target site recognition. EMBO J. 11, 741–750 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Tavazoie, S. & Church, G.M. Quantitative whole-genome analysis of DNA–protein interactions by in vivo methylase protection in E. coli. Nat. Biotechnol. 16, 566–571 (1998).

    Article  CAS  PubMed  Google Scholar 

  15. Robison, K., McGuire, A.M. & Church, G.M. A comprehensive library of DNA-binding site matrices for 55 proteins applied to the complete Escherichia coli K-12 genome. J. Mol. Biol. 284, 241–254 (1998).

    Article  CAS  PubMed  Google Scholar 

  16. Arigoni, F. et al. A genome-based approach for the identification of essential bacterial genes. Nat. Biotechnol. 16, 851–856 (1998).

    Article  CAS  PubMed  Google Scholar 

  17. Kanehisa, M. & Goto, S. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 28, 27–30 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Edwards, J.S. & Palsson, B.O. The Escherichia coli MG1655 in silico metabolic genotype: its definition, characteristics, and capabilities. Proc. Natl. Acad. Sci. USA 97, 5528–5533 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Edwards, J.S., Ibarra, R.U. & Palsson, B.O. In silico predictions of Escherichia coli metabolic capabilities are consistent with experimental data. Nat. Biotechnol. 19, 125–130 (2001).

    Article  CAS  PubMed  Google Scholar 

  20. Zwaal, R.R., Broeks, A., van Meurs, J., Groenen, J.T. & Plasterk, R.H. Target-selected gene inactivation in Caenorhabditis elegans by using a frozen transposon insertion mutant bank. Proc. Natl. Acad. Sci. USA 90, 7431–7435 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Giaever, G. et al. Genomic profiling of drug sensitivities via induced haploinsufficiency. Nat. Genet. 21, 278–283 (1999).

    Article  CAS  PubMed  Google Scholar 

  22. Hicks, G.G. et al. Functional genomics in mice by tagged sequence mutagenesis. Nat. Genet. 16, 338–344 (1997).

    Article  CAS  PubMed  Google Scholar 

  23. Zambrowicz, B.P. et al. Disruption and sequence identification of 2,000 genes in mouse embryonic stem cells. Nature 392, 608–611 (1998).

    Article  CAS  PubMed  Google Scholar 

  24. Sambrook, J., Fritsch, E.F. & Maniatis, T. Molecular cloning: a laboratory manual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; 1989).

    Google Scholar 

  25. Ausubel, F.M. et al. Current protocols in molecular biology. (Wiley Interscience, New York, NY; 1994).

    Google Scholar 

  26. Marton, M.J. et al. Drug target validation and identification of secondary drug target effects using DNA microarrays. Nat. Med. 4, 1293–1301 (1998).

    Article  CAS  PubMed  Google Scholar 

  27. Altschul, S.F., Gish, W., Miller, W., Myers, E.W., & Lipman, D.J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990).

    Article  CAS  PubMed  Google Scholar 

  28. Livesey, F.J., Furukawa, T., Steffen, M.A., Church, G.M. & Cepko, C.L. Microarray analysis of the transcriptional network controlled by the photoreceptor homeobox gene Crx. Curr. Biol. 10, 301–310 (2000).

    CAS  PubMed  Google Scholar 

  29. Tavazoie, S., Hughes, J.D., Campbell, M.J., Cho, R.J. & Church, G.M. Systematic determination of genetic network architecture. Nat. Genet. 22, 281–285 (1999).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We would like to thank Martin Steffen and Xiaohua Huang for their tireless efforts to produce the E. coli microarrays, Barak Cohen and Tzachi Pilpel for helpful discussion, and members of the Church lab for their support and encouragement. We would also like to thank the referees for their insightful comments. This work was supported by grants from the US Department of Energy and the National Science Foundation.

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Author notes

  1. Jeremy Edwards

    Present address: Department of Chemical Engineering, University of Delaware, Newark, DE, 19716

Authors and Affiliations

  1. Department of Genetics, Harvard Medical School, Boston, 02115, MA

    Vasudeo Badarinarayana, Preston W. Estep III, Jay Shendure, Jeremy Edwards, Felix Lam & George M. Church

  2. Department of Molecular Biology, Princeton University, Princeton, 08544, NJ

    Saeed Tavazoie

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  1. Vasudeo Badarinarayana
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Correspondence to George M. Church.

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Badarinarayana, V., Estep, P., Shendure, J. et al. Selection analyses of insertional mutants using subgenic-resolution arrays. Nat Biotechnol 19, 1060–1065 (2001). https://doi.org/10.1038/nbt1101-1060

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