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J Bacteriol. 1984 July; 159(1): 71–79.
PMCID: PMC215594

Phase variation and the Hin protein: in vivo activity measurements, protein overproduction, and purification.

Abstract

The alternate expression of the Salmonella flagellin genes H1 and H2 is controlled by the orientation of a 995-base-pair invertible segment of DNA located at the 5' end of the H2 gene. The hin gene, which is encoded within the invertible region, is essential for the inversion of this DNA segment. We cloned the hin gene into Escherichia coli and placed it under the control of the PL promoter of bacteriophage lambda. These cells overproduced the Hin protein. In vivo inversion activity was measured by using a recombinant lambda phage which contains the H2 and lacZ genes under the control of the invertible region. Using this phage, we showed that the amount of inversion activity is proportional to the amount of Hin protein in the cell. An inactive form of the protein was purified by using the unusual solubility properties of the overproduced protein. The amino acid composition of the protein agreed with the DNA sequence of the hin gene. Antibodies were made to the isolated protein. These antibodies cross-reacted with two other unidentified E. coli proteins.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Birnboim HC, Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. [PMC free article] [PubMed]
  • Blattner FR, Williams BG, Blechl AE, Denniston-Thompson K, Faber HE, Furlong L, Grunwald DJ, Kiefer DO, Moore DD, Schumm JW, et al. Charon phages: safer derivatives of bacteriophage lambda for DNA cloning. Science. 1977 Apr 8;196(4286):161–169. [PubMed]
  • Enomoto M, Oosawa K, Momota H. Mapping of the pin locus coding for a site-specific recombinase that causes flagellar-phase variation in Escherichia coli K-12. J Bacteriol. 1983 Nov;156(2):663–668. [PMC free article] [PubMed]
  • Gill R, Heffron F, Dougan G, Falkow S. Analysis of sequences transposed by complementation of two classes of transposition-deficient mutants of Tn3. J Bacteriol. 1978 Nov;136(2):742–756. [PMC free article] [PubMed]
  • Gottesman ME, Adhya S, Das A. Transcription antitermination by bacteriophage lambda N gene product. J Mol Biol. 1980 Jun 15;140(1):57–75. [PubMed]
  • Hawkes R, Niday E, Gordon J. A dot-immunobinding assay for monoclonal and other antibodies. Anal Biochem. 1982 Jan 1;119(1):142–147. [PubMed]
  • Hedgpeth J, Ballivet M, Eisen H. Lambda phage promoter used to enhance expression of a plasmid-cloned gene. Mol Gen Genet. 1978 Jul 11;163(2):197–203. [PubMed]
  • Hunkapiller MW, Lujan E, Ostrander F, Hood LE. Isolation of microgram quantities of proteins from polyacrylamide gels for amino acid sequence analysis. Methods Enzymol. 1983;91:227–236. [PubMed]
  • Komeda Y, Silverman M, Simon M. Genetic analysis of Escherichia coli K-12 region I flagellar mutants. J Bacteriol. 1977 Sep;131(3):801–808. [PMC free article] [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • March SC, Parikh I, Cuatrecasas P. A simplified method for cyanogen bromide activation of agarose for affinity chromatography. Anal Biochem. 1974 Jul;60(1):149–152. [PubMed]
  • Plasterk RH, Brinkman A, van de Putte P. DNA inversions in the chromosome of Escherichia coli and in bacteriophage Mu: relationship to other site-specific recombination systems. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5355–5358. [PubMed]
  • Reed RR. Transposon-mediated site-specific recombination: a defined in vitro system. Cell. 1981 Sep;25(3):713–719. [PubMed]
  • Scott TN, Simon MI. Genetic analysis of the mechanism of the Salmonella phase variation site specific recombination system. Mol Gen Genet. 1982;188(2):313–321. [PubMed]
  • Silverman M, Zieg J, Hilmen M, Simon M. Phase variation in Salmonella: genetic analysis of a recombinational switch. Proc Natl Acad Sci U S A. 1979 Jan;76(1):391–395. [PubMed]
  • Silverman M, Simon M. Phase variation: genetic analysis of switching mutants. Cell. 1980 Apr;19(4):845–854. [PubMed]
  • Silverman M, Zieg J, Mandel G, Simon M. Analysis of the functional components of the phase variation system. Cold Spring Harb Symp Quant Biol. 1981;45(Pt 1):17–26. [PubMed]
  • Silverman M, Zieg J, Simon M. Flagellar-phase variation: isolation of the rh1 gene. J Bacteriol. 1979 Jan;137(1):517–523. [PMC free article] [PubMed]
  • Simon M, Zieg J, Silverman M, Mandel G, Doolittle R. Phase variation: evolution of a controlling element. Science. 1980 Sep 19;209(4463):1370–1374. [PubMed]
  • Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. [PubMed]
  • Zieg J, Hilmen M, Simon M. Regulation of gene expression by site-specific inversion. Cell. 1978 Sep;15(1):237–244. [PubMed]
  • Zieg J, Silverman M, Hilmen M, Simon M. Recombinational switch for gene expression. Science. 1977 Apr 8;196(4286):170–172. [PubMed]
  • Zieg J, Simon M. Analysis of the nucleotide sequence of an invertible controlling element. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4196–4200. [PubMed]

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