Functional analysis of putative restriction-modification system genes in the Helicobacter pylori J99 genome - PubMed (original) (raw)

Functional analysis of putative restriction-modification system genes in the Helicobacter pylori J99 genome

H Kong et al. Nucleic Acids Res. 2000.

Abstract

Helicobacter pylori is a gram-negative bacterium, which colonizes the gastric mucosa of humans and is implicated in a wide range of gastroduodenal diseases. The genomic sequences of two H.pylori strains, 26695 and J99, have been published recently. About two dozen potential restriction-modification (R-M) systems have been annotated in both genomes, which is far above the average number of R-M systems in other sequenced genomes. Here we describe a functional analysis of the 16 putative Type II R-M systems in the H. pylori J99 genome. To express potentially toxic endonuclease genes, a unique vector was constructed, which features repression and antisense transcription as dual control elements. To determine the methylation activities of putative DNA methyltransferases, we developed polyclonal antibodies able to detect DNA containing N6-methyladenine or N4-methylcytosine. We found that <30% of the potential Type II R-M systems in H.pylori J99 strain were fully functional, displaying both endonuclease and methyltransferase activities. Helicobacter pylori may maintain a variety of functional R-M systems, which are believed to be a primitive bacterial 'immune' system, by alternatively turning on/off a subset of numerous R-M systems.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Map of plasmid pLT7K. Ori, origin of replication (colE1 derived from pBR322); ampR, β-lactamase gene (ampicillin resistance gene); KanR, kanamycin resistance gene which is flanked by multiple restriction sites suitable for cloning; cI857, the gene for a mutant form of the λ repressor; _lac_I, the gene for the lactose repressor protein, LacI; PR, bacteriophage λ major rightward promoter; PT7, bacteriophage T7 promoter; OcI, λ operator bound by λ CI repressor; Olac, lac operator bound by _Lac_I.

Figure 2

Figure 2

Agarose gel (1%) showing the electrophoresis patterns of bacteriophage λ DNA digested by different amounts of restriction endonuclease, _Hpy_99II, encoded by JHP46. Lane 1, bacteriophage λ DNA + _Hin_dIII, φX174 + _Hae_III, size standard; lane 2, λ DNA digested with 9 µl _Hpy_99I; lane 3, λ DNA digested with 3 µl _Hpy_99I; lane 4, λ DNA digested with 1 µl _Hpy_99I; lane 5, λ DNA digested with 0.3 µl _Hpy_99I.

Figure 3

Figure 3

Agarose gel (1%) showing the electrophoresis patterns of λ DNA digested by restriction endonuclease _Hpy_99III, encoded by JHP1049, from consecutive fractions of a heparin column. Lane 1, λ DNA + _Hin_dIII, φX174 + _Hae_III, size standard; lanes 2–12, λ DNA digested with 5 µl of _Hpy_99III eluted from a heparin column.

Figure 4

Figure 4

Dot blot assay to measure methylase activities using rabbit primary antibodies that react specifically with DNA containing m6A or m4C modifications. The dot blot assay was performed as described in Materials and Methods. The ORF from which each DNA sample was derived is marked above each set of dots. (A) Methylase dot blot assay using m6A antibody with dilution of 1:750 000. (B) Methylase dot blot assay using m4C antibody with dilutions of 1:150 000. Three dilutions of DNA samples were spotted: 0.45, 0.15 and 0.05 µg of DNA (top to bottom on each panel).

Similar articles

Cited by

References

    1. Wilson G.G. and Murray,N.E. (1991) Restriction and modification systems. Annu. Rev. Genet., 25, 585–627. - PubMed
    1. Roberts R.J. (1990) Restriction enzymes and their isoschizomers. Nucleic Acids Res., 18, 2331–2365. - PMC - PubMed
    1. Smith H.O. and Wilcox,K.W. (1970) A restriction enzyme from Haemophilus influenzae. I. Purification and general properties. J. Mol. Biol., 51, 379–391. - PubMed
    1. Roberts R.J., Breitmeyer,J.B., Tabachnik,N.F. and Myers,P.A. (1975) A second specific endonuclease from Haemophilus aegyptius. J. Mol. Biol., 91, 121–123. - PubMed
    1. Schildkraut I.S. (1984) Screening for and characterizing restriction endonucleases. In Setlow,J.K. and Hollaender,A. (eds), Genetic Engineering, Principles and Methods, Vol. 6. Plenum Press, New York, NY, pp. 117–140.

Publication types

MeSH terms

Substances

LinkOut - more resources