Cloning the<i>Dde</i>I restriction-modification system using a two-step method

Howard, Katherine; Card, Charles O.; Benner, Jack S.; Callahan, Hilary; Maunus, Robert; Silber, Karen R.; Wilson, Geoffrey G.; Brooks, Joan E.

doi:10.1093/nar/14.20.7939

Cited by 46 publications

(13 citation statements)

References 34 publications

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“…Sometimes, introducing both endonuclease and methylase genes into a new host cell at the same time could cause problems, because the endogenous DNA may not be sufficiently modified by the incoming MTase if the endonuclease gene is expressed immediately (22). To reduce the possibility of obtaining false-negative results, those putative H. pylori endonuclease genes whose gene products showed no detectable endonuclease activity in the pUC19 system were cloned into pLT7K.…”

Section: Resultsmentioning

confidence: 99%

Comparative genomics of the restriction-modification systems in Helicobacter pylori

Lin

Pósfai

Roberts

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

153

164

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Helicobacter pylori is a Gram-negative bacterial pathogen with a small genome of 1.64 -1.67 Mb. More than 20 putative DNA restrictionmodification (R-M) systems, comprising more than 4% of the total genome, have been identified in the two completely sequenced H. pylori strains, 26695 and J99, based on sequence similarities. In this study, we have investigated the biochemical activities of 14 Type II R-M systems in H. pylori 26695. Less than 30% of the Type II R-M systems in 26695 are fully functional, similar to the results obtained from strain J99. Although nearly 90% of the R-M genes are shared by the two H. pylori strains, different sets of these R-M genes are functionally active in each strain. Interestingly, all strain-specific R-M genes are active, whereas most shared genes are inactive. This agrees with the notion that strain-specific genes have been acquired more recently through horizontal transfer from other bacteria and selected for function. Thus, they are less likely to be impaired by random mutations. Our results also show that H. pylori has extremely diversified R-M systems in different strains, and that the diversity may be maintained by constantly acquiring new R-M systems and by inactivating and deleting the old ones.H elicobacter pylori is one of the most common bacterial pathogens that colonizes the gastric mucosa of humans. H. pylori is implicated in a wide range of gastroduodenal diseases (1, 2). H. pylori is commonly believed to be a very diverse species. It is believed that, in addition to genetic recombination, de novo mutation could have a role in generating the high level of genetic variation in H. pylori (3). MutH and MutL homologues cannot be found in H. pylori genomes, which suggests H. pylori may not have a functional mismatch repair system (4, 5). Recent analysis of the complete genomic sequences of two unrelated H. pylori isolates reveals that although intraspecies variation does exist, the overall genomic organization, gene order, and predicted proteins of the two strains are quite similar (5, 6). Approximately 6-7% of the genes are specific to each strain (5). The 26695 and J99 strains have a relatively small genome size of 1.67 and 1.64 megabase pairs (4, 5). However, more than twenty DNA restriction-modification (R-M) systems can be identified in each strain based on sequence similarities. The biological significance of this large complement of R-M systems is not clear. The majority of the H. pylori R-M systems are of Type II, which consist of two separate enzymes: the restriction endonucleases, which are responsible for degrading unmodified foreign DNA, and the modification DNA methyltransferases (methylase or M), which protect endogenous DNA from endonucleolytic digestion by methylating them at the endonuclease recognition sites (7).Interesting observations have been reported regarding H. pylori R-M genes. A novel H. pylori gene, iceA (induced when the bacteria contact the host epithelium), was identified recently (8). DNA sequences have revealed two alleles of the iceA locu...

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Section: Resultsmentioning

confidence: 99%

Comparative genomics of the restriction-modification systems in Helicobacter pylori

Lin

Pósfai

Roberts

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

153

164

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“…1) from our wild-type strain, ER1370, and was shown to complement seven mcrB mutations by several different tests for restriction activity (48). These included restriction of phage X that had been modified by M -MspI (methylated sequence mrCCGG [59]; hereafter called A MspI), M -HaeII (recognition site RGCGCY; position of methylation unknown; hereafter called A * HaeII), or M DdeI (meC TNAG [24]; hereafter called X * DdeI) in addition to T4gt. McrB+C+ restriction in a wild-type strain reduces the titer of the first three test phage by factors of 5 to 100 relative to the titer on an McrB-control strain, while it reduces the titer of T4gt by a factor of 106 to 107.…”

Section: Methodsmentioning

confidence: 99%

“…Plasmids carrying modification methylase genes were pMspI 1-30 (41); pER82 (48), which carries the gene for M -HaeII; pAluIM12.0 (T. Jaeger and G. Wilson, unpublished data); pHpaIIM (C. Card and G. Wilson, unpublished data); pPvuIIM1.9 (7); pBanIIH3-19 (G. Wilson, unpublished data); and pDdeIMl.6 (24). All of these constructs are based on pBR322 and confer ampicillin resistance.…”

Section: Methodsmentioning

confidence: 99%

Genetic and sequence organization of the mcrBC locus of Escherichia coli K-12

et al. 1990

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The mcrB (rglB) locus of Escherichia coli K-12 mediates sequence-specific restriction of cytosine-modifled DNA. Genetic and sequence analysis shows that the locus actually comprises two genes, mcrB and mcrC. We show here that in vivo, McrC modifies the specificity of McrB restriction by expanding the range of modified sequences restricted. That is, the sequences sensitive to McrB+-dependent The locus known as mcrB was one of the first restriction systems to be discovered (33), by virtue of its action on special variants of T-even bacteriophage that incorporate 5-hydroxymethylcytosine (hm`C) into their DNA without further modification (see reference 50 for a review). This locus, formerly known as rglB (or r2,4) (48), was rediscovered because of difficulties encountered in cloning the genes for site-specific modification methylases associated with type II restriction-modification systems (7,26,40,49). In addition to hm5C-DNA, many but not all sequences methylated by site-specific cytosine modification methylases are restricted by the system in vivo, and the consensus recognition sequence 5'GmC was proposed (49). McrB is thus a sequence-specific, modification-requiring restriction system. We show here that the mcrB locus described above actually comprises two genes and that both are required for restriction of most the sequences previously characterized as sensitive. Thus, we will refer to the complete system as the McrBC system.The genes encoding the system are contained within the immigration control region of the Escherichia coli K-12 genome. Three restriction systems are encoded within 14 kilobases (kb) here (48). The well-studied hsdRMS locus (20, 31, 55) encodes the multisubunit type I system EcoK, which recognizes a seven-base sequence and cleaves the target when the sequence is not modified. The other two systems are the flanking loci mcrBC, described above, and mrr, which mediates site-specific restriction of adenine-modified DNA (22). The sequence organization of this region, judged by Southern blot analysis of chromosomal DNA, is highly variable in enteric bacteria (12), both in the hsd genes specifically and in the flanking sequences. Sequence analysis presented here is consistent with recent acquisition of the mcrBC genes by E. coli, possible accounting for some of the observed variability. * Corresponding author.At the molecular level, restriction systems consist of sequence-specific double-stranded endonucleases, usually accompanied by a sequence-specific modification methylase. So far, four classes of endonucleases have been described. The simplest are the type II enzymes, in which the endonuclease and protective methylase activities reside in separate enzymes. These endonucleases typically act as dimers of identical subunits and require only Mg2" for activity (38).One group of type II isoschizomers, typified by DpnI, recognizes a modified site (28), as McrBC appears to do. In contrast, type I and type III enzymes have separate specificity subunits that recognize the DNA site and require ATP in a...

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“…One well-documented problem has been caused by insufficient methyltransferase expression in the clone, which then fails to protect against endonuclease activity. This situation invariably results in mutation and/or deletion of the R gene from the clone (Brooks et al 1989;Du¨sterho¨ft et al 1991;Erdmann et al 1991Erdmann et al , 1992Hammond et al 1990;Howard et al 1986). For other RM systems, particularly those from Actinomycetales, the cloning problems encountered have been associated with failures of expression (Roberts and Halford 1993).…”

Section: Discussionmentioning

confidence: 99%

Comparative analysis of expression of the Sal I restriction-modification system in Escherichia coli and Streptomyces

Álvarez

Gómez

et al. 1996

Mol Gen Genet

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The salIR and salIM genes encode the endonuclease and methyltransferase components of the SalI restriction-modification system from Streptomyces albus G. Expression of the salI genes in Escherichia coli was investigated and major differences with Streptomyces were found. In E. coli there is no detectable expression of the salI R gene due to inactivity of the sal-pR promoter region. In the natural host of the system this region directs transcription of the salI genes as a bicistronic message. In contrast to salIR, salIM is transcribed in the heterologous host from a promoter within the salI DNA. Since sal-pR is not active, the gene cannot be expressed as part of the salI operon. It is probably transcribed from sal-pM, a promoter internal to the operon which allows independent expression of the modification gene in Streptomyces. Replacement of sal-pR by the strong pLac promoter allows expression of salIR in E. coli and enhances expression of salIM. The resulting strain produces about 10 times more endonuclease than a Streptomyces clone containing the SalI system under the control of sal-pR.

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Cloning theDdeI restriction-modification system using a two-step method

Cited by 46 publications

References 34 publications

Comparative genomics of the restriction-modification systems in Helicobacter pylori

Comparative genomics of the restriction-modification systems in Helicobacter pylori

Genetic and sequence organization of the mcrBC locus of Escherichia coli K-12

Comparative analysis of expression of the Sal I restriction-modification system in Escherichia coli and Streptomyces

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