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PubMed1992

DNA diagnosis with mutation-specific artificial methylation sites: application to rapid screening of delta F508.

Many polymerase chain reaction (PCR)-based methods for diagnosis of minute mutations are suboptimal for automated screening because of their reliance on gel electrophoresis or probe hybridization. In the method reported here, PCR products containing artificial methylation sites are analyzed by measuring incorporation of radiolabeled methyl groups. Primers are designed to amplify the possible mutation-containing region such that the 3' end of one primer lies adjacent to the possible mutation. Sequence modification near that end creates either a mutation- or wild type (WT)-specific artificial methylation site in the PCR product. The product is briefly incubated with an appropriate DNA methylase and tritiated S-adenosylmethionine ([3H]SAM), separated from free SAM by column chromatography, and analyzed for incorporation of tritium. Applying this technique to the cystic fibrosis delta F508 deletion, we accurately diagnosed five homozygotes, five heterozygotes, and five normal individuals within 40 min of PCR completion. The method can be generalized to rapid, automated detection of a variety of point mutations and small deletions.

Base Sequence↗

PubMedDate not supplied

[A system of EcoRV restriction-modification: genes, enzymes and synthetic substrates].

The genes, encoding the restriction endonuclease and modification methylase EcoRV have been cloned from the natural plasmid pLG13 into pBR32 derivative vector pIL233. A resultant clone, expressing both enzyme activities, was used as a source of DNA for sequencing these genes by a procedure, that employed construction of deletion derivatives used to locate borders (by means of a functional test) and to sequence ca. 300 bp near the deletion breakpoint. From the sequence data, we infer that the endonuclease, a 29 KDa protein, and the methylase, a 36 KDa protein, are transcribed from a 310 bp intergenic region in opposite directions. There is no apparent homology between the enzymes and genes of the EcoRI and the EcoRV systems. A synthetic decamer, containing the EcoRV endonuclease recognition sequence and a phosphoamide bond at the cleavage point, is not cleaved by the highly purified endonuclease; the unmodified synthetic decamer is cleaved at the same conditions, only that the cleavage occurs to produce a blunt end--GAT/ATC, and not in a place previously reported (GATAT/C).

Base Sequence↗

PubMed1999

Molecular evolution of DNA-(cytosine-N4) methyltransferases: evidence for their polyphyletic origin.

DNA N4-cytosine methyltransferases (N4mC MTases) are a family of S-adenosyl-L-methionine (AdoMet)-dependent MTases. Members of this family were previously found to share nine conserved sequence motifs, but the evolutionary basis of these similarities has never been studied in detail. We performed phylogenetic analysis of 37 known and potential new family members from the multiple sequence alignment using distance matrix, parsimony and maximum likelihood approaches to infer the evolutionary relationship among the N4mC MTases and classify them into groups of orthologs. All the treeing algorithms employed as well as results of exhaustive sequence database searching support a scenario, in which the majority of N4mC MTases, except for M. Bal I and M. Bam HI, arose by divergence from a common ancestor. Interestingly, MTases M. Bal I and M. Bam HI apparently originated from N6-adenine MTases and represent the most recent addendum to the N4mC MTase family. In addition to the previously reported nine sequence motifs, two more conserved sequence patches were detected. Phylogenetic analysis also provided the evidence for massive horizontal transfer of MTase genes, presumably with the whole restriction-modification systems, between Bacteria and Archaea.

Amino Acid Sequence↗

PubMed1997

The hsd loci of Mycoplasma pulmonis: organization, rearrangements and expression of genes.

Two paralogous, site-specific invertible loci, designated hsd1 and hsd2 (host specificity determinant), have been identified in the Mycoplasma pulmonis genome. They encode putative type I restriction and modification (R-M) systems with maximum sequence homology to the type IC family, which includes EcoR124II and EcoDXXI. Each locus encodes an endonuclease subunit (HsdR), a methylase subunit (HsdM) and two DNA specificity subunits (HsdS). The gene organization at each locus is such that hsdR and hsdM are flanked by two hsdS genes. Within each locus, one of the hsdS genes, hsdR and hsdM, is encoded in tandem by the same DNA strand, while the second hsdS gene is encoded by the complementary strand but without overlap with the other three hsd genes. The hsdR and hsdM sequences of one locus are almost identical to their counterparts in the other. The four hsdS genes (two per locus) are highly homologous at their 5' ends and also share sequence similarities in the 3' ends of their corresponding coding regions. Owing to the disposition of and sequence similarities among the hsdS genes, they form inverted repeats at each locus. Analysis by polymerase chain reaction (PCR) has shown that both loci behave as site-specific DNA invertible elements with multiple inversion sites, termed 'vipareetus', occurring within the hsdS genes. The inversions lead to a reassortment of hsdS sequences, generating an array of recombinant genes that probably encode S subunits possessing alternative DNA-binding specificities. Sequence information obtained from the analysis of hsd2 transcripts by 5' RACE (rapid amplification of cDNA ends) indicates that inversion induces the transcription of alternative hsdS genes by the relocation of coding sequences downstream of a promoter and ribosome-binding site (RBS) situated at one end of each locus.

Amino Acid Sequence↗

PubMed1989

Nucleotide sequence and genetic organization of the NgoPII restriction-modification system of Neisseria gonorrhoeae.

The NgoPII restriction endonuclease, which recognizes the sequence 5'-GG decreases CC-3', differs from its isoschizomer HaeIII in being sensitive to methylation at the external cytosine residue. The entire nucleotide sequence of a cloned 3.3 kb segment of Neisseria gonorrhoeae strain P9 chromosomal DNA which harbours the NgoPII restriction-modification system has been determined. This data, coupled with sub-cloning experiments, indicates that the restriction endonuclease (R.NgoII) and modification (M.NgoII) genes are transcribed from separate promoters but are arranged in tandem, with the R.NgoPII gene being located on the 5' side of the M.NgoPII gene. Unlike all previously reported restriction systems the 3' end of the endonuclease open reading frame overlaps the 5' end of the methylase open reading frame by 8 codons. This overlap may have implications for the regulation of the NgoPII restriction-modification system.

Amino Acid Sequence↗

PubMed1981

Cloning and expression of the Pst I restriction-modification system in Escherichia coli.

Here we report the cloning and preliminary characterization of the Pst I restriction-modification system of Providencia stuartii 164. Transformants of Escherichia coli carrying the Pst I gene system inserted into the cloning vector pBR322 were selected on the basis of acquired resistance to bacteriophage lambda infection. Pst I endonuclease was detected in osmotic shock fluid from each of the resistant clones. Plasmid and chromosomal DNA from these clones could not be digested by Pst I, indicating that the gene for the corresponding modification enzyme had also been cloned and was being expressed. The smallest recombinant plasmid encoding both activities, pPst201, contains an insert of approximately 4000 base pairs. In vitro transcription studies indicate that this DNA fragment also contains the endogenous promoter(s) of the system. When pPst201 was introduced into a minicell-producing strain of E. coli, two new proteins, 32,000 and 35,000 daltons, were synthesized. We have assigned these to the Pst I modification (methylase) and restriction enzymes, respectively. The active form of the restriction enzyme is a dimer, as determined by gel filtration. Constructed transformants of P. stuartii 164 that carry the Pst I system inserted into pBR322 produce approximately 10 times more Pst I endonuclease activity than does the native strain.

Base Sequence↗

PubMed1992

Cloning and sequence analysis of the StsI restriction-modification gene: presence of homology to FokI restriction-modification enzymes.

StsI endonuclease (R.StsI), a type IIs restriction endonuclease found in Streptococcus sanguis 54, recognizes the same sequence as FokI but cleaves at different positions. A DNA fragment that carried the genes for R.StsI and StsI methylase (M.StsI) was cloned from the chromosomal DNA of S.sanguis 54, and its nucleotide sequence was analyzed. The endonuclease gene was 1,806 bp long, corresponding to a protein of 602 amino acid residues (M(r) = 68,388), and the methylase gene was 1,959 bp long, corresponding to a protein of 653 amino acid residues (M(r) = 76,064). The assignment of the endonuclease gene was confirmed by analysis of the N-terminal amino acid sequence. Genes for the two proteins were in a tail-to-tail orientation, separated by a 131-nucleotide intercistronic region. The predicted amino acid sequences between the StsI system and the FokI system showed a 49% identity between the methylases and a 30% identity between the endonucleases. The sequence comparison of M.StsI with various methylases showed that the N-terminal half of M.StsI matches M.NIaIII, and the C-terminal half matches adenine methylases that recognize GATC and GATATC.

Amino Acid Sequence↗

PubMed2002

Transformation using in vivo and in vitro methylation in Streptomyces griseus.

Streptomyces griseus does not readily take up foreign DNA isolated from other Streptomyces species or Escherichia coli, presumably due to its unique restriction-modification systems that function as a barrier for interspecific DNA transfer. To efficiently transform S. griseus by avoiding the restriction barriers, we methylated incoming DNA in vivo and in vitro and treated protoplasts with heat prior to transformation. Whereas heat treatment of protoplasts or methylation of the E. coli-Streptomyces shuttle vectors (pXE4 and pKK1443) did not prominently improve the transformation efficiency, HpaII methylation of the vectors from any E. coli strains tested in this study highly increased the transformation efficiency. The highest transformation efficiency was observed when the shuttle vectors were isolated from the dam, hsd strain of E. coli (GM161) and methylated by AluI and HpaII methyltransferases, and the efficiency was approximately the same as that of the vectors from S. griseus. We identified several restriction-modification systems that decrease the transformation efficiency. This research also led us to understand methylation profiles and restriction-modification systems in S. griseus.

DNA Methylation↗

PubMed1987

[Chemical synthesis and properties of oligonucleotide substrates for restriction endonuclease BamHI and methyltransferase Eco dam].

Oligodeoxyribonucleotides which form a number of duplexes, containing the recognition sequences for endonuclease BamHI and DNA methylase Eco dam, were synthesised by the phosphotriester approach. Furthermore, synthesis of 3'-phosphorylated oligodeoxyribonucleotides from corresponding S-methyl phosphorothioate triester oligomers is described. The synthetic duplexes are characterized by some defects in the recognition sequences for endonuclease BamHI and methylase Eco dam, viz. nick, absence of an internucleotide phosphate, modifications (including partial single-strandedness) of the recognition site. Interaction of the enzymes with these synthetic substrates was investigated.

Base Sequence↗

PubMed1995

Restriction-modification systems as genomic parasites in competition for specific sequences.

Restriction-modification (RM) systems are believed to have evolved to protect cells from foreign DNA. However, this hypothesis may not be sufficient to explain the diversity and specificity in sequence recognition, as well as other properties, of these systems. We report that the EcoRI restriction endonuclease-modification methylase (rm) gene pair stabilizes plasmids that carry it and that this stabilization is blocked by an RM of the same sequence specificity (EcoRI or its isoschizomer, Rsr I) but not by an RM of a different specificity (PaeR7I) on another plasmid. The PaeR7I rm likewise stabilizes plasmids, unless an rm gene pair with identical sequence specificity is present. Our analysis supports the following model for stabilization and incompatibility: the descendants of cells that have lost an rm gene pair expose the recognition sites in their chromosomes to lethal attack by any remaining restriction enzymes unless modification by another RM system of the same specificity protects these sites. Competition for specific sequences among these selfish genes may have generated the great diversity and specificity in sequence recognition among RM systems. Such altruistic suicide strategies, similar to those found in virus-infected cells, may have allowed selfish RM systems to spread by effectively competing with other selfish genes.

Binding, Competitive↗

PubMed1987

EcoR124 and EcoR124/3: the first members of a new family of type I restriction and modification systems.

We have purified the EcoR124 and EcoR124/3 restriction enzymes and shown that they are type I enzymes by several criteria: subunit composition, DNA and S-adenosylmethionine-dependent ATPase activity, and site-specific DNA methylase activity. By immunochemical criteria these enzymes are related to each other but are unrelated to the two previously investigated families of type I restriction enzymes. They form therefore a new family which we call type IC. The arrangement of the structural genes coding for these enzymes and their transcriptional organisation have been determined. These are different from the common arrangement found for the other two families of type I enzymes.

Adenosine Triphosphatases↗

PubMed1985

The EcoDXX1 restriction and modification system of Escherichia coli ET7. Purification, subunit structure and properties of the restriction endonuclease.

The Escherichia coli plasmid pDXX1 codes for a new restriction-modification system. The specific restriction endonuclease coded by this system has been purified by a procedure that includes phosphocellulose and heparin-agarose chromatography. Sedimentation on glycerol gradients showed one peak of activity with a value of about 12 S. The highly purified enzyme require ATP and Mg2+ for activity as well as S-adenosylmethionine, although some S-adenosylmethionine molecules are probably bound to the enzyme. The enzyme does not cleave lambda DNA at well-defined sites and has a strong non-modified DNA-dependent ATPase activity. The enzyme has also methylase activity acting against non-modified DNA.

Adenosine Triphosphatases↗

PubMed1995

Dam methylase from Escherichia coli: kinetic studies using modified DNA oligomers: hemimethylated substrates.

We have measured steady-state kinetics of the N6-adenine methyltransferase Dam Mtase using as substrates non-selfcomplementary tetradecamer duplexs (d[GCCGGATCTAGACG]-d[CGTCTAGATCC-GGC]) containing the hemimethylated GATC target sequence in one or the other strand and modifications in the GATC target sequence of the complementary strands. Modifications included substitution of guanine by hypoxanthine (I), thymine by uracil (U) or 5-ethyl-uracil (E) and adenine by 2,6-diamino-purine (D). Thermodynamic parameters were obtained from the concentration dependence of the melting temperature (Tm) of the duplexes. Large differences in DNA methylation of duplexes containing single dI for dG substitution of the Dam recognition site were observed compared with the canonical substrate, if the substitution involved the top strand (on the G.C rich side). Substitution in either strand by uracil (dU) or 5-ethyluracil (dE) resulted in small perturbation of the methylation patterns. When 2,6-diamino-purine (dD) replaced the adenine to be methylated, small, but significant methylation was observed. The kinetic parameters of the methylation reaction were compared with the thermodynamic free energies and significant correlation was observed.

2-Aminopurine↗

PubMed2001

Restriction and modification systems of ruminal bacteria.

A high frequency of type II restriction endonuclease activities was detected in Selenomonas ruminantium but not in other rumen bacteria tested. Eight different restriction endonucleases were characterized in 17 strains coming from genetically homogeneous local population. Chromosomal DNA isolated from S. ruminantium strains was found to be refractory to cleavage by various restriction enzymes, implying the presence of methylase activities additional to those required for protection against the cellular endonucleases. The presence of Dam methylation was detected in S. ruminantium strains as well as in several other species belonging to the Sporomusa subbranch of low G + C Gram-positive bacteria (Megasphaera elsdenii, Mitsuokella multiacidus).

Animals↗

PubMed2006

Molecular characterization of geminivirus-derived small RNAs in different plant species.

DNA geminiviruses are thought to be targets of RNA silencing. Here, we characterize small interfering (si) RNAs-the hallmarks of silencing-associated with Cabbage leaf curl begomovirus in Arabidopsis and African cassava mosaic begomovirus in Nicotiana benthamiana and cassava. We detected 21, 22 and 24 nt siRNAs of both polarities, derived from both the coding and the intergenic regions of these geminiviruses. Genetic evidence showed that all the 24 nt and a substantial fraction of the 22 nt viral siRNAs are generated by the dicer-like proteins DCL3 and DCL2, respectively. The viral siRNAs were 5' end phosphorylated, as shown by phosphatase treatments, and methylated at the 3'-nucleotide, as shown by HEN1 miRNA methylase-dependent resistance to beta-elimination. Similar modifications were found in all types of endogenous and transgene-derived siRNAs tested, but not in a major fraction of siRNAs from a cytoplasmic RNA tobamovirus. We conclude that several distinct silencing pathways are involved in DNA virus-plant interactions.

Arabidopsis↗

PubMed1981

Overproduction of the EcoRII endonuclease and methylase by Escherichia coli strains carrying recombinant plasmids constructed in vitro.

Recombinant DNA molecules were constructed from the plasmid pIL203 and the EcoRI-fragment of N3 plasmid containing EcoRII endonuclease and methylase genes and also a gene for resistance to sulfanilamide. The pIL203 plasmid, used as a vector, consisted of the Bam HI-EcoRI-fragment of the plasmid pBR322 conferring resistance to ampicillin and the Bam HI-EcoRI-fragment of lambda phage containing promoters, a thermosensitive mutation in the cI gene and a suppressible amber mutation in the cro gene. Ampicillin-sulfanilamide-resistant clones were selected and tested for their restriction and modification phenotype. The recombinant plasmid DNA, isolated from ApRSuR-resistant clones, which restricted and modified phage lambda imm21 with EcoRII specificity, had the EcoRI-fragment with EcoRII genes in a single orientation. The recombinant plasmid pSK323 was transferred into E. coli strains with su-, su1, su2 or su3 phenotypes. The synthesis of products of EcoRII genes by these strains grown at 37 degrees C is increased by 10--50-fold.

Cloning, Molecular↗

PubMed2000

Binding of EcoP15I DNA methyltransferase to DNA reveals a large structural distortion within the recognition sequence.

EcoP15I DNA methyltransferase, a member of the type III restriction-modification system, binds to the sequence 5'-CAGCAG-3' transferring a methyl group from S-adenosyl-l-methionine to the second adenine base. We have investigated protein-DNA interactions in the methylase-DNA complex by three methods. Determination of equilibrium dissociation constants indicated that the enzyme had higher affinity for DNA containing mismatches at the target base within the recognition sequence. Potassium permanganate footprinting studies revealed that there was a hyper-reactive permanganate cleavage site coincident with adenine that is the target base for methylation. More importantly, to detect DNA conformational alterations within the enzyme-DNA complexes, we have used a fluorescence-based assay. When EcoP15I DNA methyltransferase bound to DNA containing 2-aminopurine substitutions within the cognate sequence, an eight to tenfold fluorescent enhancement resulting from enzymatic flipping of the target adenine base was observed. Furthermore, fluorescence spectroscopy analysis showed that the changes attributable to structural distortion were specific for only the bases within the recognition sequence. More importantly, we observed that both the adenine bases in the recognition site appear to be structurally distorted to the same extent. While the target adenine base is probably flipped out of the DNA duplex, our results also suggest that fluorescent enhancements could be derived from protein-DNA interactions other than base flipping. Taken together, our results support the proposed base flipping mechanism for adenine methyltransferases.

2-Aminopurine↗

PubMed1985

The mom gene of bacteriophage mu: a unique regulatory scheme to control a lethal function.

The mom gene of bacteriophage Mu encodes a DNA modification function which converts adenine to acetamido adenine in a sequence-specific manner. The mom gene itself is subject to a complex regulation: gene expression requires methylation by the Escherichia coli Dam methylase of specific sites upstream of the mom promoter and transactivation of the promoter by a Mu gene product. The requirement for transactivation can be overcome when mom is transcribed from foreign promoters. When cloned into various sites in pBR322, the mom gene is always found in an orientation where transcription from vector promoters is excluded. The productive orientation is lethal to the cell. This effect is mediated by the concerted action of the mom gene product and the product of gene com (control of mom, previously termed ORF-x) whose coding region overlaps the 5-coding region of the mom gene. When mom is expressed from its own promoter, internal deletions in com completely abolish expression of the mom gene. Fragments lacking the 5' end of com can be cloned downstream of constitutive plasmid promoters. The com gene product itself is not lethal to the cell. The region encoding mom has been cloned in pL expression vectors. The mom gene product, a peptide of 27 kDal, has been visualized on gels. Efficient expression of Mom from pL requires gene com. A fusion between MS-2 polymerase and com has been generated. The fusion product is made in large amounts, whereas the mom gene product is not overproduced although the gene is present on the same transcriptional unit.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophage mu↗