New restriction enzymes discovered from Escherichia coli clinical strains using a plasmid transformation method

The presence of restriction enzymes in bacterial cells has been predicted by either classical phage restriction-modification (R-M) tests, direct in vitro enzyme assays or more recently from bacterial genome sequence analysis. We have applied phage R-M test principles to the transformation of plasmid DNA and established a plasmid R-M test. To validate this test, six plasmids that contain BamHI fragments of phage lambda DNA were constructed and transformed into Escherichia coli strains containing known R-M systems including: type I (EcoBI, EcoAI, Eco124I), type II (HindIII) and type III (EcoP1I). Plasmid DNA with a single recognition site showed a reduction of relative efficiency of transformation (EOT = 10(-1)-10(-2)). When multiple recognition sites were present, greater reductions in EOT values were observed. Once established in the cell, the plasmids were subjected to modification (EOT = 1.0). We applied this test to screen E.coli clinical strains and detected the presence of restriction enzymes in 93% (14/15) of cells. Using additional subclones and the computer program, RM Search, we identified four new restriction enzymes, Eco377I, Eco585I, Eco646I and Eco777I, along with their recognition sequences, GGA(8N)ATGC, GCC(6N)TGCG, CCA(7N)CTTC, and GGA(6N)TATC, respectively. Eco1158I, an isoschizomer of EcoBI, was also found in this study.     

A new restriction endonuclease from Spirulina platensis.

Three restriction endonucleases, Sp1I, Sp1II and Sp1III have been purified partially from Spirulina platensis subspecies siamese and named. Sp1I cleaves bacteriophage lambda DNA at one site, phi X 174 RF DNA at two sites, but does not cleave pBR322 DNA. This enzyme recognizes the sequence 5'CGTACG3' 3'GCATCG5' and cuts the site indicated by the arrows. Sp1II is an isoschizomer of Tth111I and Sp1III is an isoschizomer of HaeIII.     

The leftward promoter of bacteriophage lambda. Isolation on a small restriction fragment and deletion of adjacent regions

As part of our investigations on the relationship between DNA structure and gene regulation, a 352-base pair Hae III fragment was cloned containing the leftward operator-promoter (PL) region of bacteriophage lambda. This was accomplished without the aid of a phenotypic assay for the cloned fragment. A Hae III digest of a segment of the lambda genome was first fractionated by RPC-5 column chromatography. The partially purified PL fragment was then ligated into the Eco RI site of the pBR322 plasmid vector and cloned into the recBC+ Escherichia coli host C600(R-M-) using a technique that converts the Hae III ends of the fragment into Eco RI sites. Similar cloning attempts into a recBC- host (C600-SF8) were unsuccessful. The cloned fragment has the PL promoter oriented toward the tetracycline resistance genes of the vector, and is isolated from the plasmid (pRW601) by digestion with Eco RI followed by sucrose gradient sedimentation. The fragment was identified as PL by restriction mapping, repressor binding, sequencing, and promoter location. The now complete sequence of this fragment, part of which was known previously, reveals a large A/T-rich region immediately adjacent to the PL promoter. We have generated deletions in this region in order to study the influence of this sequence on promoter function.     

Purification and properties of a site-specific restriction endonuclease AaaI from Acetobacter aceti subsp. aceti No. 1023

Abstract A type II restriction endonuclease, named Aaa I, was purified from Acetobacter aceti subsp. aceti No. 1023. The optimum pH and temperature were determined to be 8.5 and 37°C, respectively. The enzyme activity was stimulated by the addition of either NaCl or KCl and their optimum concentrations were 100 mM for both cations. Aaa I recognized the hexanucleotide sequence and cleaved it at the positions indicated by the arrows. Aaa I was an isoschizomer of Xma III from Xanthomonas malvacaerum and Eco 52I from Escherichia coli .     

Restriction endonucleases in Azospirillum

Azospirillum brasilense, A. amazonense, and A. lipoferum strains were screened for restriction endonucleases using phage lambda DNA. The extract of A. brasilense 29711 cleaved lambda DNA into specific fragments. It was concluded that this strain possesses a class II restriction endonuclease which was named AbrI. AbrI has a single recognition site on lambda DNA at position of approx. 33 500 bp. AbrI was characterized as an isoschizomer of XhoI, which cuts lambda DNA at 33 498 bp and cleaves double-stranded DNA at the sequence 5'-C TCGAG-3'. From other Azospirilla strains only A. amazonense QRZ42 extracts (AamI activity) cleaved DNA into specific fragments under certain conditions.     

Mucopolysaccharidosis I: a comparative study of haplotypes Eco47III-NspI sites frequencies in patients and healthy subjects of Mexican population

BACKGROUND: Mucopolysaccharidosis I (MPS-I) is an autosomal recessive disorder, which is caused by mutations in the IDUA gene. It induces the deficiency of glycosidase alpha-L-duronidase. The enzyme that is required for the degradation of heparan and dermatan sulfate. This disorder expresses a wide range of clinical symptoms (severe mental retardation, skeletal deformations, hepatosplenomegaly, corneal clouding and mild visceral organ involvement). In the present paper, we report the frequencies of haplotypes of the Eco47III-NspI sites, in the IDUA gene, in Mexican healthy and in MPS-I individuals. METHODS: Eco47III and NspI intragenic polymorphisms in IDUA gene were studied in 262 (524 chromosomes) Mexican healthy subjects and in 53 (106 chromosomes) MPS-I patients. RESULTS: The genotypes for IDUA Eco47III and NspI sites in Mexicans were in agreement with Hardy-Weinberg expectations. Allele frequency distributions for individual sites differed (P < 0.05) in both groups. Haplotype Eco47III-NspI frequencies of Mexican MPS-I patients also differed from those of the normal Mexican population. The data provide evidence of linkage disequilibrium, since the MPS-I group constitutes a subset of the Mexican control population. The disequilibrium in Mexican MPS-I patients was defined by an increase in the haplotype A1B2, and deficiency in A2B1, with respect to normal population (P < 0.05). CONCLUSIONS: Our results support that these polymorphisms can be associated to mutations in IDUA gene, which leads to MPS-I in Mexican patients. On the other hand, these polymorphisms can be used to identify heterozygosity when they are informative.     

Prevalence of CTGCAG recognizing restriction and modification systems in ruminal selenomonades

Analysis of restriction and modification activities in natural population of Selenomonas ruminantium have revealed the prevalence of CTGCAG (Pst I isoschizomers) recognizing restriction and/or modification systems in these bacteria. Pst I isoschizomeric restriction endonucleases were detected in 4 out of 15 strains tested. In one strain, the Pst I isoschizomeric restriction system was accompanied by another restriction and modification system recognizing GAATTC sequence (Eco RI isoschizomer). Four other strains contained CTGCAG specific methylases which lacked cognate endo-nuclease activities. Presence of identical restriction and modification systems in both of subspecies of S. ruminantium, as well as the occurrence of Pst I isoschizomers in various combinations, indicate the possibility of horizontal transfer of genes coding for these systems.     

Substrate specificity of restriction endonucleases Eco471 and Eco521

Cleavage sites for Eco47I and Eco52I restriction endonucleases, which are isoschizomers of Ava II and Xma III, respectively, have been structurally elucidated.     

Distribution of 5-methylcytosine in phage lambda genome methylated by DNA methylase Eco RII

The distribution of 5-methylcytosine in Eco RI-Bam HI fragments of phage lambda DNA in vitro methylated by Eco RII methylase has been studied. The general picture of distribution of methylated sites in phage lambda DNA is slightly different from the statistical distribution. However, the sites have been found, where the distribution of 5-methylcytosine is not accidental. A complete absence of 5-methylcytosine in the J-fragment, a genome lambda area essential for site-specific recombination, has been found. The absence of Eco RII is supposed to be the best protection of this area of phage genome from the increased mutagenesis, characteristic for nucleotide sequences methylated by DNA-methylated Eco RII and Eco RII type.     

Substrate specificity of restriction endonuclease Eco781

The recognition sequence and cleavage point of restriction endonuclease Eco781 have been determined as 5'-GGCGCC-. There are several known enzymes recognizing the same sequence, although the prototype NarI and isoschizomers NdaI and NunII cleave the substrate to produce 5'-protruding ends, whereas cleavage with isoschizomer BbeI results in 3'-protruding ends. Therefore, restrictase Eco78I, generating flush ends, may be regarded as an enzyme with new specificity among the restriction endonucleases recognizing the 5'-GGCGCC-sequence.     

A new restriction endonuclease Eco31I recognizing a non-palindromic sequence

A restriction endonuclease with a novel site-specificity has been isolated from the Escherichia coli strain RFL31. The nucleotide sequences around a single Eco31I cut on pBR322 DNA and two cuts of lambda DNA have been compared. A common 5'GAGACC 3'CTCTGG sequence occurs near each cleavage site. Precise mapping of the cleavages in both DNA strands places the cuts five nucleotides to the left of the upper sequence and one nucleotide to the left of the lower sequence. This enabled us to deduce the following recognition and cleavage specificity of Eco31I: 5' GGTCTCN decreases 3' CCAGAGN NNNN increases.     

Type II restriction endonucleases from Helicobacter pylori include an enzyme with a novel recognition sequence

Type II restriction endonuclease activities of Helicobacter pylori strain Roberts and of the type strain H. pylori NCTC 11637 were detected and analysed by conventional techniques. The endonucleases were partially purified, their optima for activity and their recognition and cleavage sites were determined. H. pylori (Roberts) contained at least two enzymes: HpyBI was an isoschizomer of RsaI (GT/AC) and HpyBII was of a novel specificity (GTN/NAC). H. pylori NCTC 11637 was found to contain an isoschizomer of EcoRV (HpyCI: GAT/ATC) and at least one other enzyme which was too unstable to characterise.     

Isolation and purification of restriction endonuclease BpeI from Bordetella pertussis

New restriction endonuclease has been isolated from Bordetella pertussis vaccine strain 305 and purified in 1 stage on Sepharose covalently bound with blue dextran. The isolated restrictase has been found capable of breaking down lambda-phag DNA into 7 fragments. According to its specificity, Bpe I is the isoschizomer of Hind III obtained from Haemophilus influenzae strain Rd.     

Construction of a hybrid col E1 plasmid carrying the gene for bacteriophage lambda repressor.

A biologically active hybrid DNA molecule was constructed from plasmid Col E1 and the Eco R1 fragment of lambda DNA containing the gene for lambda repressor. The presence of this gene in the hybrid molecule was demonstrated genetically. The hybrid plasmid contains two closely located targets for restriction endonuclease Hind 111 in the integrated fragment. Thus, the plasmid may be used as a vector not only for Eco R1 fragments but also for Hind 111 fragments.     

Recognition sequences of restriction endonucleases and methylases--a review

The properties and sources of all known endonucleases and methylases acting site-specifically on DNA are listed. The enzymes are crossindexed (Table I), classified according to homologies within their recognition sequences (Table II), and characterized within Table II by the cleavage and methylation positions, the number of recognition sites on the DNA of the bacteriophages lambda, phi X174 and M13mp7, the viruses Ad2 and SV40, the plasmids pBR322 and pBR328 and the microorganisms from which they originate. Other tabulated properties of the restriction endonucleases include relaxed specificities (Table III), the structure of the restriction fragment ends (Table IV), and the sensitivity to different kinds of DNA methylation (Table V). Table VI classifies the methylases according to the nature of the methylated base(s) within their recognition sequences. This table also comprises those restriction endonucleases, which are known to be inhibited by the modified nucleotides. Furthermore, this review includes a restriction map of bacteriophage lambda DNA based on sequence data. Table VII lists the exact nucleotide positions of the cleavage sites, the length of the generated fragments ordered according to size, and the effects of the Escherichia coli dam- and dcmI-coded methylases M X Eco dam and M X Eco dcmI on the particular recognition sites.     

Cloning of the restriction-modification genes of Bacillus centrosporus in Escherichia coli

A genomic library of Bacillus centrosporus was obtained using pBR327 as a vector. The total plasmid DNA of the library was cleaved by the BcnI restriction endonuclease and then transformed in Escherichia coli RR1. Two clones possessing restriction and DNA modification profiles of BcnI were identified among the transformants. Their respective plasmids were 13.3 and 9.05 kbp in size. Restriction mapping of both plasmids showed each of them to contain two sites for HindIII and one for both Eco31I and Eco47III, located at the same distance. This was assumed to be the location region of the BcnI restriction-modification genes. Confirmation of the assumption was obtained by deletion mapping of the recombinant plasmids. Special features concerning cloning of the restriction-modification genes are discussed on the basis of the results obtained.     

Isolation of specialized lambda transducing bacteriophages for flagellar genes (fla) of Escherichia coli K-12.

Specialized transducing lambda phages carrying the region III flagellar genes (fla) of Escherichia coli K-12 were isolated by a new method. A strain carrying both a cryptic lambda prophage near the his genes and a deletion of the attlambda gene was used as a starting strain. The lysogen of lambdacI857pga18-bio69 was isolated in which the prophage was integrated within the lambda cryptic genes by means of recombination with the residual lambda DNA. The strains with deletions starting within the prophage and ending in these fla genes were selected from among the heat-resistant survivors of the lysogen. They were then infected with heat-inducible and lysis-defective lambda phages and, thus, specialized transducing phage lines for hag and fla were obtained. High-frequency transfer lines of rare phages carrying the fla genes were isolated by inducing a strain carrying a heat-inducible lambda prophage near the his genes and selecting by transduction of a fla deletion strain. Preliminary characterization of these transducing phages is also reported.     

Comparative evaluation of plasmid profiling and ribotyping in the analysis of Lactobacillus plantarum strain heterogeneity in silage

F. DUFFNER AND M. O'CONNELL. 1995. Seventy-two Lactobacillus plantarum isolates were recovered from six uninoculated grass silages for the purposes of firstly evaluating the usefulness of (1) restriction endonuclease digestion of total genomic DNA, (2) plasmid profiling and (3) ribotyping in Lact. plantarum strain differentiation and secondly, examining the strain heterogeneity in well preserved silage.
The three methods for differentiation were applied to 72 of the isolates and allowed at least 32 different strains to be identified. Twenty-five different plasmid profiles were detected (26 if the absence of plasmids is included as a profile). Ribotyping with Eco RI identified only 11 patterns among the silage isolates. A variety of restriction enzymes was screened to increase the sensitivity of ribotyping to detect strain differences and Bam HI was used successfully for this purpose, differentiating all of the strains tested.
Two dominant strains (I and II) were identified in one particular silage, comprising 47% and 17% respectively of the isolates, while strains III and V comprised 37% and 25% of the Lact. plantarum population isolated from another of the silages.