Cloning and nucleotide sequences of the BanI restriction-modification genes in Bacillus aneurinolyticus

The genes of the BanI restriction-modification system specific for GGPyPuCC were cloned from the chromosomal DNA of Bacillus aneurinolyticus IAM1077, and the coding regions were assigned on the nucleotide sequence on the basis of the N-terminal amino acid sequences and molecular weights of the enzymes. The restriction and modification genes coded for polypeptides with calculated molecular weights of 39,841 and 42,637, respectively. Both the enzymes were coded by the same DNA strand. The restriction gene was located upstream of the methylase gene, separated by 21 bp. The cloned genes were significantly expressed in E. coli cells, so that the respective enzymes could be purified to homogeneity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration indicated that the catalytically active form of the endonuclease was dimeric and that of the methylase was monomeric. Comparison of the amino acid sequences revealed no significant homology between the endonuclease and methylase, though both enzymes recognize the same target sequence. Sequence comparison with other related enzymes indicated that BanI methylase contains sequences common to cytosine-specific methylases.     

New restriction endonucleases from Acetobacter aceti and Bacillus aneurinolyticus.

Two restriction endonucleases with new sequence specificities have been isolated from Acetobacter aceti IFO 3281 and Bacillus aneurinolyticus IAM 1077 and named AatII and BanII, respectively. Based on analysis of the sequences around the restriction sites, the recognition sequences and cleavage sites of these endonucleases were deduced as below: (formula; see text)     

Cloning and expression in Escherichia coli of the BanI and BanIII restriction-modification systems from Bacillus aneurinolyticus

The genes coding for the GGPyPuCC-specific (BanI) and ATCGAT-specific (BanIII) restriction-modification systems of Bacillus aneurinolyticus IAM1077 were cloned and expressed in Escherichia coli using pBR322 as a vector. The plasmids carrying the BanI and BanIII restriction-modification genes were designated pBanIRM8 and pBanIIIRM12, respectively. The restriction maps of these recombinant plasmids were constructed. These two plasmids were stably maintained in E. coli HB101. However, when E. coli JM109 was used as a host, pBanIIIRM12 was efficiently propagated but pBanIRM8 was not. The HB101 cells carrying only the restriction gene of BanIII were viable, but the BanI restriction gene carrier could not form colonies on agar plates. The growth of bacteriophage λ was strongly restricted only in the F. coli HB101 cells harboring pBanIRM8. These facts indicate that the BanI restriction enzyme is expressed and functions more efficiently than BanI modification enzyme in E. coli.     

Purification, properties and specificity of the restriction endonuclease from Bacillus stearothermophilus.

The restriction endonuclease BstI was purified from 70kg of Bacillus stearothermophilus. The final product is at least 97% pure as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; this major protein species co-migrates with the enzyme activity on native polyacrylamide-gel electrophoresis and isoelectric focusing. Pure restriction endonuclease BstI has a subunit mol.wt. of 26,000 and is probably a loosely associated dimer. The enzyme shows maximum activity at pH values between 7 and 9.5, and in the presence of 0.5-2mM-Mg2+. NaCl inhibits the restriction enzyme activity. Restriction endonuclease BstI cleaves DNA in a position identical with that cleaved by endonuclease BamHI (for Bacillus amyloliquefaciens), i.e.: (formula: see text). In the presence of high concentrations of enzyme, DNA cleavage occurs at secondary sites. This side-specificity is enhanced by the addition of glycerol. Preliminary studies indicate that these sites are of the type: (formula: see text).     

Substrate specificity of restriction endonuclease VspI

The recognition sequence and cleavage point of restriction endonuclease VspI have been determined as 5'-AT decreases TAAT. This enzyme is not isoschizomer of any known restriction endonucleases. DNA pBR322 contains a single VspI recognition sequence in position 3539. Therefore this enzyme may be used for cloning DNA in the VspI site in AmpR-gene of pBR322.     

BliI, a restriction endonuclease from Bacillus licheniformis

From Bacillus licheniformis a site-specific restriction endonuclease, named BliI, has been purified and characterized. BliI was able to digest lambda DNA at pH 9.1 over a wide temperature range (25-65 degrees C). Digestion of lambda and psi X174 DNAs with BliI produced banding patterns identical to those seen with HaeIII. Therefore, BliI and HaeIII endonculeases are isoschizomers.     

The SalGI restriction endonuclease. Enzyme specificity.

We have analysed the kinetics of DNA cleavage in the reaction between the SalGI restriction endonuclease and plasmid pMB9. This reaction is subject to competitive inhibition by DNA sequences outside the SalGI recognition site; we have determined the Km and Vmax. for the reaction of this enzyme at its recognition site and the KI for its interaction at other DNA sequences. We conclude that the specificity of DNA cleavage by the enzyme is only partly determined by the discrimination it shows for binding at its recognition sequence compared with binding to other DNA sequences.     

Substrate specificity of restriction endonuclease SfeI

The recognition sequence and cleavage site C decreases TRYAG of a new restriction endonuclease SfeI have been determined.     

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.     

The Type II restriction endonuclease MvaI has dual specificity

The MvaI restriction endonuclease cuts 5′-CC↓AGG-3′/5′-CC↑TGG-3′ sites as indicated by the arrows. N4-methylation of the inner cytosines (C^m4CAGG/C^m4CTGG) protects the site against MvaI cleavage. Here, we show that MvaI nicks the G-strand of the related sequence (CCGGG/CCCGG, BcnI site) if the inner cytosines are C5-methylated: C^m5C↓GGG/CC^m5CGG. At M.SssI-methylated SmaI sites, where two oppositely oriented methylated BcnI sites partially overlap, double-nicking leads to double-strand cleavage (CC^m5C↓GGG/CC^m5C↑GGG) generating fragments with blunt ends. The double-strand cleavage rate and the stringency of substrate site recognition is lower at the methylation-dependent site than at the canonical target site. MvaI is the first restriction endonuclease shown to possess, besides the ‘normal’ activity on its unmethylated recognition site, also a methylation-directed activity on a different sequence.     

Relaxed specificity of the EcoRV restriction endonuclease

The EcoRV restriction endonuclease normally shows a high specificity for its recognition site on DNA, GATATC. In standard reactions, it cleaves DNA at this site several orders of magnitude more readily than at any alternative sequence. But in the presence of dimethyl sulphoxide and at high pH, the EcoRV enzyme cleaves DNA at several sites that differ from its recognition site by one nucleotide. Of the 18 (3 X 6) possible sequences that differ from GATATC by one base, all were cleaved readily except for the following 4 sites: TATATC, CATATC, GATATA and GATATG. However, two of the sites that could be cleaved by EcoRV in the presence of dimethyl sulphoxide, GAGATC and GATCTC, were only cleaved on DNA that lacked dam methylation: both contain the sequence GATC, the recognition site for the dam methylase of Escherichia coli.     

Purification of cohesive-end-producing restriction endonuclease from Bacillus subtilis G.

A new restriction endonuclease was partially purified from Bacillus subtilis G (IAM1247). This restriction endonuclease (endonuclease RBsuG) seems to produce cohesive ends at its cleavage site.     

Site-specific restriction endonuclease BtcI from Bacillus thuringiensis var. canadensis

Efficiency of bacteriophage Tp4 plating on Bacillus thuringiensis var. canadensis H5 (Can) is decreased 10(7)-fold as compared with the efficiency of plating on Bacillus thuringiensis var. galleriae H5 (Gal). Bacteriophage Tp4 having propagated for one cycle in Can cells might be further grown in this strain without restriction. The sitespecific restriction endonuclease BtcI isolated from Bacillus thuringiensis var. canadensis recognises the same nucleotide sequence GATC in DNA as recognised by restriction endonuclease Sau3A.     

Recognition sequence for the restriction endonuclease BglI from Bacillus globigii

Restriction endonuclease BglI recognizes the DNA sequence (Formula: see text) and cleaves each strand at the site indicated, thus generating 3' protruding ends. The recognition sequence was deduced by correlating mapping data with nucleotide sequence information and the position of cleavage was unambiguously determined by 32P labeling of 5' termini produced by BglI digestion.     

Determination of substrate specificity of restriction endonuclease FauI

The recognition sequence and cleavage point of restriction endonuclease FauI have been determined as 5'-CCCGC(4/6). Not being isoschisomer of any known restriction endonuclease, this enzyme may be used in genetic engineering.     

Purification and substrate specificity of BcmI restriction endonuclease

A strain producing the new specific restriction endonuclease BcmI has been found in the Bacillus generum. The enzyme has been purified by chromatography on the blue sepharose, phosphocellulose PII, heparin sepharose. The analogous purification has been obtained when the blue sepharose has been substituted for the orange sepharose, the home produced sorbent. The BcmI enzyme has been shown by the substrate specificity definition to be an isoschizomer of the restriction endonuclease ClaI.