The specific non-symmetrical sequence recognized by restriction endonuclease MboII

The restriction endonuclease MboII, isolated from Moraxella bovis (ATCC 10900), cleaves bacteriophage φX174am3 replicative form I DNA into ten fragments. The physical map of these fragments has been aligned with the sequence of φX174 DNA. There is no sequence with 2-fold rotational symmetry common to the region of all ten cleavage sites. However, the non-symmetrical sequence 5′-G-A-A-G-A-3′ 3′-C-T-T-C-T-5′ occurs near to each cleavage site. Precise mapping of the cleavages in both DNA strands at several sites places the cuts eight nucleotides to the right of the upper sequence and seven nucleotides to the right of the lower sequence.     

A specific endonuclease from Haemophilus haemolyticus.

A restriction-like endonuclease, HhaI, has been partially purified from Haemophilus haemolyticus. This enzyme cleaves bacteriophage lambda DNA and adenovirus-2 DNA at many sites, and cleaves simian virus 40 DNA at only two sites. It recognizes the sequence 5′ -G-C-G-↓C-3′ 3′ -C-↑G-C-G-5′, and cuts at the sites indicated by the arrows.     

A specific endonuclease from Brevibacterium albidum

A new restriction-like endonuclease, BalI, has been partially purified from Brevibacterium albidum. This enzyme cleaves bacteriophage λ DNA at least 18 times and adenovirus-2 DNA at least 16 times, but does not cleave simian virus 40 DNA. All sites cleaved by BalI are also cut by the specific endonuclease HaeIII from Haemophilus aegyptius. The recognition sequence of BalI is 5′-T-G-G ↓ C-C-A-3′ 3′-A-C-C ↓ G-G-T-5′ and the cleavage site is indicated by the arrows.     

Characterization of a site-specific restriction endonuclease SphI from Streptomyces phaeochromogenes

A new type-II restriction endonuclease SphI, has been partially purified from Streptomyces phaeochromogenes. SphI recognizes the hexanucleotide sequence 5′-GCATG↓C and cleaves it at the position marked by the arrow. This nucleotide sequence is present twice in SV40 DNA, four times in λ DNA and only once in the cloning vehicles pBR322, pBR325, pBR327 and pBR328.     

Sth132I,a novel class-IIS restriction endonuclease of Streptococcus thermophilus ST132

The Sth132I restriction endonuclease (R.Sth132I) was detected in Streptococcus thermophilus ST132 and purified to near homogeneity by heparin Sepharose CL-6B affinity chromatography. Fragments from Sth132I digestion of plasmid DNA were subcloned into pUC19 in Escherichia coli DH5α and sequenced. Sequence analysis of inserts and their ligation junction sites revealed that Sth132I is a novel class-IIS restriction endonuclease, which recognizes the non-palindromic sequence5′-CCCG(N)₄-3′3′-GGGC(N)₈-5′.     

Cloning of human mitochondrial DNA in Escherichia coli

In order to determine its nucleotide sequence, human mitochondrial DNA (mtDNA) purified from term placentae was cloned in Escherichia coli using the plasmid vector pBR322. The products of an mtDNA MboI digestion (23 fragments ranging in size from 2800 to 25 base-pairs (bp)) were ligated with BamHI-cut pBR322. The ampicillin-resistant tetracycline-sensitive colonies obtained upon transformation of E. coli χ1776 were screened by agarose gel electrophoresis of colony lysates, colony hybridization and restriction analysis. All but MboI fragment 2 were obtained in this way. MboI fragments 5 and 8 were each found only once among the 705 clones screened. All other MboI fragments were approximately equally represented in the population of clones except for a slight bias towards smaller fragments. MboI fragment 2 overlaps with the mtDNA BamHI/EcoRI (1.7 kb³) and the 0.9 kb HinIII fragments. These were cloned in similarly restricted pBR322 to provide a set of clones covering most of the mtDNA molecule. Clones representative of each MboI fragment were shown to be complementary to mtDNA by hybridization to Southern blots of mtDNA digests and were thereby partially mapped. Further mapping was obtained by restriction analysis of mtDNA sequentially degraded by exonuclease III. A collection of recombinant clones has thus been obtained using the mtDNA isolated from a single placenta and is now being used to obtain a complete nucleotide sequence of human mtDNA.     

Characterization of DNA restriction and modification activities in Neisseria species

Type II restriction endonuclease activities detected in various Neisseria species were characterized for sequence specificity and precise site of cleavage. NsiCI isolated from N. sicca C351 cleaves the sequence 5′-GAT↓ATC-3′ (EcoRV isoschizomer); NmeCI from N. meningitidis C114 and NphI from N. pharyngis C245 cleave 5′-N↓GATCN-3′ (MboI isoschizomers); NgoPII and NgoPIII from N. gonorrhoeae P9-2 cleave at 5′-CC↓GCGG-3′ (SacII isoschizomer) and 5′-GG↓CC-3′ (HaeIII isoschizomer), respectively. Chromosomal DNA isolated from these strains and two other N. meningitidis strains (which lacked detectable endonuclease activities), was found to be refractive to cleavage by various restriction enzymes, implying the presence of methylase activities additional to those required for protection against the cellular endonucleases.     

A sequence specific endonuclease from Micrococcus radiodurans

A new sequence specific endonuclease, MraI has been purified from Micrococcus radiodurans. This enzyme cleaves bacteriophage λ DNA at three sites, adenovirus type 2 DNA at more than 12 sites and has a unique site on ΦX174 DNA. It has no sites on SV40, PM2 and pBR322 DNA. The three sites on phage λ DNA are different from those cleaved by SmaI, XmaI and XorII. The sites of cleavage are located at 0.424, 0.447 and 0.834 fractional lengths on the physical map of λ DNA. MraI is shown to be an isoschizomer of SacII and SstII recognizing the palindromic nucleotide sequence ′5-CCGC↓GG-3′. The enzyme shows an absolute requirement of Mg²⁺, but is active in the absence of added 2-mercaptoethanol. The enzyme shows activity at a broad range of temperature and pH with an optimum at 45°C and pH 7.0. MraI represents the first restriction enzyme from a bacterium whose DNA lacks modified methylated bases.     

A specific endonuclease from Arthrobacter luteus.

A new restriction-like endonuclease, AluI, has been partially purified from Arthrobacter luteus. This enzyme cleaves bacteriophage λ DNA, adenovirus-2 DNA and simian virus 40 DNA at many sites including all sites cleaved by the endonuclease HindIII from Haemophilus influenzae serotype d. Radioactive oligonucleotides in pancreatic DNAase digests of (5′-³²P)-labelled fragments of phage λ DNA released by the action of AluI had the 5′ terminal sequence pC-T-N-. The enzyme recognises the tetranucleotide sequence

and cleaves it at the position marked by the arrows.     

A new site-specific endonuclease BbeI from Bifidobacterium breve: Restriction endonucleases; ; 3′-cohesive termini; DNA sequence analysis

A new site-specific endonuclease, BbeI, has been partially purified from the anaerobic bacterium, Bifidobac-terium breve. BbeI recognizes the hexanucleotide sequence

and cleaves it at the sites indicated by the arrows, producing 3′-cohesive termini four bases long.     

A new restriction endonuclease from Streptomyces albus G.

A restriction endonuclease, SalI, has been partially purified from Streptomyces albus G. This enzyme cleaves adenovirus-2 DNA at three sites, bacteriophage λ DNA at two sites, but does not cleave simian virus 40 DNA or φX174 DNA. It recognizes the sequence

and cuts at the sites indicated by the arrows. An endonuclease (XamI) with similar specificity has also been isolated from Xanthomonas amaranthicola.     

Paucity of the Sau3AI recognition sequence (GATC) in the genome of Methanococcus voltae

Summary High molecular weight genomic DNA isolated from the archaebacterium Methanococcus voltae by alkaline-SDS lysis was not effectively digested with the restriction enzyme Sau3AI, which recognizes the base sequence GATC. Mc. voltae DNA was also resistant to digestion by MboI and BamHI which recognize sites containing the same GATC sequence. Examination of a Mc. voltae genomic library prepared in Escherichia coli JM83 with a pUC vector revealed that the 5–10 kb inserts were still resistant to Sau3AI digestion, indicating a likely lack of the GATC sequence in Mc. voltae DNA.     

Use of a new retrieving adaptor in the cloning of a synthetic human insulin A-chain gene

A DNA duplex encoding the A-chain of human insulin was constructed from eight chemically synthesized oligomers by enzymatic ligation to form a partial duplex followed by repair synthesis to complete the complementary strands. After sequential addition of translation start and stop signal adaptors the assembly was cloned in pBR322. To regenerate the end of the coding sequence by precise removal of extraneous nucleotides a new method using a synthetic retrieval adaptor was developed. The procedure included filling in the cohesive ends of the EcoRI site by repair synthesis, ligating a symmetrical adaptor having an MboII recognition sequence to the resulting blunt end, cutting with MboII and removing the single protruding 3′-nucleotide using the 3′ exonuclease activity of DNA polymerase I. Synthetic oligomers useful for ligation to a synthetic insulin C-chain gene were added to the retrieved end of the gene. Sequence analysis established that retrieval adaptors of this type may be used for precise excision of up to eight nucleotides from the end of a cloned DNA fragment.     

Structural Requirements forFokI-DNA Interaction and Oligodeoxyribonucleotide-instructed Cleavage

TheFokI restriction endonuclease recognizes the double-stranded (ds) 5′-GGATG-3′ site and cuts at the 9th and 13th nucleotides downstream from the 5′-3′ and 3′-5′ strands, respectively. To elucidate the interaction betweenFokI and DNA, and the effect of Mg²⁺on this interaction, we usedFokI with various combinations of dsDNA, single-stranded (ss) DNA and oligodeoxyribonucleotides (oligos) containing a double-stranded hairpin carrying theFokI recognition site. Oligo- and dsDNA-FokI interactions showed that for fully effective recognition, two or more base-pairs were required outside the 5′-GGATG-3′ site. When usingFokI with ssDNA and oligos, precise cutting with no observable byproducts was observed at the 9th or 13th nucleotide. This was independent of whether the region between the recognition and cut sites was perfectly complementary or whether there were up to four mismatches in this region, or a single mismatch within the cut site. Moreover,FokI cleavage, when followed by step-wise filling-in ofFokI cohesive ends in the dsDNA, allowedFokI to recleave such sites when two or more nucleotides were added, releasing 2-mer, 3-mer, or 4-mer single-stranded chains. Electrophoretic mobility shift assays showed that the DNA helix was bent when complexed withFokI (without Mg²⁺). Such a complex, when formed in the absence of Mg²⁺, did not accept the subsequently added Mg²⁺for several minutes. This suggests a tight, diffusion-resistant contact between the enzyme and the cognate DNA sequence. In the presence of Mg²⁺, the half-life of the complexFokI and dsDNA was 12 minutes at 22°C. In the absence of Mg²⁺, such a complex, possessing a terminally located 5′-GGATG-3′ site, had a half-life of 1.5 to 2 minutes. However, if magnesium ions were present, this complex had a stability similar to that of a complex formed with dsDNA containing a centrally located 5′-GGATG-3′ site.     

A site-specific endonuclease from Caulobacter crescentus CB-13: restriction endonuclease CcrI

A site-specific restriction endonuclease (CcrI) has been identified from Caulobacter crescentus CB-13. This enzyme has been purified to homogeneity and the cleavage patterns with various DNAs and sequence data show that CcrI recognizes the same sequence as the XhoI restriction endonuclease (5′-C^↓-T-C-G-A-G-3′). Ccr has an absolute requirement for magnesium ions with an optimum concentration of 4 mM. The enzyme is optimally active at pH 8.0 and is stable up to 70°C. CcrI has a molecular weight of 65300 and exists as a monomer in its native state. Most of the physical characteristics observed for CcrI were similar to those observed for XhoI. Kinetic studies on CcrI and XhoI suggest that the enzymes interact with λ DNA in the same manner; however, with ?X-174 R.F. DNA, CcrI has a greater affinity for the supercoiled molecule than XhoI.     

A third restriction endonuclease from Xanthomonas malvacearum.

An additional sequence-specific endonuclease, XmaIII, has been partially purified from Xanthomonas malvacearum. XmaIII recognizes ten cleavage sites in adenovirus 2 DNA, two sites in bacteriophage lambda and no site in either simian virus 40 DNA or φX174 DNA. It recognizes the sequence

and cleaves at the sites indicated by the arrows. No other endonuclease with this particular nucleotide sequence specificity has been reported.     

A new specific endonuclease present in Xanthomonas holcicola, Xanthomonas papavericola and Brevibacterium luteum

A new specific endonuclease, XhoI, has been partially purified from Xanthomonas holcicola. This enzyme cleaves adenovirus-2 DNA at five sites, bacteriophage λ DNA at one site, φX174 DNA at one site, but does not cleave simian virus 40 DNA. It recognizes the sequence

and cuts at the sites indicated by the arrows. Enzymes with identical specificity have also been found in Xanthomonas papavericola and Brevibacterium luteum.     

The relaxation of specificity of BanI restriction endonuclease from Bacillus aneurinolyticus IAM 1077

The restriction endonuclease BanI from Bacillus aneurinolyticus IAM 1077, which recognizes 5′-GGPyPuCC-3′ and cleaves between G and G within this sequence, has decreased substrate specificity at high nuclease concentrations. The relaxation of its specificity was enhanced during modified reactions: digestion of pBR322 DNA or lambda DNA in the presence of high glycerol and dimethyl-sulfoxide (DMSO) produced additional fragments in addition to the inherent fragments. Therefore, it is required to check the reaction conditions carefully for generation of inherent fragments.     

Cartographie de Restriction Comparée du Satellite DNA chez l'Homme et le Chimpanzé

Highly repeated DNA satellite α sequences from man and chimpanzee (Pan troglodytes) have been compared, using restriction endonucleases. The two species share a 340 base pairs tandemly represented DNA, that is cut once by EcoRt. Pan troglodytes differ from man by loss of the two MboI and EcoRI star sites and by the gain of an Hae III site in the repeated sequence.