Molecular cloning of the three base restriction endonuclease R.CviJI from eukaryotic Chlorella virus IL-3A.

R.CviJI is unique among site-specific restriction endonucleases in that its activity can be modulated to recognize either a two or three base sequence. Normally R.CviJI cleaves RGCY sites between the G and C to leave blunt ends. In the presence of ATP R.CviJI* cleaves RGCN and YGCY sites, but not YGCR sites. The gene encoding R.CviJI was cloned from the eukaryotic Chlorella virus IL-3A and expressed in Escherichia coli. The primary E.coli cviJIR gene product is a 278 amino acid protein initiated from a GTG codon, rather than the expected 358 amino acid protein initiated from an in-frame upstream ATG codon. Interestingly, the 278 amino acid protein displays the normal restriction activity but not the R.CviJI* activity of the native enzyme. Nine restriction and modification proteins which recognize a central GC or CG sequence share short regions of identity with R.CviJI amino acids 144-235, suggesting that this region is the recognition and/or catalytic domain.     

IL-3A virus infection of a Chlorella-like green alga induces a DNA restriction endonuclease with novel sequence specificity.

A type II restriction endonuclease, named CviJI, was isolated from a eukaryotic Chlorella-like green alga infected with the dsDNA containing virus IL-3A. CviJI is the first restriction endonuclease to recognize the sequence PuGCPy; CviJI cleaves DNA between the G and C. Methylation of the cytosine in PuGCPy sequences prevents cleavage by CviJI. CviJI cleaved DNA into smaller but defined fragments in the presence of ATP. This "star" activity was stimulated by dithiothreitol and/or S-adenosylmethionine but did not occur under conditions which favor "star" activity of other restriction endonucleases.     

Rapid shotgun cloning utilizing the two base recognition endonuclease CviJI.

A new approach has been developed for the rapid fragmentation and fractionation of DNA into a size suitable for shotgun cloning and sequencing. The restriction endonuclease CviJI normally cleaves the recognition sequence PuGCPy between the G and C to leave blunt ends. Atypical reaction conditions which alter the specificity of this enzyme (CviJI**) yield a quasi-random distribution of DNA fragments from the small molecule pUC19 (2686 base pairs). To quantitatively evaluate the randomness of this fragmentation strategy, a CviJI** digest of pUC19 was size fractionated by a rapid gel filtration method and directly ligated, without end repair, to a lacZ minus M13 cloning vector. Sequence analysis of 76 clones showed that CviJI** restricts PyGCPy and PuGCPu, in addition to PuGCPy sites, and that new sequence data is accumulated at a rate consistent with random fragmentation. Advantages of this approach compared to sonication and agarose gel fractionation include: smaller amounts of DNA are required (0.2-0.5 micrograms instead of 2-5 micrograms), fewer steps are involved (no preligation, end repair, chemical extraction, or agarose gel electrophoresis and elution are needed), and higher cloning efficiencies are obtained (CviJI** digested and column fractionated DNA transforms 3-16 times more efficiently than sonicated, end-repaired, and agarose fractionated DNA).     

Chemically-induced affinity star restriction specificity: a novel TspGWI/sinefungin endonuclease with theoretical 3-bp cleavage frequency

The type IIS/IIC restriction endonuclease TspGWI recognizes the sequence 5'-ACGGA-3', cleaving DNA 11/9 nucleotides downstream. Here we show that sinefungin, a cofactor analog of S-adenosyl methionine, induces a unique type of relaxation in DNA recognition specificity. In the presence of sinefungin, TspGWI recognizes and cleaves at least 12 degenerate variants of the original recognition sequence that vary by single base pair changes from the original 5-bp restriction site with only a single degeneracy per variant appearing to be allowed. In addition, sinefungin was found to have a stimulatory effect on cleavage at these nondegenerate TspGWI recognition sites, irrespective of their number or the DNA topology. Interestingly, no fixed "core" could be identified among the new recognition sequences. Theoretically, TspGWI cleaves DNA every 1024 bp, while sinefungin-induced activity cleaves every 78.8 bp, corresponding to a putative 3-bp long recognition site. Thus, the combination of sinefungin and TspGWI represents a novel frequent cutter, next only to CviJI/CviJI*, that should prove useful in DNA cloning methodologies.     

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.     

Cloning of CviPII nicking and modification system from chlorella virus NYs-1 and application of Nt.CviPII in random DNA amplification

The cloning and expression of the CviPII DNA nicking and modification system encoded by chlorella virus NYs-1 is described. The system consists of a co-linear MTase encoding gene (cviPIIM) and a nicking endonuclease encoding gene (cviPIINt) separated by 12 nt. M.CviPII possesses eight conserved amino acid motifs (I to VIII) typical of C5 MTases, but, like another chlorella virus MTase M.CviJI, lacks conserved motifs IX and X. In addition to modification of the first cytosine in CCD (D = A, G or T) sequences, M.CviPII modifies both the first two cytosines in CCAA and CCCG sites as well. Nt.CviPII has significant amino acid sequence similarity to Type II restriction endonuclease CviJI that recognizes an overlapping sequence (RG--CY). Nt.CviPII was expressed in Escherichia coli with or without a His-tag in a host pre-modified by M.CviPII. Recombinant Nt.CviPII recognizes the DNA sequence CCD and cleaves the phosphodiester bond 5' of the first cytosine while the other strand of DNA at this site is not affected. Nt.CviPII displays site preferences with CCR (R = A or G) sites preferred over CCT sites. Nt.CviPII is active from 16 to 65 degrees C with a temperature optimum of 30-45 degrees C. Nt.CviPII can be used to generate single-stranded DNAs (ssDNAs) for isothermal strand-displacement amplification. Nt.CviPII was used in combination with Bst DNA polymerase I large fragment to rapidly amplify anonymous DNA from genomic DNA or from a single bacterial colony.     

Synthesis of DNA coding for human proinsulin

A chemical-enzymatic synthesis of 271- and 286-bp DNA duplexes, each of which contains the entire sequence coding for human proinsulin has been accomplished. In addition to the coding sequence, the 271-bp fragment carries translation initiation and termination signals plus EcoRI-HindIII restriction enzyme sites for insertion into an appropriate plasmid vector. The 286-bp fragment also contains a Shine-Dalgarno (SD) sequence preceding an ATG codon. Employing the 286-bp polynucleotide, the 568-bp tandem proinsulin gene has been obtained. The synthesis of these DNA fragments involved preparation of 42 oligonucleotides by a rapid N-methylimidazolide phosphotriester method and enzymatic conversion of the oligonucleotides into the gene subfragments, which were cloned separately and fused to yield the desired DNAs coding for proinsulin. The proinsulin gene fragments were cloned in Escherichia coli and shown to have the correct sequences.     

A spite specific endonuclease from thermus thermophilus 111, Tth111I.

A site specific endonuclease with novel specificity has been isolated from Thermus thermophilus strain 111 and named Tth111I. Tth111I cleaves lambda DNA into three fragments of 23.5, 25.7 and 50.8% of the total length, and ColE1 DNA into two fragments of nearly equal length. The sequences around Tth111I cleavage sites of ColE1 and lambda DNA were determined by the Maxam and Gilbert method and the two dimensional mapping method. The results suggest that Tth111I recognizes the DNA sequence (formula: see text) and cleaves the site as indicated by the arrows. Assuming that the first T.A pair in the sequence can be replaced for any base pair, the Tth111I recognition sequence has the symmetry with the two-fold axis as most type II restriction endonucleases do.     

Organization of the yeast ribosomal RNA gene cluster via cloning and restriction analysis.

The restriction endonuclease EcoR1 cleaves Saccharomyces cerevisiae DNA, which codes for ribosomal RNA (rRNA), into seven fragments, A second restriction endonuclease, HindIII, cleaves the same yeast ribosomal DNA into two fragments. These two restriction enzymes each yield DNA segments that total about 5.9 megadaltons. The "repeat unit" of the yeast genes coding for rRNA is thus about 5.9 megadaltons or about 9000 base pairs long. The two HindIII-cleaved DNA fragments as well as one of the EcoR1-cleaved DNA fragments were purified and amplified by cloning in Escherichia coli. Three of the seven EcoR1-generated DNA fragments could then be ordered by treating the two cloned HindIII DNA fragments with EcoR1. This led the assignment of the two HindIII restriction sites. The various restriction DNA fragments were hybridized directly from the gel utilizing 32P-labeled 5 S, 5.8 S, 18 S, and 25 S rRNA. Identification of the various DNA restriction segments then led to the final ordering of the DNA fragments. The gene coding for the 5 S RNA is adjacent to the gene coding for the 35 S precursor rRNA. These two groups of genes thus occur as a cluster in the following sequence: [5 S-spacer]-[spacer-18 S-5.8 S-25 S-spacer]-[spacer-5 S]. The actual map of the DNA restriction fragments is presented.     

Cleavage of single strand oligonucleotides and bacteriophage phi X174 DNA by Msp I endonuclease

Type II restriction endonucleases cleave duplex DNA at nucleotide sequences displaying 2-fold symmetry. Our data show that Msp I cleaves single strand oligonucleotides, d(G-A-A-C-C-G-G-A-G-A) and d(T-C-T-C-C-G-G-T-T) at 4 degrees, 25 degrees, and 37 degrees C reaction temperatures. The rate of cleavage of d(G-A-A-C-C-G-G-A-G-A) is several-fold faster than that of d(T-C-T-C-C-G-G-T-T). Single strand phi X174 DNA is also, cleaved by Msp I endonuclease giving well defined fragments. 5'-Nucleotide analysis of the fragments generated from single strand and replicating form DNA suggest that cleavage occurs at the recognition sequence d(C-C-G-G). The data show that Msp I endonuclease cleaves single strand oligonucleotides and prefers a recognition sequence surrounded by purine nucleotides. A general model for endonuclease cleavage of single strand and duplex DNA is presented.     

Creation of a type IIS restriction endonuclease with a long recognition sequence

Type IIS restriction endonucleases cleave DNA outside their recognition sequences, and are therefore particularly useful in the assembly of DNA from smaller fragments. A limitation of type IIS restriction endonucleases in assembly of long DNA sequences is the relative abundance of their target sites. To facilitate ligation-based assembly of extremely long pieces of DNA, we have engineered a new type IIS restriction endonuclease that combines the specificity of the homing endonuclease I-SceI with the type IIS cleavage pattern of FokI. We linked a non-cleaving mutant of I-SceI, which conveys to the chimeric enzyme its specificity for an 18-bp DNA sequence, to the catalytic domain of FokI, which cuts DNA at a defined site outside the target site. Whereas previously described chimeric endonucleases do not produce type IIS-like precise DNA overhangs suitable for ligation, our chimeric endonuclease cleaves double-stranded DNA exactly 2 and 6nt from the target site to generate homogeneous, 5′, four-base overhangs, which can be ligated with 90% fidelity. We anticipate that these enzymes will be particularly useful in manipulation of DNA fragments larger than a thousand bases, which are very likely to contain target sites for all natural type IIS restriction endonucleases.     

Catalytic domain of restriction endonuclease BmrI as a cleavage module for engineering endonucleases with novel substrate specificities

Creating endonucleases with novel sequence specificities provides more possibilities to manipulate DNA. We have created a chimeric endonuclease (CH-endonuclease) consisting of the DNA cleavage domain of BmrI restriction endonuclease and C.BclI, a controller protein of the BclI restriction-modification system. The purified chimeric endonuclease, BmrI198-C.BclI, cleaves DNA at specific sites in the vicinity of the recognition sequence of C.BclI. Double-strand (ds) breaks were observed at two sites: 8 bp upstream and 18 bp within the C-box sequence. Using DNA substrates with deletions of C-box sequence, we show that the chimeric endonuclease requires the 5′ half of the C box only for specific cleavage. A schematic model is proposed for the mode of protein–DNA binding and DNA cleavage. The present study demonstrates that the BmrI cleavage domain can be used to create combinatorial endonucleases that cleave DNA at specific sequences dictated by the DNA-binding partner. The resulting endonucleases will be useful in vitro and in vivo to create ds breaks at specific sites and generate deletions.     

Nonrandom DNA sequencing of exonuclease III-deleted complementary DNA

The nonrandom DNA sequence analysis procedure of Poncz et al. [Proc. Natl. Acad. Sci. USA 79, 4298-4302 (1982)] was extensively modified to permit the determination of complementary DNA (cDNA) sequences containing G-C homopolymer regions. The recombinant cDNA plasmid was cleaved at a unique restriction enzyme site close to the cDNA and treated with Exonuclease III under controlled conditions to generate a set of overlapping fragments having deletions 50-1500 bases in length at the free 3' termini. After removal of single-stranded DNA regions by Bal31 and DNA polymerase I large fragment, the unique restriction enzyme site was recreated by blunt end ligation of synthetic oligonucleotides to the deleted DNA fragments and restriction enzyme digestion. The cDNA fragment was excised from the cloning vector using a second different restriction enzyme having a unique site that flanks the cDNA fragment and subsequently force-cloned into either M13 mp10 or mp11. This method should also be particularly useful for the sequencing of other types of DNA molecules with lengths 1500 bp or smaller.     

Restriction endonuclease activity induced by NC-1A virus infection of a Chlorella-like green alga.

A type II restriction endonuclease, CviBI, was isolated from a eukaryotic, Chlorella-like green alga infected with the dsDNA containing virus NC-1A. The enzyme recognizes the sequence GANTC and cleaves DNA between the G and A. Methylation of deoxyadenosine in the GANTC sequence probably inhibits enzyme activity. In vitro CviBI cleaves host nuclear DNA but not viral DNA. A survey of 18 other viruses which infect the same Chlorella sp. revealed that infection with 5 of these viruses also induced a restriction endonuclease which cleaves DNA into the same size fragments as CviBI.     

Mung bean nuclease cleaves preferentially at the boundaries of variant surface glycoprotein gene transpositions in trypanosome DNA

Telomere-linked genes coding for the variant surface glycoproteins (VSGs) of African trypanosomes have been difficult to clone because their flanking regions frequently lack restriction sites. Therefore, we constructed a genomic DNA library of fragments generated by digestion of purified trypanosome DNA with mung bean nuclease, an enzyme that cleaves before and after genes in Plasmodium falciparum DNA (McCutchan, T. F., Hansen, J. L., Dame, J. B., and Mullins, J. A. (1984) Science 225, 625-628). Southern hybridizations with several gene probes showed that under the appropriate conditions mung bean nuclease produces discrete trypanosome DNA fragments that are as clearly resolved on an agarose gel as restriction fragments. The majority of VSG genes are on fragments of about 1.7 kilobase pairs. To examine the sites of mung bean nuclease cleavage, the insert boundary sequences of eight recombinant clones in the library containing VSG genes were determined. In general, mung bean nuclease cleaved 300-800 base pairs in front of the VSG start codon and within 50 base pairs on either side of the termination codon. These regions also form the boundaries of VSG gene conversion events indicating that the enzyme recognizes, in part, a conformational structure rather than a specific sequence. The analyzed clones included both telomere-linked and interior basic copy VSG genes indicating that the library potentially contains all of the telomere-linked VSG genes in the genome.     

A sequence-specific endonuclease (Xmn I) from Xanthomonas manihotis.

A type II restriction endonuclease Xmn I with a novel site specificity has been isolated from Xanthomonas manihotis. Xmn I does not cleave SV40 DNA, but cleaves phi X174 DNA into three fragments, which constitute 76.61%, 18.08% and 5.31% of the total length of 5386 base pairs, and cleaves pBR322 DNA into two fragments of 55.71% and 44.29% of the entire 4362 base pairs. The nucleotide sequences around the cleavage sites made by Xmn I are not exactly homologous, but they have a common sequence of 5' GAANNNNTTC 3' according to a simple computer program analysis on nucleotide sequences of phi X174 DNA, pBR322 DNA and SV40 DNA. The results suggest that the cleavage site of Xmn I is located within its recognition sequence of 5' GAANNNNTTC 3'.     

Total synthesis of multi-kilobase DNA sequences from oligonucleotides

A method for synthesizing DNA from 40-mer oligonucleotides, which we used to generate a 32-kb DNA fragment, is explained. DNA sequences are synthesized as approximately 500 bp fragments (synthons) in a two-step PCR reaction and cloned using ligation-independent cloning (LIC). Synthons are then assembled into longer full-length sequences in a stepwise manner. By initially synthesizing smaller fragments (synthons), the number of clones sequenced is low compared with synthesizing complete multi-kilobase DNA sequences in a single step. LIC eliminates the need for purification of fragments before cloning, making the process amenable to high-throughput operation and automation. Type IIs restriction enzymes allow seamless assembly of synthons without placing restrictions on the sequence being synthesized. Synthetic fragments are assembled in pairs to generate the final construct using vectors that allow selection of desired clones with two unique antibiotic resistance markers, and this eliminates the need for purification of fragments after digestion with restriction endonucleases.     

Simple repeated sequences in human satellite DNA.

In an extensive analysis, using a range of restriction endonucleases, HinfI and TaqI were found to differentiate satellites I, II and III & IV. Satellite I is resistant to digestion by TaqI, but is cleaved by HinfI to yield three major fragments of approximate size 770, 850 and 950bp, associated in a single length of DNA. The 770bp fragment contains recognition sites for a number of other enzymes, whereas the 850 and 950bp fragments are "silent" by restriction enzyme analysis. Satellite II is digested by HinfI into a large number of very small (10-80bp) fragments, many of which also contain TaqI sites. A proportion of the HinfI sites in satellite II have the sequence 5'GA(GC)TC. The HinfI digestion products of satellites III and IV form a complete ladder, stretching from 15bp or less to more than 250bp, with adjacent multimers separated by an increment of 5bp. The ladder fragments do not contain TaqI sites and all HinfI sites have the sequence 5'GA(AT)TC. Three fragments from the HinfI ladder of satellite III have been sequenced, and all consist of a tandemly repeated 5bp sequence, 5'TTCCA, with a non-repeated, G+C rich sequence, 9bp in length, at the 3' end.     

Preferential uptake of restriction fragments from a gonococcal cryptic plasmid by competent Neisseria gonorrhoeae

Factors involved in the specificity of DNA uptake by competent Neisseria gonorrhoeae were examined. Host-controlled modification did not affect uptake. Certain restriction fragments of the 4.2 kb gonococcal cryptic plasmid pFA1 and of the replicative form of the bacteriophage M13 were taken up in preference to others, independent of differences in fragment size. A 600 bp fragment from the 4.2 kb plasmid was cloned into pLES2, a gonococcal-Escherichia coli shuttle vector; the 600 bp fragment was taken up into a DNAase-I-resistant state in preference to the vector fragment. A second 370 bp fragment in pFA1 was also taken up preferentially. The 600 bp and 370 bp fragments share a 10 bp sequence, which is found in pFA1 only on fragments that were taken up readily. However, a fragment from M13 which was efficiently taken up did not contain this 10 bp sequence. In addition, this sequence was not sufficient to direct preferential DNA uptake by gonococci, since a recombinant plasmid containing this 10 bp sequence was not taken up appreciably better than the vector plasmid or another recombinant plasmid containing an unrelated 10 bp sequence. Sequence comparisons of the three restriction fragments which were preferentially taken up did not yield any consensus sequences greater than 7 bp. Although it is likely that efficient uptake of DNA by gonococci is determined by DNA structure, a single short sequence could not be found that accounted for specific uptake.     

Recognition sequence of restriction endonuclease KpnI from Klebsiella pneumoniae.

We have determined the recognition sequence of the restriction endonuclease KpnI, previously isolated from Klebsiella pneumoniae. The enzyme cleaves the twofold rotationally symmetric sequence (see book for formula) at the positions indicated by the arrows, producing 3' protruding cohesive ends, four nucleotides in length. The specific cleavage site was unambiguously deduced using both 3' and 5' end analyses of KpnI generated restriction fragments of simian-virus 40 (SV40) DNA (1 site), adenovirus-2 (Ad-2) DNA (8 sites), and a plasmid (pCRI) DNA (2 sites).