Restriction endonucleases HindII and TaqI cleave DNA with mismatched nucleotides within their recognition sequences.

Restriction endonucleases HindII and TaqI, but not SalI, were found to efficiently cleave synthetic hexadecanucleotide duplexes which contained either an A/C or a G/T mismatch within their respective restriction sites. Double-stranded M13 DNAs with identical mismatches were also cleaved under the assay conditions. These results suggest that the distortion of the DNA duplex, caused by these purine/pyrimidine mismatches is not sufficiently large so as to interfere with the recognition and the subsequent cleavage of the DNA by these two enzymes. HindII and SalI, but not TaqI, were furthermore shown to hydrolyze the two strands of the duplex with different rates. The differences between the mode of recognition of their respective restriction sites by these three enzymes are discussed.     

Purification and characterization of two new modification methylases: MClaI from Caryophanon latum L and MTaqI from Thermus aquaticus YTI.

A method for detecting Type II modification methylases and determining their methylation site by assaying the ability of methylated DNA to be cleaved by heterologous restriction enzymes is described and applied to the isolation of the restriction modification methylases from Thermus thermophilus HB8, Thermus aquaticus YTI and Caryophanon latum L. M.TaqI is shown to have a methylation specificity identical to M.ThI (TCGmeA). M.ClaI methylates at adenine and protects a subset of TthI sites indicating that it methylates the sequence ATCGmeAT. Methylation by M.ThI also protects against cleavage by SalI, XhoI and at some HindII, AccI and MboI sites.     

Cleavage of DNA.RNA hybrids by type II restriction enzymes.

The action of a number of restriction enzymes on DNA.RNA hybrids has been examined using hybrids synthesised with RNAs of cucumber mosaic virus as templates. The enzymes EcoRI, HindII, SalI, MspI, HhaI, AluI, TaqI and HaeIII cleaved the DNA strand of the hybrids (and possible also the RNA strand) into specific fragments. For four of these enzymes, HhaI, AluI, TaqI and HaeIII, comparison of the restriction fragments produced with the known sequences of the viral RNAs confirmed that they were recognising and cleaving the DNA strand of the hybrids at their correct recognition sequences. It is likely that the ability to utilise DNA.RNA hybrids as substrates is a general property of Type II restriction enzymes.     

The effect of Z-conformation on enzymatic activity of restriction endonucleases

Recombinant plasmid pGC20 containing (GC)9-insert into SmaI site of pUC19 has been used to study the inhibition of cleavage by six restriction endonucleases; KpnI, SacI, EcoRI and also BamHI, XbaI and SalI, due to Z-DNA formation in negatively supercoiled plasmid. The recognition sites of these enzymes were located at different distances on both sides of the (CG)10-sequence. It was shown that the inhibition of the cleavage by KpnI, SacI and EcoRI was decreased in this series as fast as the distance between recognition site and B-Z junction was increased, and no inhibition of cleavage by EcoRI was found. However, such a correlation was not found in the series of BamHI, XbaI and SalI. In contrast with EcoRI the cleavage by SalI was inhibited completely. These results indicate the difference for "sensitivity" of restriction endonucleases to the structural perturbations of DNA associated with B-Z junctions. It seems to depend on features of the enzyme-substrate interaction mechanisms and also on recognition and flanking sequences of DNA. Consequently, experiments with the inhibition of the cleavage by any enzyme can not help to determine the dimension of the region of DNA with altered structure.     

Construction of a recombinant bacterial plasmid containing DNA sequences for a mouse embryonic globin chain.

Messenger RNAs for mouse embryonic globins were purified from yolk sac derived eyrthroid cells in mouse fetuses. Double stranded DNAs complementary to these messengers were synthesized and blunt end ligated to a EcoRI digested and DNA polymerase I repaired pBR322 plasmid. Of the ampicillin resistant transformants, one contained a plasmid with globin-specific cDNA. The inserted sequence is about 350 base pairs long. It contains one restriction site for EcoRI and one restriction site for HinfI about 170 and 80 base pairs from one end. The insert is not cleaved by HindIII, HindII, BamHI, PstI, SalI, AvaI, TaqI, HpaII, BglI. A mixture of purified messengers coding for alpha chains and for x, y and z embryonic chains was incubated with the recombinant plasmid and the hybridized messenger was translated in a mRNA depleted reticulocyte lysate protein synthesizing system. The product of translation was identified as a z chain by carboxymethylcellulose cromatography. The recombinant plasmid is named "pBR322-egz" after embryonic globin z.     

The TaqI 'star' reaction: strand preferences reveal hydrogen-bond donor and acceptor sites in canonical sequence recognition

TaqI endonuclease recognizes and cleaves its canonical sequence, TCGA, with complete fidelity under standard conditions. In the presence of some organic solvents, TaqI endonuclease introduced additional single-strand and double-strand cuts at sequences termed TaqI 'star' sites. Using 'middle-labeled' DNA, the relative rates of cleavage of each strand were simultaneously determined for several star sites. These star recognition sequences differed from the canonical sequence by a single base, and all potential star sites were either nicked or cleaved. Star sites within the middle labeled substrate represented ten of the twelve possible star sequences for each strand. For each group of identical star sites, one strand was consistently preferred for cleavage. Based on these preferences, a model for TaqI recognition of the TCGA sequence is proposed. According to this model, sequence discrimination is mediated by eight hydrogen bonds formed between TaqI and the cognate nucleotides within the major groove.     

Single-strand and double-strand cleavage at half-modified and fully modified recognition sites for the restriction nucleases Sau3a and Taqi

The influence of cytosine methylation on the cleavage of DNA by the restriction nucleases Sau3A and TaqI has been investigated. Bovine satellite DNA fragments containing a GATCGA sequence, i.e. a Sau3A site overlapping with a TaqI site have been used in this study. The methylation of these fragments has been determined by sequence analysis. It has been found that a TaqI site (TCGA) methylated at cytosine in both DNA strands is still sensitive to double-strand cleavage. A Sau3A site (GATC), however, is rendered resistant to double-strand cleavage by methylation of a single cytosine. Fragments containing the "half-modified" Sau3A site are nicked in the unmethylated DNA strand. It has been shown by sequence analysis of nicked DNA that the single-strand break occurs at the same position which is cleaved in unmodified DNA.     

Construction and mapping of recombinant plasmids used for the preparation of DNA fragments containing the Escherichia coli lactose operator and promoter.

Three DNA restriction fragments of established sequence containing the Escherichia coli lac genetic controlling regions were cloned. In each case a recombinant plasmid was constructed which was suitable for the subsequent large scale purification of the lac fragment. A 789-base pair HindII fragment, containing the lac operator, promoter, and cyclic AMP receptor protein binding site, was ligated into the single HindII site of the amplifiable plasmid minicolicin E1 DNA (pVH51). A 203-base pair Hae III fragment containing the same genetic sites was ligated into the single Eco RI site of pVH51 which had been "filled in" by the Micrococcus luteus DNA polymerase. Thus, the lac fragment was inserted between two Eco RI sites. Plasmids containing multiple copies of this Eco RI fragment were then constructed. A 95-base pair Alu I fragment containing the lac promoter and operator was cloned similarly. Also, the 203-base pair fragment was cloned into the Eco RI site of pVH51 using a 300-base pair linker fragment (isolated by RPC-5 column chromatography) which permitted retention of its Hae III ends. Mapping studies on pVH51 DNA with a number of DNA restriction endonucleases, including Alu I, Taq I, and Hpa II, are described.     

Vectors with hidden cloning sites

A general strategy is described for using the cleavage site of restriction enzymes in vectors for cloning regardless of how many sites the given enzymes have in the vector. The application of this method allows one to open any vector at its cloning site with protruding ends which can be compatible with almost every commercially available Class II restriction enzyme. By employing this method, the laborious construction of new vectors can be simplified considerably. This general strategy is based on the known ability of Class IIS restriction enzymes to cut any sequence located outside of their recognition site; the introduction of a linker containing recognition site(s) for Class IIS restriction enzyme(s), not present originally in the vector, gives rise to the possibility of opening the vector so as to produce overhangs of arbitrary sequence. In particular, when a symmetrical short sequence representing the protruding end of any Class II enzyme is situated at the cutting position of the Class IIS enzyme, cleavage with the Class IIS enzyme exposes the hitherto hidden, "unique" cloning site. This technique is demonstrated by cloning the cDNA of the multidrug resistance protein to an expression vector.     

Bovine mitochondrial DNA polymorphism in restriction endonuclease cleavage patterns and the location of the polymorphic sites

Cleavage patterns of mitochondrial DNA (mtDNA) by restriction endonuclease analysis were examined in four Japanese Black cows, three Japanese Shorthorn cows, and six Holstein cows. Seventeen restriction enzymes which recognize six base pairs and two restriction enzymes which recognize four base pairs were used in this study. Polymorphism was observed with three restriction enzymes, HindIII, TaqI, and MspI, and was detected within the breeds. Nucleotide substitution was determined in the HindIII polymorphic site by DNA cloning and sequencing; this is C----T at position 10126 of the URF-3 region. Furthermore, the MspI and TaqI polymorphic sites were located on the physical map.     

Partial characterization of a DNA restriction endonuclease from Ruminococcus flavefaciens FD-1 and its inhibition by site-specific adenine methylation.

The principal DNA restriction-modification system of the cellulolytic ruminal bacterium Ruminococcus flavefaciens FD-1 is described. The restriction endonuclease RflFI could be separated from cell extracts by phosphocellulose and heparin-sepharose chromatography. Restriction enzyme digests utilizing RflFI alone or in combination with SalI, a restriction enzyme isolated from Streptomyces albus G, showed that the DNA sequence recognized by RflFI either overlapped or was the same as that recognized by SalI. DNA sequence analysis confirmed that RflFI was identical in activity to SalI, with the recognition sequence being 5'-GTCGAC-3' and cleavage occurring between G and T. Adenine methylation within this sequence can be catalyzed in vitro by TaqI methylase, and this inhibited the cleavage of plasmid DNA molecules by RflFI and SalI. Chromosomal DNA from R. flavefaciens FD-1 is also methylated within this DNA sequence because neither restriction endonuclease could degrade this DNA substrate. These findings provide a means to protect plasmid molecules from degradation prior to gene transfer experiments with R. flavefaciens FD-1.     

Partial characterization of a DNA restriction endonuclease from Ruminococcus flavefaciens FD-1 and its inhibition by site-specific adenine methylation.

The principal DNA restriction-modification system of the cellulolytic ruminal bacterium Ruminococcus flavefaciens FD-1 is described. The restriction endonuclease RflFI could be separated from cell extracts by phosphocellulose and heparin-sepharose chromatography. Restriction enzyme digests utilizing RflFI alone or in combination with SalI, a restriction enzyme isolated from Streptomyces albus G, showed that the DNA sequence recognized by RflFI either overlapped or was the same as that recognized by SalI. DNA sequence analysis confirmed that RflFI was identical in activity to SalI, with the recognition sequence being 5'-GTCGAC-3' and cleavage occurring between G and T. Adenine methylation within this sequence can be catalyzed in vitro by TaqI methylase, and this inhibited the cleavage of plasmid DNA molecules by RflFI and SalI. Chromosomal DNA from R. flavefaciens FD-1 is also methylated within this DNA sequence because neither restriction endonuclease could degrade this DNA substrate. These findings provide a means to protect plasmid molecules from degradation prior to gene transfer experiments with R. flavefaciens FD-1.     

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.     

pUR222, a vector for cloning and rapid chemical sequencing of DNA.

A multipurpose plasmid, pUR222, was constructed. It contains six unique cloning sites (PstI, SalI, AccI, HindII, BamHI and EcoRI) in a small region of its lac Z-gene part. Insertion of foreign DNA into the plasmid can be easily detected. Bacteria harbouring recombinant plasmids generally give rise to white colonies, while those containing only vector DNA form blue colonies on indicator plates. Plasmid DNA purified by a rapid method (Birnboim, H.C. and Doly, J. (1979) Nucl. Acids. Res. 7, 1513-1523) can be used for chemical sequencing of the cloned insert DNA. Labeled fragments need not be isolated after cutting with the proper restriction enzymes and are treated directly according to the sequencing protocol of Maxam and Gilbert.     

Physical mapping of Streptomyces coelicolar A3(3) actinophages. III. Restriction analysis

It has been shown that endonucleases HindII, HindIII, SalI and BsuI treatment of phiC62, or phiC43 and phiC31 DNAs forms more than 20 fragments. EcoRI cleaves phiC62, phiC31, phiC31c5 and phiC31c28 into seven fragments, but phiC311yg33 into six fragments. Comparison of molecular weights of DNA restricts obtained after hydrolysis of phage DNAs containing deletions by endonuclease EcoRI made it possible to determine the location of four fragments on restriction map and to orientate this map in relation to the molecule's ends. BamHI cleaves phiC43 DNA into two fragments. By heteroduplexing BamHi site was mapped within the phiC43 insertion sequence.     

Hydrolysis by restriction endonucleases at their DNA recognition sequences substituted with mismatched base pairs.

Restriction endonucleases were tested for their ability to catalyze the cleavage of mismatch-containing recognition sites in DNA. These mismatched base pairs were T.G, U.G, or A.C in covalently closed, circular heteroduplexes prepared by in vitro extension of chemically synthesized oligonucleotide primers annealed to a bacteriophage M13-derived viral DNA. None of the restriction enzymes was able to completely cleave the mismatch-containing recognition sites under standard conditions. However, three of them, SmaI, SalI, and SstI, catalyzed partial digestion leading to an accumulation of DNA singly nicked at the mismatched recognition site. The ability of SmaI and SstI to partially cleave at a mismatch was shown to depend on the nature and position of the mismatch within the corresponding recognition site. In contrast, little or no digestion was obtained with AccI, HincII, HindIII, and KpnI at mismatch-containing sites. Therefore, in some cases a transition-type substitution in only one strand of a recognition site inhibits restriction endonuclease-catalyzed digestion at that site although in others partial digestion occurs.     

alpha-Putrescinylthymine and the sensitivity of bacteriophage phi W-14 DNA to restriction endonucleases.

The modified base alpha-putrescinylthymine (putT) in phi W-14 DNA blocks cleavage of the DNA by 17 of 32 Type II restriction endonucleases. The enzymes cleaving the DNA do so to widely varying extents. The frequencies of cleavage of three altered forms of the DNA show that putT blocks recognition sites either when it occurs within the site or when it is in a sequence flanking the site. The blocking is dependent on both charge and steric factors. The charge effects can be greater than the steric effects for some of the enzymes tested. All the enzymes cleaving phi W-14 DNA release discrete fragments, showing that the distribution of putT is ordered. The cleavage frequencies for different enzymes suggest that the sequence CAputTG occurs frequently in the DNA. Only TaqI of the enzymes tested appeared not to be blocked by putT, but it was slowed down. TaqI generated fragments are joinable by T4 DNA ligase.     

Construction of plasmid vectors that facilitate subcloning and recovery of yeast and Escherichia coli DNA fragments

We have constructed a plasmid vector (pNF2) which is a derivative of the multicopy yeast cloning vehicle YEp24. This derivative contains a single BamHI site flanked immediately on each side by SalI sites. The latter site was selected because it appears to be infrequent in yeast nuclear DNA. Thus, DNA fragments produced by partial digestion with enzymes (such as Sau3A) that cut at frequent intervals and leave single-stranded ends that have sequence homology with BamHI sites, can be conveniently subcloned into this site. Such fragments can then be excised intact by digestion with SalI enzyme. Plasmid pNF2 also contains the kanamycin-resistance (kanR) gene derived from Tn903 and confers resistance in yeast to the antibiotic G418. pNF2 was converted into an integrating vector (pNF3) by deleting a 2.2-kb EcoRI fragment containing a sequence that determines autonomous replication in yeast. Further deletion of a HindIII fragment containing the yeast URA3 gene converts the plasmid into one containing only pBR322 sequences plus the kanR gene (pNF4).     

Cleavage of phosphorothioate-substituted DNA by restriction endonucleases

M13 RF DNA was synthesized in vitro in the presence of various single deoxynucleoside 5'-O-(1-thiotriphosphate) phosphorothioate analogues, and the three other appropriate deoxynucleoside triphosphates using a M13 (+)-single-stranded template, Escherichia coli DNA polymerase I and T4 DNA ligase. The resulting DNAs contained various restriction endonuclease recognition sequences which had been modified at their cleavage points in the (-)-strand by phosphorothioate substitution. The behavior of the restriction enzymes AvaI, BamHI, EcoRI, HindIII, and SalI towards these substituted DNAs was investigated. EcoRI, BamHI, and HindIII were found to cleave appropriate phosphorothioate-substituted DNA at a reduced rate compared to normal M13 RF DNA, and by a two-step process in which all of the DNA is converted to an isolable intermediate nicked molecule containing a specific discontinuity at the respective recognition site presumably in the (+)-strand. By contrast, SalI cleaved substituted DNA effectively without the intermediacy of a nicked form. AvaI, however, is only capable of cleaving the unsubstituted (+)-strand in appropriately modified DNA.     

HindII, HindIII, and HpaI restriction fragment maps of bacteriophage lambda DNA

The site-specific restriction endonucleases isolated from Hemophilus influenzae strains Rc (HincII) and Rd (HindII + III), and Hemophilus parainfluenzae (HpaI) were used to digest bacteriophage lambda DNA into 34, 40, and 15 specific fragments, respectively. The sites cleaved by each of these enzymes were localized on the lambda physical map and the fragments resulting from these cleavages were electrophoretically identified on gels by (1) analysis of the digestion profiles of deletion and transducing derivatives of lambda; and (2) digesting individual fragments produced by one restriction endonuclease with another restriction endonuclease. This paper presents the HindII, HindIII, and HpaI restriction fragment maps for the entire lambda genome, and the data used to derive these maps for the region of the lambda genome between the attachment site (at 57.3% lambda) and the right vegetative end (100% lambda). The data for mapping the left arm of lambda may be found in the accompanying paper (Robinson and Landy, 1977).