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.     

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.     

Identity of Phosphodiesterase and Phosphomonoesterase Activities with Nuclease P1 (a Nuclease from Penicillium citrinum)

Enzymatic properties of a purified Penicillium nuclease (designated as nuclease P₁) were investigated. The enzyme activities for RNA, heat-denatured DNA, native DNA, 3′-AMP and 2′-AMP showed a great degree of similarity with respect to the following properties: a) Range of stable pH (5~8), b) temperature optima (at around 70°C), c) thermostability (about 50% inactivation at 67°C, pH 6.0 for 15 min, d) effect of metal ions and SH inhibitors, e) requirement of Zn²⁺, f) protection from the heat-inactivation by albumin and Zn²⁺, g) inactivation on standing in the cold and reactivation on heating, h) sensitivity to protease, and i) competitive relationship between substrates in the enzyme reaction. Moreover, the ratio of enzyme activities in several mutants of Penicillium citrinum was constant. From these results, together with constant ratio of the specific activities throughout purification, it is concluded that a single enzyme might be responsible for both phosphodiesterase and phosphomonoesterase functions.     

The DNA sequence recognised by the HinfIII restriction endonuclease

HinfIII is a type III restriction enzyme (Kauc &; Piekarowicz, 1978) isolated from Haemophilus influenzae Rf. Like other type III restriction endonucleases, the enzyme also catalyses the modification of susceptible DNA. It requires ATP for DNA cleavage and S-adenosyl methionine for DNA methylation. We have determined the DNA sequence recognised by HinfIII to be:

$\text{5′-C-G-A-A-T-3′}\text{·····3′-G-C-T-T-A-5′}$

In restriction, the enzyme cleaves the DNA about 25 base-pairs to the right of this sequence. In the modification reaction only one of the strands is methylated, that containing the 5′-C-G-A-A-T-3′ sequence.     

Regulatory mutants of simian virus 40: constructed mutants with base substitutions at the origin of DNA replication.

Mutants of simian virus 40 (SV40) with base substitutions at or near the origin of replication of the viral genome have been constructed by bisulfite mutagenesis at the BglI restriction site of SV40 DNA, followed by transfection of cells with the BglI-resistant (BglI^r) DNA so generated. Based on plaque morphology at different temperatures, the resulting BglI^r mutants could be classified into four-groups. Class I mutants (designated ar for “altered restriction”) were indistinguishable from wild-type SV40; class II mutants (designated shp for “sharp plaque”) produced small, sharp-edged plaques; class III mutants (designated sp for “small plaque”) produced small plaques at 32 °C, 37 °C and 40 °C; and class IV mutants (designated cs for “cold sensitive”) produced small plaques at 32 °C and wild-type plaques at 37 °C and 40 °C. That the altered plaque morphology of sp and cs mutants was related to mutation at the BglI restriction site was demonstrated by co-reversion to wild-type of the plaque phenotype and BglI sensitivity. The nucleotide sequence around the original BglI site was determined in the DNA from one mutant of each class. In each case a different base-pair substitution was found, at a site outside sequences coding for SV40 proteins. When rates of replication of mutant DNAs were measured during productive infection, ar mutant DNA was synthesized at a rate comparable to that of wild-type SV40 DNA, shp mutant DNA was made at a rate exceeding that of wild-type, sp mutant DNA was synthesized at a lower rate than that of wild type. and cs mutant DNA synthesis was reduced at 32 °C, but about the same as the wild-type rate at 40 °C. These patterns of mutant DNA synthesis were unaltered in cells co-infected with mutant and wild-type virus, i.e. the defects in DNA synthesis were not trans-complementable. We conclude that the defective mutants have single base-pair changes in a cis element that determines the rate of viral DNA replication, presumably within the origin signal itself.     

The Fsp4HI restriction-modification system: Gene cloning, comparison of protein structures, and biochemical properties of recombinant DNA methyltransferase M.Fsp4HI

Genes coding for the Flavobacterium sp. 4H restriction-modification (RM) system, which recognizes the sequence 5′-GCNGC-3′, were cloned in Escherichia coli ER2267 and sequenced. The Fsp4HI RM system includes two genes: one for DNA methyltransferase (M.) and the other for restriction endonuclease (R.), immediately following the former in the same direction. The genes partly overlap. According to the deduced amino acid sequences, M.Fsp4HI belongs to C5 DNA methyltransferases, whereas R.Fsp4HI is only slightly similar to some restriction enzymes recognizing similar sequences. M.Fsp4HI was purified by column chromatography. The optimal conditions for the enzyme are 30°C and pH 7.5. M.Fsp4HI modifies the first cytosine in 5′-GCNGC-3′.     

Purification,Properties and Recognition Sequence of Site-specific Restriction Endonuclease from Gluconobacter cerinus IFO 3285

A type II restriction endonuclease, designated as GceGLI, was purified from cells of Gluconobacter cerinus IFO 3285. The purified enzyme was found to be homogeneous on Polyacrylamide gel disc electrophoresis. The enzyme worked best at 37°C and pH 7.5 and required 7 mM MgCl₂ and 100 mM NaCl. The purified enzyme was stable when preincubated over a pH range of 7.5 to 9.5 for 12 hr at 4°C and a temperature range of 37 to 40°C for 5 min at pH 7.5. The enzyme was shown to cleave λ φX174 RF, SV40, pBR322, M13 mp7 RF and Ad2 DNAs at 4, 1,0, 0, 0 and 25 or more sites, respectively, and to recognize the DNA sequence of 5′-C-C-G-C-G-G-3′ and to cut between C and G on the right side of the sequence, being an isoschizomer of SacII of Streptomyces achromogenes ATCC 12767.     

Comparison of the Restriction Endonuclease Digestion Patterns of Mitochondrial DNA from Normal and Male Sterile Cytoplasms of ZEA MAYS L

High resolution gel electrophoresis has allowed the assignment of fragment number and molecular weight to EcoRI, SalI and PstI restriction fragments of mitochondrial DNA from B37 normal (N) and B37 T, C and S male sterile cytoplasmic types of maize. A minimum complexity of 450-475 kb has been established. Hybridization of cloned EcoRI fragments to restriction digests of total mitochondrial DNA suggests that at least 80% of the genome is composed of unique sequences. Restriction fragments of identical size in N, T, C and S contain similar sequence information as evidenced by their hybridization behavior.—The total SalI digest and the larger PstI fragments representing 80% of the total complexity were used to calculate the fraction of shared fragments of each pairwise combination of cytoplasmic types. The C type mtDNA is most closely allied with the other mtDNAs and shares 67% of fragments with S, 65% with N, and 60% with T. The S type mtDNA is quite divergent from N (53% shared fragments) and T (56% shared fragments). N and T share 59% of the fragments. These results are discussed in terms of the origin of mtDNA diversity in maize.     

A detailed physical map of HeLa cell mitochondria DNA and its alignment with the positions of known genetic markers

Twenty-one fragments have been identified among the products of digestion of HeLa cell mtDNA with the restriction enzyme Hpa II. The sum of the molecular sizes of these fragments, estimated from their mobility relative to that of known markers, accounts, within experimental error, for the total length of HeLa cell mtDNA. The 21 fragments have been ordered in a physical map by two approaches: (1) sequential digestion with Hpa II of the fragments produced by Eco RI, Hind III, andHpa I enzymes, and (2) fragment-primed DNA synthesis. The Hpa II map has been aligned with the maps constructed with the other three enzymes and with the unique cutting site produced by Bam I. The combined map thus obtained has resolved HeLa cell mtDNA into 27 recognizable segments in the molecular size range between 75 and 1950 base pairs. This physical map has been aligned with the known positions of the rRNA and 4 S RNA genes on the two mtDNA strands by RNA-DNA hybridization experiments utilizing purified ³²P-labeled 12 and 16 S rRNA.     

Cloning and sequence comparison ofAvaI andBsoBI restriction-modification systems

AvaI andBsoBI restriction endonucleases are isoschizomers which recognize the symmetric sequence 5′CYCGRG3′ and cleave between the first C and second Y to generate a four-base 5′ extension. TheAvaI restriction endonuclease gene (avaIR) and methylase gene (avaIM) were cloned intoEscherichia coli by the methylase selection method. TheBsoBI restriction endonuclease gene (bsoBIR) and part of theBsoBI methylase gene (bsoBIM) were cloned by the “endo-blue” method (SOS induction assay), and the remainder ofbsoBIM was cloned by inverse PCR. The nucleotide sequences of the two restriction-modification (RM) systems were determined. Comparisons of the predicted amino acid sequences indicated thatAvaI andBsoBI endonucleases share 55% identity, whereas the two methylases share 41% identity. Although the two systems show similarity in protein sequence, their gene organization differs. TheavaIM gene precedesavaIR in theAvaI RM system, while thebsoBIR gene is located upstream ofbsoBIM in theBsoBI RM system. BothAvaI andBsoBI methylases contain motifs conserved among the N⁴ cytosine methylases.     

ThermotolerantCampylobacter with no or weak catalase activity isolated from dogs

ThermotolerantCampylobacter strains isolated from dog feces were characterized by phenotypical tests, DNA base composition, and DNA-DNA-hybridization. Out of 98 strains, 63 were catalase negative or weakly reacting (CNW); they were found in diarrheic as well as in healthy dogs. The CNW strains were all nalidixic-acid sensitive, hippurate negative, and grew at 42°C but not at 25°C. Seven strains were further investigated. They were sensitive to 2,3,5-triphenyl-tetrazolium chloride, reduced nitrate, and produced H₂S in the lead acetate test but not in triple-sugar-iron agar. The mol% G+C for five CNW isolates ranged from 35.2–35.8, which is higher than reported for any thermotolerantCampylobacter species. The strains also formed a well-delimitated DNA homology group with 80% or more intragroup relatedness and about 40% related toC. coli andC. jejuni.     

Purification and properties of a glycoprotein adenosine 5′-monophosphatase from the plasma membrane fraction of Arachis hypogaea cotyledons

An adenosine 5′-monophosphatase (AMPase) has been purified from the plasma membrane fraction of germinating cotyledons of peanut (Arachis hypogaea L.) by selective solubilization of the membrane-bound enzyme with 0.5% n-octyl β-glucoside at a protein-to-detergent ratio of 2:3 in the presence of Mg²⁺ and EDTA, followed by ion exchange chromatography on DEAE-cellulose. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis it showed a single protein band with a molecular weight of 55 000. The enzyme is a glycoprotein with 42.7% carbohydrate content. It had a broad pH optimum of 5.0–6.0. The K_m and V_max values were 1.08·10⁻³ M and 8.5 μmol/min per mg protein, respectively, with 5′-AMP as substrate. The enzyme is specific for 5′-AMP. Other nucleotides (GMP, UMP, CMP, ADP, GDP, ATP, GTP and UTP) as well as phosphorylated sugars were not hydrolyzed. p-Nitrophenyl phosphate was hydrolyzed at a relatively much lower rate (15%) and the substrate affinity (1/Km was only one-tenth that of AMP. The purified enzyme is competitively inhibited by ADP (K_i = 2.4 mM) and is also inhibited by NaF in a non-competitive manner with a K_i value of 35 mM. Divalent cations, Ca²⁺, Mg²⁺, Hg²⁺, Zn²⁺, Mn²⁺, Ni²⁺ and monovalent cations, K⁺, Li⁺ and Na⁺ had no effect on the enzyme activity. The purified enzyme was highly unstable, losing its total activity within 24 h at −20°C, or 0°C, while under these conditions the unpurified solubilized enzyme (octyl glucoside extract) was stable for several days, indicating that some stabilizing factors, most likely phospholipids, were lost during the enzyme purification.     

DNA-Triesters - The Synthesis and Absolute Configuration Assignments at P-Stereogenic Centres

Abstract

Decadeoxyribonucleotide GGGAATTCCC and nine diastereomeric pairs of its mono-O-ethyl ester analogues were synthesized via phosphoramidite approach using the combination of 5′-DMT-base protected (except T) nucleoside 3′-(2-cyanoethyl N,N-diisopropyl phosphoramidites) and 3′-(0-ethyl N,N-diisopropyl phosphoramidites). Under conditions of release from solid support and removal of base-protecting groups (25% NH₄OH, 25°C, 48 h) 2-cyanoethyl groups were removed while O-ethyl phosphate triester functions were practically intact. Isolation of products and separation of diastereomers were performed by means of RP-HPLC. Absolute configuration at P-stereogenic centres was established via degradation of decamers into corresponding dinucleoside O-ethyl phosphates and stereochemical correlation with dinucleoside phosphorothioates of known configuration at phosphorus. Decadeoxyribonucleotide mono-O-ethyl esters were used for mapping the contact points between DNA and Eco RI endonuclease - the restriction enzyme which recognizes canonical sequence. GAATTC and cleaves unmodified DNA strands giving G and p AATTC.     

Identification of Arsenobetaine as a Major Arsenic Compound in the Ivory Shell Buccinum striatissimum

A restriction endonuclease, designated as DmaI, was purified from cell-free extracts of Deleya marina IAM 14114 by streptomycin treatment, ammonium sulfate fractionation and two steps of chromatographies on heparin-Sepharose CL-6B and Mono Q (HR 5/5, FPLC). The purified enzyme was homogeneous on SDS-polyacrylamide gel disk electrophoresis and a ligation-recutting test. The relative molecular mass measurements of the purified enzyme gave 28,000 daltons by SDS-polyacrylamide gel disk electrophoresis and 56,000 daltons by gel filtration. These data indicated that the purified enzyme (56,000 daltons) has a dimeric structure composed of two 28,000-dalton subunits. The isoelectric point was 5.5. The purified enzyme worked best at 37°C in a reaction mixture (50 μl) containing 1.0 μg λDNA, 10 mm Tris–HCl, 7 mm 2-mercaptoethanol, 7 mm MgCl₂ and 100 mm NaCl (pH 7.5). The enzyme was stable up to 55°C and between pH 7.0 and 9.0. The purified enzyme recognizes the palindromic hexanucleotide DNA sequence 5′-CAGCTG-3′, cuts between G and C and produces a flush end (isoschizomer of PvuII).     

Isolation and Structures of Gibberellins from Immature Seeds of Calonyction aculeatum

A deoxyribonuclease was purified about 500 times from a Rhizopus product “Gluczyme.” The enzyme attacks native DNA and produces oligonucleotides terminated with pG, pA, or pC at the 5′ end and with G at the 3′ end. A small amount of mononucleotides was found in the digestion products when the hydrolysis was continued for a long period. The pH and temperature optima for the action were found to be 7.8~8.0 and 50°C, respectively, and the enzyme was activated three fold in the presence of 5 × 10^?3 m Mg²⁺ or Mn²⁺. This enzyme was named DNase Rh.     

Extended TAQing system for large‐scale plant genome reorganization

We previously developed a large‐scale genome restructuring technology called the TAQing system. It can induce genomic rearrangements by introducing transient and conditional formation of DNA double‐strand breaks (DSBs) via heat activation of a restriction enzyme TaqI, which can cleave DNA at 5′‐TCGA‐3′ sequences in the genome at higher temperatures (37–42°C). Such heat treatment sometimes confers lethal damage in certain plant species and TaqI cannot induce rearrangements in AT‐rich regions. To overcome such problems we developed an extended TAQing (Ex‐TAQing) system, which enables the use of a wider range of restriction enzymes active at standard plant‐growing temperatures. We established the Ex‐TAQing system using MseI that can efficiently cleave DNA at room temperature (at temperatures ranging from 22 to 25°C) and the 5′‐TTAA‐3′ sequence which is highly abundant in the Arabidopsis genome. A synthetic intron‐spanning MseI gene, which was placed downstream of a heat‐shock‐inducible promoter, was conditionally expressed upon milder heat treatment (33°C) to generate DSBs in Arabidopsis chromosomes. Genome resequencing revealed various types of genomic rearrangements, including copy number variations, translocation and direct end‐joining at MseI cleavage sites. The Ex‐TAQing system could induce large‐scale rearrangements in diploids more frequently (17.4%, n = 23) than the standard TAQing system. The application of this system to tetraploids generated several strains with chromosomal rearrangements associated with beneficial phenotypes, such as high salinity stress tolerance and hypersensitivity to abscisic acid. We have developed the Ex‐TAQing system, allowing more diverse patterns of genomic rearrangements, by employing various types of endonucleases and have opened a way to expand the capacity for artificial genome reorganization.     

The satellite bands of the DNA of Drosophila virilis

Purified DNA has been prepared from Drosophila virilis using a modification of the method derived for bacteria (Marmur, 1961). Some physical properties have been examined, a new hidden satellite discovered, and a difference found in the satellite banding pattern of different tissues. — In addition to the three satellite bands lighter than the main band previously reported (Gall et al., 1970), a new satellite heavier than the main band has been detected after thermal denaturation of the DNA (which substantially shifts the buoyant density of the main band but not that of the satellites indicating that all are fast-annealing). The satellite pattern of DNA extracted from heads alone differed from that of the entire animals: the amount of satellite I was decreased and II increased; III was unaffected; IV was increased relative to the amount in the main band. The total content of satellite material in the heads (assumed to be entirely diploid) was 42%, the highest amount reported for any organism. — Thermal transitions were determined for the DNA from adults and larvae. After preparative CsCl density gradient fractionation of adult DNA, two sets of bimodal thermal curves were obtained (in SSC) with agreement between the initial position in the preparative gradient, the thermal transitions, and the G+C content from density except for satellite III for which the T_m gave a more accurate G+C amount. DNA from satellites I and II together generated a T_m of 81.2° which was similar to a calculated T_m of 81.9° making the naive assumption that the thermal components of the two satellites would interact in a simple additive fashion. A T_m of 71.9° was ascribed to satellite III which indicates that it is not the equivalent of the poly (A-T) band found at the same density in D. melanogaster (Fansler et al., 1970). The calculated overall base composition from the density equivalents (using the value for satellite III from thermal data) gave an expected G+C content of 36.6%. The measured value was 36.0%. The possible significance of the differential satellite pattern has been discussed.     

The T4336C Variant Is a Marker of Mitochondrial Subhaplogroup H1, a Common Component of the Russian and German Gene Pools

Analysis of mitochondrial DNA (mtDNA) restriction polymorphism carried out in a sample of Russians from Magadan (n= 150) showed that the frequency of the +4332AvaII variant (a T–C transition at nucleotide position 4336) in this population was 4.7%. All +4332AvaII types of mtDNA belonged to the mitochondrial group H. They were characterized by a back of the AluI restriction endonuclease site at position 7025. According to hypervariable segment 1 sequencing data, they contained the 16304C variant, and thus belong to the subgroup H1. Thus, the +4332AvaII (T4336C) variant is a marker of the mitochondrial subgroup H1, chiefly occurring in German-speaking populations. Utilization of the H1-mtDNA markers for the investigation of the genetic history and the origin of Slavs is discussed.     

Physical mapping and characterization of Chlamydomonas mitochondrial DNA molecules: Their unique ends,sequence homogeneity,and conservation

Sixteen site-specific endonucleases were used to characterize the mitochondrial (mt)-DNA of Chlamydomonas reinhardtii. Recognition sites for SmaI, XhoI, and BglII were absent in the mtDNA. mtDNA fragments appeared in stoichiometric proportions in every nuclease digest indicating that C. reinhardtii mtDNA consists of a homogenous population of molecules devoid of either inter- or intramolecular heterogeneity. Six DNA fragment maps were derived for those endonucleases that produced discrete and readily measurable DNA fragments. These maps, which exhibited marked internal consistency, also suggested that the linear mtDNA molecules possessed unique ends. This was subsequently confirmed by in vitro 5′-end labeling of mtDNA molecules prior to endonuclease digestion. These results indicate that (1) the linear mtDNA isolated under our experimental conditions possessed not only unique ends but also a nonpermuted gene sequence and (2) such mtDNA molecules were generated by a site-specific cleavage of the closed circular mtDNA molecules shown to exist in vivo. mtDNA sequence conservation in Chlamydomonas is quite striking. No difference in endonuclease cleavage pattern has yet been detected among a number of C. reinhardtii strains or between mating types.