Properties of the plasmid pFT15/10-1 isolated from the vaccine strain of Francisella tularensis

Plasmid, designated pFT15/10-1, was isolated from Francisella tularensis vaccine strain 15/10. The plasmid is presented by the homogeneous 5.02 +/- 0.054 Md monomeric circular DNA molecules in electron microscopic preparations. Plasmid size is 7-7.3 kb as defined by electrophoresis in agarose gel. The restriction analysis has revealed that plasmid pFT15/10-1 possesses a single specific cleavage site for restriction endonuclease EcoRI, two sites for restriction endonucleases BamHI, BgIII, HincII, HindIII, PstI, three sites for BglI and SalI, some for AluI, TagI, MvaI, CfrI. Plasmid is not digested by restriction endonucleases SmaI, XmaI, KpnI, MluI. Restriction map of the plasmid was constructed for most frequently used restriction endonucleases.     

Nicking endonucleases

Nicking endonucleases are a new type of enzymes. Like restriction endonucleases, they recognize short specific DNA sequence and cleave DNA at a fixed position relatively to the recognition sequence. However, unlike restriction endonucleases, nicking endonucleases cleave only one predetermined DNA strand. Until recently, nicking endonucleases were suggested to be naturally mutated restriction endonucleases which had lost their ability to dimerize and as a result the ability to cleave the second strand. We have shown that nicking endonucleases are one of the subunits of heterodimeric restriction endonucleases. Mechanisms used by various restriction endonucleases for double-stranded cleavage, designing of artificial nicking endonucleases on the basis of restriction endonucleases, and application of nicking endonucleases in molecular biology are reviewed.     

Restriction mapping of plasmid pSa1, isolated from Streptomyces albus G strain

A novel plasmid designated pSa1 has been isolated from Streptomyces albus G strain producing SalGI restriction endonuclease. Molecular weight of the plasmid is 3.4 +/- 0.2 mD. The action of 12 restriction endonucleases on the plasmid DNA was studied. Restriction map of pSa1 DNA was established for SmaI, HindII, XbaI and KpnI endonucleases.     

Mesophilic cyanobacteria producing thermophilic restriction endonucleases

When searching for the site-specific endonucleases in several strains of Phormidium we made the following observations. Among the 16 strains that originated from 15 species of Phormidium, 12 produced one or more restriction enzymes, of which two produced the highly thermophilic restriction endonucleases PtaI and PpaAII with their optimum activity at 65-80 degrees C, which is far above the lethal temperature for the host microorganism (40 degrees C). These two temperature-resistant enzymes are isoschizomers of known BspMII and TaqI endonucleases, respectively. The presence of the thermophilic TaqI isoschizomer does not seem to play any role in the mesophilic host microorganism, which does not even contain an active cognate methyltransferase. Among the remaining 10 strains, six produced isoschizomers of endonucleases which we first described in cyanobacteria, namely: PfaAII (NdeI), PinBII and PtaI (BspMII), PlaAII (RsalI), PpaAII, PpeI (ApaI). Two enzymes, PauAII (AhaIII) and PfaAII (NdeI), belong to a group of a very rarely occurring isoschizomers. Out of 21 cyanobacterial endonucleases investigated by us, four were active in a wide range of temperatures (from 15 to 60 degrees C) which also extended the optimal growth temperature of the hosts. We assume that our observation on the presence of temperature-resistant restriction enzymes in mesophilic hosts supports the idea of horizontal gene transfer. Restriction modification systems may be an excellent tool for investigation of that phenomenon.     

Restriction endonucleases and their applications

Restriction endonucleases are deoxyribonucleases which cleave double-stranded DNA into fragments. With only one exception, all restriction endonucleases recognize short, non-methylated DNA sequences. Restriction endonucleases can be divided into two groups based on the position of the cleavage site relative to the recognition sequence. Class I restriction endonucleases cleave double-stranded DNA at positions outside the recognition sequence and generate fragments of random size. The cleavage sites of Class II restriction endonucleases are located, in most cases, within the recognition sequence. Most of the Class II restriction endonucleases recognize 4, 5, or 6 base pair palindromes and generate fragments with either flush ends or staggered ends. DNA fragments with staggered ends contain 3, 4, or 5 nucleotide single-stranded tails called ‘sticky ends’. DNA fragments produced by Class II restriction endonuclease cleavage can be separated on gels according to their molecular weight. The fragments can be isolated from the gel and used for sequence analysis to elucidate genetic information stored in DNA. Further, an isolated fragment can be inserted into a small extrachromosomal DNA, e.g. plasmid, phage or viral DNA, and its replication and expression can be studied in clones of prokaryotic or eukaryotic cells. Restriction endonucleases and cloning technology are powerful modern tools for attacking genetic problems in medicine, agriculture and industrial microbiology.     

Netropsin, distamycin A, bis-netropsins as selective inhibitors of restrictases and DNAse I

The simultaneous analysis of DNAase I "footprinting" data and restriction endonucleases inhibition data was performed on the same DNA end-labelled fragment. The inhibition induced by netropsin, a number of bis-netropsins and distamycin A was investigated. These experiments led us to the following conclusions. The restriction endonucleases inhibition by the ligands is caused by the ligand molecules binding in the close vicinity to the restriction endonuclease recognition sequence. The zone of +/- 4 bp from the center of the restriction endonuclease recognition sequence can be defined as the zone of the influence of the bounded ligand on the restriction endonuclease. But in this case the intersection of recognition sequence and the binding site occupied by a single ligand molecule is not sufficient for the inhibition to occur. Restriction endonuclease cutting sites protected by netropsin can be predicted basing upon known nucleotide sequence specificity of netropsin. Netropsin and bis-netropsins show different nucleotide sequence specificity. This fact can be used for selective inhibition of restriction endonucleases.     

Restriction map of the temperate phage E105 from the polylysogenic culture of Erwinia carotovora 268

DNA structure of the temperate bacteriophage E105 from polylysogenic culture of Erwinia carotovora 268 has been studied. The viral 29.12-29.17 MD DNA has been shown to be linear and nonpermuted. The complete restriction map of the viral DNA has been constructed for MvaI and HpaI and partial for Eco31 restriction endonucleases based on the pair hydrolysis of the native DNA as well as its fragments. Altogether, 19 sites for restriction endonucleases have been localized on bacteriophage DNA.     

WebFARM: web server for finite automated restriction mapping

Restriction endonucleases are indispensable tools in molecular biology and biotechnology. Type II restriction endonucleases are part of restriction modification systems. DNA fragment extraction and restriction mapping are the basis for several biotechnological activities. WebFARM is a server application for identifying restriction endonuclease recognition sites and to give information regarding restriction mapping for given nucleotide sequences. WebFARM analyses given nucleotide sequence and identify restriction site for selected restriction endonucleases. It will also provide frequency of restriction for each restriction endonuclease. AVAILABILITY: http://webfarm.bioinfoindia.org/     

The Fidelity Index provides a systematic quantitation of star activity of DNA restriction endonucleases

Restriction endonucleases are the basic tools of molecular biology. Many restriction endonucleases show relaxed sequence recognition, called star activity, as an inherent property under various digestion conditions including the optimal ones. To quantify this property we propose the concept of the Fidelity Index (FI), which is defined as the ratio of the maximum enzyme amount showing no star activity to the minimum amount needed for complete digestion at the cognate recognition site for any particular restriction endonuclease. Fidelity indices for a large number of restriction endonucleases are reported here. The effects of reaction vessel, reaction volume, incubation mode, substrate differences, reaction time, reaction temperature and additional glycerol, DMSO, ethanol and Mn²⁺ on the FI are also investigated. The FI provides a practical guideline for the use of restriction endonucleases and defines a fundamental property by which restriction endonucleases can be characterized.     

A method for generating selective DNA probes for the analysis of C-negative regions in human chromosomes

Linker-adapter polymerase chain reaction (LA-PCR) is among the most efficient techniques for whole genome DNA amplification. The key stage in LA-PCR is the hydrolysis of a DNA sample with restriction endonucleases, and the choice of a restriction endonuclease (or several endonucleases) determines the composition of DNA probes generated in LA-PCR. Computer analysis of the localization of the restriction sites in human genome has allowed us to propose an efficient technique for generating DNA probes by LA-PCR using the restriction endonucleases HaeIII and RsaI. In silico hydrolysis of human genomic DNA with endonucleases HaeIII and RsaI demonstrate that 100- to 1,000-bp DNA fragments are more abundant in the gene-rich regions. Applying in situ hybridization to metaphase chromosomes, we demonstrated that the produced DNA probes predominantly hybridized to the C-negative chromosomal regions, whereas the FISH signal was almost absent in the C-positive regions. The described protocol for generating DNA probes may be successfully used in subsequent cytogenetic analysis of the C-negative chromosomal regions.     

Restriction endonucleases: general survey procedure and survey of gliding bacteria.

Among 120 strains of gliding bacteria which were screened for restriction endonucleases, 27 were found positive. Additionally, three strains carried enzymes able to release the supercoiled state of closed circular DNA. By using a new rapid method, restriction endonuclease activity was released by stirring about 0.5 g of cells (fresh weight) in a motor-driven glass homogenizer in buffer containing Triton X-101, ethylenediaminetetraacetic acid, and mercaptoethanol. A yield from 60 to 80% of the total activity present in the cells was obtained with minimal destruction of the cells. The enzyme activity in the crude extract was measured semi-quantitatively by digestion of DNA and subsequent separation of the fragments on an agarose slab gel. The method appears to be generally applicable for the extraction of restriction endonucleases from gram-negative bacteria on an analytical scale and in a modified form for large-scale preparation of restriction enzymes.     

Type II restriction modification systems of Prevotella bryantii TC1-1 and Prevotella ruminicola 23 strains and their effect on the efficiency of DNA introduction via electroporation

The restriction endonucleases PbrTI and Pru2I, isoschizomers of Sau3AI and HaeIII, were partially purified and characterized from anaerobic rumen bacteria Prevotella bryantii TC1-1 and Prevotella ruminicola 23, respectively. These are the first type II restriction endonucleases discovered in strains of the genus Prevotella, and they represent one of the barriers hindering gene transfer in these microorganisms. Heterologous DNA was protected against the action of the PbrTI or Pru2I by incubation in a cell-free extract of the respective strain which contained 20 mM EDTA. This led to the development of a protocol enabling successful electrotransformation of the P. bryantii TC1-1 strain with a pRH3 Bacteroides--Escherichia coli shuttle vector containing up to 7-kb long DNA inserts. Plasmid DNA isolated from the transformed strain facilitated the transfer with further increased efficiency and made possible the introduction of ligation reaction products directly to P. bryantii TC1-1 without passing them first through E. coli.     

Detection of tandem repeats in the genome of Citellus genus using restrictases

DNA from ground squirrels of the Citellus genus (Rodentia, Sciuridae) were analysed by centrifugation in the presence of CsCl followed by digestion by restriction endonucleases. Digestion of DNA of two species C. undulatus and C. fulvus by 10 of the 16 restriction endonucleases used led to formation of electrophoretically discrete fragments that are multiple to 330 b.p. in length which points out the tandem organization of repetitive sequences similar to the satellite DNA of many mammal species. However, upon centrifugation we failed to reveal a satellite band in these species; hence the tandem repeats refer to the class of cryptic satellites in the ground squirrels and do not differ in base composition from the remaining part of DNA. The main fraction of the genome was revealed in the form of discrete fragments by cleavage with HindIII and AluI. Both of these restriction endonucleases were used for comparative analysis of DNA of 12 Citellus species. It has been shown that DNA of all species can be digested by HindIII and yields a series of fragments that are multiple to 330-30 b.p. in length and the total content of which varies from species to species within 4-22%. The fraction of the tandem repeats does not correlate with the systematic position of species nor with the amount of heterochromatin in the chromosomes. AluI cuts the DNA of 11 species yielding 110 and 220 b.p. fragments compared to only 60 and 280 b.p. in the DNA of C. dauricus. Under HindIII digestion we can also reveal the tandem repeats in marmot, which is phylogenetically close to the Citellus of the Marmota genus, but they have another periodicity--180 b.p. We propose that the age of ground squirrels repeats is 2-3 million years and they are significantly younger than the marmot repeats.     

Restriction endonuclease mapping of Co1E2-P9 and Co1E3-CA38 plasmids

Using single and double restriction-endonuclease digestions, 16 and 17 cleavage sites have been mapped for the ColE2-P9 and ColE3-CA38 plasmids, respectively. One or more sites for AvaI, BglI, EcoRI, HincII, PvuI, PvuII, SmaI and XhoI endonucleases were found in both plasmids, two BglII sites were found only in ColE2-P9, and one KpnI site was unique to ColE3-CA38. ColE2-P9 was found to be slightly smaller than ColE3-CA38,4.4 Md compared to 4.6 Md. Eleven restriction sites are common to both plasmids in that they are identically placed relative to each other. These sites define a continuous DNA segment equal to over 60% of each plasmid. The remaining portions of the plasmids, which contain the non-homologous regions identified by Inselburg and Johns (1975) have no restriction sites in common, and differ in size by about 0.2 Md.     

A new Thermus sp. class-IIS enzyme sub-family: isolation of a 'twin' endonuclease TspDTI with a novel specificity 5'-ATGAA(N(11/9))-3', related to TspGWI,TaqII and Tth111II

The TspDTI restriction endonuclease, which shows a novel recognition specificity 5'-ATGAA(N(11/9))-3', was isolated from Thermus sp. DT. TspDTI appears to be a 'twin' of restriction endonuclease TspGWI from Thermus sp. GW, as we have previously reported. TspGWI was isolated from the same location as TspDTI, it recognizes a related sequence 5'-ACGGA(N(11/9))-3' and has conserved cleavage positions. Both enzymes resemble two other class-IIS endonucleases from Thermus sp.: TaqII and Tth111II. N-terminal amino acid sequences of TspGWI tryptic peptides exhibit 88.9-100% similarity to the TaqII sequence. All four enzymes were purified to homogeneity; their polypeptide sizes (114.5-122 kDa) make them the largest class-IIS restriction endonucleases known to date. The existence of a Thermus sp. sub-family of class-IIS restriction endonucleases of a common origin is herein proposed.     

Expression of ultraviolet-induced restriction alleviation in Escherichia coli K-12. Detection of a lambda phage fraction with a retarded mode of DNA injection

Ultraviolet-induced restriction alleviation is an SOS function which partially relieves the K-12-specific DNA restriction in Escherichia coli. Restriction alleviation is determined by observing elevated survival of unmodified phage lambda in cells irradiated with ultraviolet prior to infection. We demonstrate that restriction of lambda is also relieved when log-phase cells are irradiated as late as 50 min after adsorption of lambda. At this time more than 60% of the lambda DNA is already released as acid-soluble material from the cells. Experiments involving reextraction of lambda DNA from infected cells and a mild detergent treatment removing absorbed phages from the cellular surface showed that only a small specific fraction of all lambda infections is destined to escape restriction due to restriction alleviation. This fraction (10-20%) has a retarded mode of DNA injection (60 min or longer) after adsorption which allows the expression of the restriction alleviation function before the phage DNA is exposed to restriction endonucleases. This behaviour of a fraction of lambda phages explains why the SOS function restriction alleviation could initially be discovered. We show that the retarded mode of DNA injection is not required for another SOS function acting on lambda DNA, the increased repair of ultraviolet-irradiated DNA (Weigle reactivation).     

Detection of aquatic microorganisms from the Black Sea--producers of restriction endonucleases

300 clones of microorganisms isolated at different stations and from different depths in the Black Sea were screened for restriction endonucleases production. The production of restriction endonucleases was found in 17 clones screened. Three of them were identified to be Alteromonas haloplanktis B1. Restriction endonuclease AhaB1 is an isoshizomer of Sau961. An identified Alteromonas haloplanktis clone B8 produces AhaB8I restriction endonuclease the prototype to which is KpnI. Of the clones isolated three are Moraxella species B4 producing MapB4I restriction endonuclease analogous to BanI, three are Bacillus species producing BspB2I and one is Micrococcus lylae 113 producing Mly1131 analogue of NarI, six Moraxella species B6 produce MspB6I. The isolated producer strains may be used for isolation of above mentioned restriction endonucleases.     

Nicking Endonucleases as Unique Tools for Biotechnology and Gene Engineering

Russian Journal of Bioorganic Chemistry - Nicking endonucleases (NE) are a special group of the restriction endonucleases family. These unique enzymes catalyze the hydrolysis of only one DNA strand...     

The Role of Cysteine Residues in the Interaction of Nicking Endonuclease BspD6I with DNA

Molecular Biology - Nicking endonucleases (NEs) are a small, poorly studied family of restriction endonucleases. The enzymes recognize a target sequence in DNA, but catalyze the hydrolysis of only...     

Restriction endonucleases from Bifidobacteria

The sitespecific restriction endonucleases were found in four strains among the twelve strains of anaerobic bacteria of generum Bifidobacterium. Two of the restriction endonucleases studied, BadI from B. adolescentis LVA1 and BbfI from B. bifidum LVA3, are isoshizomers of XhoI and recognize the nucleotide sequence CTCGAG. The restriction endonucleases Bbf7411I from B. bifidum 7411 and Bla7920I from B. lactentis 7920 recognize and hydrolize the nucleotide sequence TCCGGA having the specifity analogous to the one of restriction endonuclease CauB3I. Like CauB3I, these restriction endonucleases are unable to hydrolyize DNA if the adenine residues in the recognition site are methylated.