[Proteolytic control of the antirestriction activity of Tn2l, Tn5053, Tn5045 Tn501 TN402 non-conjugative transposons].

Conjugative plasmids and conjugative transposons contain the genes, which products specifically inhibit the type I restriction--modification systems. Here is shown that non-conjugative transposons Tn2l, Tn5053, Tn5045, Tn501, Tn402 partially inhibit the restriction activity of the type I restriction-modification endonuclease EcoKI (R2M2S1) in Escherichia coli cells K12 (the phenomenon of restriction alleviation, RA). Antirestriction activity of the transposons is determined by the MerR and ArdD proteins. Antirestriction activity of transposons is absent in mutants E. coli K12 clpX and clpP and is decreased in mutants E. coli K12 recA, recBC and dnaQ (mutD). Induction of the RA in response to the MerR and ArdD activities is consistent with the production of unmodified target sequences following DNA repair and DNA synthesis associated with recombination repair or replication errors. RA effect is determined by the ClpXP-dependent degradation of the endonuclease activity R subunit of EcoKI (R2M2S1).

Methane dynamics in an alpine fen: a field-based study on methanogenic and methanotrophic microbial communities.

Wetlands are important sources of the greenhouse gas methane (CH4). We provide an in situ study of CH4 dynamics in the permanently submerged soil of a Swiss alpine fen. Physico-chemical pore water analyses were combined with structural and microbiological analyses of soil cores at high vertical resolution down to 50 cm depth. Methanotrophs and methanogens were active throughout the depth profile, and highest abundance of active methanotrophs and methanogens [6.1 × 10(5) and 1.1 × 10(7) pmoA and mcrA transcripts (g soil)(-1), respectively] was detected in the uppermost 2 cm of the soil. Active methanotrophic communities in the near-surface zone, dominated by viable mosses, varied from the communities in the deeper zones, but further changes with depth were not pronounced. Apart from a distinct active methanogenic community in the uppermost sample, a decrease of acetoclastic Methanosaetaceae with depth was observed in concomitance with decreasing root surface area. Overall, root surface area correlated with mcrA transcript abundance and CH4 pore water concentrations, which peaked (137.1 µM) at 10 to 15 cm depth. Our results suggest that stimulation of methanogenesis by root exudates of vascular plants had a stronger influence on CH4 dynamics than stimulation of CH4 oxidation by O2 input.

Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases.

The generation of genetically modified animals is important for both research and commercial purposes. The rat is an important model organism that until recently lacked efficient genetic engineering tools. Sequence-specific nucleases, such as ZFNs, TALE nucleases, and CRISPR/Cas9 have allowed the creation of rat knockout models. Genetic engineering by homology-directed repair (HDR) is utilized to create animals expressing transgenes in a controlled way and to introduce precise genetic modifications. We applied TALE nucleases and donor DNA microinjection into zygotes to generate HDR-modified rats with large new sequences introduced into three different loci with high efficiency (0.62%-5.13% of microinjected zygotes). Two of these loci (Rosa26 and Hprt1) are known to allow robust and reproducible transgene expression and were targeted for integration of a GFP expression cassette driven by the CAG promoter. GFP-expressing embryos and four Rosa26 GFP rat lines analyzed showed strong and widespread GFP expression in most cells of all analyzed tissues. The third targeted locus was Ighm, where we performed successful exon exchange of rat exon 2 for the human one. At all three loci we observed HDR only when using linear and not circular donor DNA. Mild hypothermic (30°C) culture of zygotes after microinjection increased HDR efficiency for some loci. Our study demonstrates that TALE nuclease and donor DNA microinjection into rat zygotes results in efficient and reproducible targeted donor integration by HDR. This allowed creation of genetically modified rats in a work-, cost-, and time-effective manner.

Production of meganucleases by cell-free protein synthesis for functional and structural studies.

Meganucleases are highly specific endonucleases that recognize and cleave long DNA sequences, making them powerful tools for gene targeting. We describe the production of active recombinant meganucleases suitable for functional and structural studies using a batch-based cell-free protein synthesis method. Isotopic labeling of the I-CreI meganuclease is demonstrated opening the way for structural and ligand binding studies in solution by nuclear magnetic resonance (NMR)(2) which was previously hampered by the problems associated with the toxicity of the enzyme for Escherichia coli limiting its growth. The method can be adapted for the synthesis of soluble engineered variants that are produced as inclusion bodies in bacterial cells, thus facilitating their purification as soluble proteins instead of using denaturing-refolding protocols.

Cell-free protein synthesis for structure determination by X-ray crystallography.

Structure determination has been difficult for those proteins that are toxic to the cells and cannot be prepared in a large amount in vivo. These proteins, even when biologically very interesting, tend to be left uncharacterized in the structural genomics projects. Their cell-free synthesis can bypass the toxicity problem. Among the various cell-free systems, the wheat-germ-based system is of special interest due to the following points: (1) Because the gene is placed under a plant translational signal, its toxic expression in a bacterial host is reduced. (2) It has only little codon preference and, especially, little discrimination between methionine and selenomethionine (SeMet), which allows easy preparation of selenomethionylated proteins for crystal structure determination by SAD and MAD methods. (3) Translation is uncoupled from transcription, so that the toxicity of the translation product on DNA and its transcription, if any, can be bypassed. We have shown that the wheat-germ-based cell-free protein synthesis is useful for X-ray crystallography of one of the 4-bp cutter restriction enzymes, which are expected to be very toxic to all forms of cells retaining the genome. Our report on its structure represents the first report of structure determination by X-ray crystallography using protein overexpressed with the wheat-germ-based cell-free protein expression system. This will be a method of choice for cytotoxic proteins when its cost is not a problem. Its use will become popular when the crystal structure determination technology has evolved to require only a tiny amount of protein.

Recombinant expression and purification of functional XorII, a restriction endonuclease from Xanthomonas oryzae pv. oryzae.

An endonuclease from Xanthomonas oryzae pathovar oryzae KACC 10331, XorII, was recombinantly produced in Escherichia coli using a T7 system. XorII was purified using a combination of ion exchange and immobilized metal affinity chromatography (IMAC). An optimized washing protocol was carried out on an IMAC in order to obtain a high purity product. The final amount of purified XorII was approximately 2.5 mg/L of LB medium. The purified recombinant XorII was functional and showed the same cleavage pattern as PvuI. The enzyme activity tested the highest at 25 degrees in 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, and 1 mM dithiothreitol at a pH of 7.9.

Highly efficient sex chromosome interchanges produced by I-CreI expression in Drosophila.

The homing endonuclease I-CreI recognizes a site in the gene encoding the 23S rRNA of Chlamydomonas reinhardtii. A very similar sequence is present in the 28S rRNA genes that are located on the X and Y chromosomes of Drosophila melanogaster. In this work we show that I-CreI expression in Drosophila is capable of causing induced DNA damage and eliciting cell cycle arrest. Expression also caused recombination between the X and Y chromosomes in the heterochromatic regions where the rDNA is located, presumably as a result of a high frequency of double-strand breaks in these regions. Approximately 20% of the offspring of males expressing I-CreI showed exceptional inheritance of X- and Y-linked markers, consistent with chromosome exchange at rDNA loci. Cytogenetic analysis confirmed the structures of many of these products. Exchange between the X and Y chromosomes can be induced in males and females to produce derivative-altered Y chromosomes, attached-XY, and attached-X chromosomes. This method has advantages over the traditional use of X rays for generating X-Y interchanges because it is very frequent and it generates predictable products.

[Endophytic bacilli--producers of type II restriction endonucleases]. / Endofitnye batsilly--produtsenty éndonukleaz restriktsii II tipa.

Two of thirteen bacillar strains isolated from the inner tissues of cotton plants were found to produce type II restriction endonucleases. The investigation of the site specificity of these enzymes showed that they are AsuI and Eco31I isoschizomers.

[Entomopathogenic bacteria Bacillus thuringiensis as producers of restriction endonucleases]. / Entomopatogennye bakterii Bacillus thuringiensis--produtsenty éndonukleaz restriktsii.

A total of 115 collection strains of Bacillus thuringiensis, belonging to various subspecies, have been studied for the presence of DNA restriction-modification systems. Restriction endonucleases of 13 strains have been isolated and characterized. No considerable correlations between the taxonomic positions of the bacteria and the specificities of the endonucleases isolated have been detected. It is concluded that the enzymes with identical specificities are present in both the crystalliferous and acrystalliferous strains of the same subspecies.

[Luminescent bacteria--producers of specific restriction endonucleases]. / Svetiashchiesia bakterii--produtsenty spetsificheskikh éndonukleaz restriktsii.

Luminescent bacteria isolated from the waters of the Black Sea and of the Indian and Pacific Oceans were tested for the presence of restriction endonucleases. For 12 of 19 restriction enzyme producers revealed, restriction sites were determined. The enzymes were identified as isoschizomers of AfIII, BanI, HaeIII, PstI, Sau3AI, and Sau96I. Possible participation of the restriction and modification enzymes in the interaction of bacterial symbionts with the animal macrosymbiont is discussed.

Restriction and modification systems of Neisseria gonorrhoeae.

An individual strain of Neisseria gonorrhoeae may produce up to 16 different DNA methytransferases (MTases). We have used a novel cloning system that is able to detect MTase clones in the absence of direct selection [Piekarowicz et al., Nucleic Acids Res. 19 (1991) 1831-1835] to identify 14 different MTase clones. Initial characterization of these clones indicates that at least seven of these MTases are linked to restriction endonuclease (ENase) systems. Six of these systems have been characterized by DNA sequence analysis, and the open reading frames encoding each of these systems have been identified. The recognition sequences for the cloned systems have the following specificities: S.NgoI, RGCGCY; S.NgoII, GGCC; S.NgoIV, GCCGCC; S.NgoV, GGNNCC; S.NgoVII, GCSGC; S.NgoVIIIA, GGTGA; and S.NgoVIIIC, TCACC. Of those systems that have been cloned, NgoI-NgoVII are typical type II R-M systems, with each encoding a DNA MTase that methylates cytosine in position 5. NgoVIII is a type IIS system, containing an ENase and two different MTases. One of these is a cytosine MTase (NgoVIIIC) and the other is an adenine MTase (NgoVIIIA). Although most of our clones encodes both the ENase and the MTase, none of the six R-M systems are genetically linked on the chromosome.

Organization and gene expression within restriction-modification systems of Herpetosiphon giganteus.

We have characterized a family of related restriction-modification (R-M) systems from the soil bacterium Herpetosiphon giganteus (Hgi). A comparison of their genetic organization reveals two types of regulatory proteins, called controlling ORF C. While one of these small reading frames derived from RM.HgiCI seems to be an enhancer of its own promoter, evidence is provided for a silencer function of the other ORF C derived from the closely related AvaII-type systems RM.HgiBI/CII/EI. The respective silencer function is detected during our various attempts to clone three isoschizomers with unusually high differences in their specific activity. Sequencing and site-directed mutagenesis revealed just two amino acids as being responsible for a massive increase in specific activity of these endonucleases.

BsuCI, a type-I restriction-modification system in Bacillus subtilis.

A type-I R-M system was identified in B. subtilis. The genes comprising the system have striking similarity to type-I R-M systems observed in Enterobacteriaceae.

A transposon-like sequence adjacent to the AccI restriction-modification operon.

We have cloned and sequenced the accIRM genes from Weeksella zoohelcum (the original identification of this strain as Acinetobacter calcoaceticus was incorrect). Our sequence differs in the coding regions from a previously published sequence by the addition of three nucleotides near the 3' end of the DNA methyltransferase-encoding gene (accIM). We have sequenced approx. 3 kb beyond this operon. Two genes were found, convergently transcribed with the R-M operon. The first of these genes encodes a protein which shows significant similarity to the recombinases of the phage integrase family. The W. zoohelcum recombinase may function as a transposon resolvase, as in Tn4430. The recombinase-encoding gene is followed by a putative transposase (Tnp), which is in turn followed by a terminator which is predicted to be Rho-dependent for the recombinase-Tnp operon and Rho-independent for the convergent R-M operon. Since the G + C content of the two operons is notably different, it is possible that the terminator is at the extremity of the mobile element and serves to protect it from incoming transcription.

Overproduction of the Hsd subunits leads to the loss of temperature-sensitive restriction and modification phenotype.

The genes hsdM and hsdS for M. EcoKI modification methyltransferase and the complete set of hsdR, hsdM and hsdS genes coding for R. EcoKI restriction endonuclease, both with and without a temperature-sensitive (ts) mutation in hsdS gene, were cloned in pBR322 plasmid and introduced into E. coli C (a strain without a natural restriction-modification (R-M) system). The strains producing only the methyltransferase, or together with the endonuclease, were thus obtained. The hsdSts-1 mutation, mapped previously in the distal variable region of the hsdS gene with C1 245-T transition has no effect on the R-M phenotype expressed from cloned genes in bacteria grown at 42 degrees C. In clones transformed with the whole hsd region an alleviation of R-M functions was observed immediately after the transformation, but after subculture the transformants expressed the wild-type R-M phenotype irrespective of whether the wild-type or the mutant hsdS allele was present in the hybrid plasmid. Simultaneous overproduction of HsdS and HsdM subunits impairs the ts effect of the hsdSts-1 mutation on restriction and modification.

[Plasmid localization and cloning of restriction modification genes from Citrobacter freundii 4111 strain]. / Plazmidnaia lokalizatsiia i klonirovanie genov sistemy restriktsii-modifikatsii iz shtamma Citrobacter freundii 4111.

Over 60 producing strains of restriction endonucleases type II have been found among 500 different strains, mostly Enterobacteriaceae. The strain Citrobacter freundii 4111 produces restriction endonuclease CfrBI, a new isoschisomer of StyI. The genes of the restriction-modification system CfrBI were located on the multicopy plasmid pZE8 containing the Co1E1-type replicon and cloned to E. coli K802. The deletion variant of 3.2-kb pZE8 which contains intact restriction-modification and a DNA fragment responsible for autonomous plasmid replication was selected among the recombinant plasmids. The strain with higher R. CfrBI production (at least 10,000,000 U/g cells, which is 500-fold higher than the wild strain) was constructed.

[Search for producers of restriction endonucleases with a given specificity]. / Poisk produtsentov éndonukleaz restriktsii zadannoi spetsifichnosti.

Six site-specific restriction endonucleases were isolated from Bacillus thuringiensis strains chosen of 58 strains ought purposefully for production of restriction enzymes. All six strains produce isoschisomers of Sau3A.

Characterization and expression of the Escherichia coli Mrr restriction system.

The mrr gene of Escherichia coli K-12 is involved in the acceptance of foreign DNA which is modified. The introduction of plasmids carrying the HincII, HpaI, and TaqI R and M genes is severely restricted in E. coli strains that are Mrr+. A 2-kb EcoRI fragment from the plasmid pBg3 (B. Sain and N. E. Murray, Mol. Gen. Genet. 180:35-46, 1980) was cloned. The resulting plasmid restores Mrr function to mrr strains of E. coli. The boundaries of the mrr gene were determined from an analysis of subclones, and plasmids with a functional mrr gene produce a polypeptide of 33.5 kDa. The nucleotide sequence of the entire fragment was determined; in addition to mrr, it includes two open reading frames, one of which encodes part of the hsdR. By using Southern blot analysis, E. coli RR1 and HB101 were found to lack the region containing mrr. The acceptance of various cloned methylases in E. coli containing the cloned mrr gene was tested. Plasmid constructs containing the AccI, CviRI, HincII, Hinfl (HhaII), HpaI, NlaIII, PstI, and TaqI N6-adenine methylases and SssI and HhaI C5-cytosine methylases were found to be restricted. Plasmid constructs containing 16 other adenine methylases and 12 cytosine methylases were not restricted. No simple consensus sequence causing restriction has been determined. The Mrr protein has been overproduced, an antibody has been prepared, and the expression of mrr under various conditions has been examined. The use of mrr strains of E. coli is suggested for the cloning of N6-adenine and C5-cytosine methyl-containing DNA.

[Detection of aquatic microorganisms from the Black Sea--producers of restriction endonucleases]. / Vyiavlenie vodnykh mikroorganizmov chernogo moria--produtsentov éndonukleaz restriktsii.

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.