A simple, general procedure for purifying restriction endonucleases.

A simple, general method for purifying restriction endonucleases is described. The method employs precipitation of nucleic acids from crude extracts with polyethyleneimine followed by affinity chromatography on columns of heparin covalently linked to agarose. Most of the sixteen enzymes tested could be purified to a degree sufficient for DNA sequencing work by this method sometimes supplemented by at most one step of ion exchange chromatography.     

Restriction endonucleases: natural and directed evolution

Type II restriction endonucleases (REs) are highly sequence-specific compared with other classes of nucleases. PD-(D/E)XK nucleases, initially represented by only type II REs, now comprise a large and extremely diverse superfamily of proteins and, although sharing a structurally conserved core, typically display little or no detectable sequence similarity except for the active site motifs. Sequence similarity can only be observed in methylases and few isoschizomers. As a consequence, REs are classified according to combinations of functional properties rather than on the basis of genetic relatedness. New alignment matrices and classification systems based on structural core connectivity and cleavage mechanisms have been developed to characterize new REs and related proteins. REs recognizing more than 300 distinct specificities have been identified in RE database (REBASE: ) but still the need for newer specificities is increasing due to the advancement in molecular biology and applications. The enzymes have undergone constant evolution through structural changes in protein scaffolds which include random mutations, homologous recombinations, insertions, and deletions of coding DNA sequences but rational mutagenesis or directed evolution delivers protein variants with new functions in accordance with defined biochemical or environmental pressures. Redesigning through random mutation, addition or deletion of amino acids, methylation-based selection, synthetic molecules, combining recognition and cleavage domains from different enzymes, or combination with domains of additional functions change the cleavage specificity or substrate preference and stability. There is a growing number of patents awarded for the creation of engineered REs with new and enhanced properties.     

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.     

Fimbriation in gliding bacteria.

Of twenty-two strains of gliding prokaryotes examined, all but three were found to possess polar fimbriae. Fimbriae were not observed on two gliders, while Chloroflexus aurantiacus bore abundant peritrichous fimbriae. In some gliding bacteria, fimbriae were associated with 'holes' surrounded by an electron-transparent collar bearing 12 spike-like projections.     

Restriction endonucleases: classification,properties, and applications

Restriction endonucleases have become a fundamental tool of molecular biology with many commercial vendors and extensive product lines. While a significant amount has been learned about restriction enzyme diversity, genomic organization, and mechanism, these continue to be active areas of research and assist in classification efforts. More recently, one focus has been their exquisite specificity for the proper recognition sequence and the lack of homology among enzymes recognizing the same DNA sequence. Some questions also remain regarding in vivo function. Site-directed mutagenesis and fusion proteins based on known endonucleases show promise for custom-designed cleavage. An understanding of the enzymes and their properties can improve their productive application by maintaining critical digest parameters and enhancing or avoiding alternative activities.     

Restriction endonucleases for pulsed field mapping of bacterial genomes.

Fundamental to many bacterial genome mapping strategies currently under development is the need to cleave the genome into a few large DNA fragments that can be resolved by pulsed field gel electrophoresis. Identification of endonucleases that infrequently cut a genome is of key importance in this process. We show that the tetranucleotide CTAG is extremely rare in most bacterial genomes with G+C contents above 45%. As a consequence, most of the sixteen bacterial genomes we have tested are cleaved less than once every 100,000 base pairs by one or more endonucleases that have CTAG in their recognition sequences: Xba I (TCTAGA), Spe I (ACTAGT), Avr II (CCTAGG) and Nhe I (GCTAGC). Similarly, CCG and CGG are the rarest trinucleotides in many genomes with G+C content of less than 45%. Thus, Sma I (CCCGGG), Rsr II (CGGWCCG), Nae I (GCCGGC) and Sac II (CCGCGG) are often suitable endonucleases for producing fragments that average over 100,000 base pairs from such genomes. Pulsed field gel electrophoresis of the fragments that result from cleavage with endonucleases that cleave only a few times per genome should assist in the physical mapping of many prokaryotic genomes.     

Screening for restriction endonucleases in methane-oxidizing bacteria.

51 methane-oxidizing bacteria strains such as Methylomonas methanica, M. rubra, Methylococcus capsulatus, M. thermophilus, M. luteus, M. ucrainicus, M. whittenburyi, Methylosinus trichosporium, M. sporium, Methylocystis parvus isolated from various ecological niches and geographical regions of the Ukraine and also the strains received from R. Whittenbury and Y. Heyer were screened for restriction endonucleases. Type II restriction endonucleases were detected in IMV B-3112 (= 12 b), IMV B-3027 (= 26), IMV B-3019 (= 9 c), IMV B-3017 (= 17 c), IMV B-3226 (= 26 v), IMV B-3033 (= Y), IMV B-3100 (= 100) and IMV B-3494 (= 1E494). The results obtained were indicative of relatively high frequency of restriction enzymes occurrence in methane-oxidizing bacteria. There were Kpn I (Asp 7181) restriction endonuclease isoschizomers in crude extracts of IMV B-3112, B-3017, B-3019, B-3027 isolated from fresh-water silt as well as in IMV B-3226 strain isolated from waste-water silt. Although these isolates had bee previously considered as untypical strains of M. ucrainicus, more detailed study of their properties allowed placing them with Methylovarius luteus (= Methylococcus luteus). IMV B-3494 strain was identified as Methylococcus capsulatus. Strain IMV B-3033 had earlier been allocated to Methylovarius whittenburyi (= Methylococcus whittenburyi). Specificity of restriction endonucleases of this strain was not tested. Therefore, for the first time restriction endonucleases were detected in methane-oxidizing bacteria. 8 strains (3 species) among 51 strains (13 species) were found to produce restriction endonucleases displaying three different types of specificity in the least. Producers of restriction endonucleases having Kpn I (Asp 7181) specificity were isolated from different water and silt samples of the Dnieper flood-land more than 20 years ago.     

Sulfonolipids of gliding bacteria. Structure of the N-acylaminosulfonates

Earlier (Godchaux, W., and Leadbetter, E. R. (1980) J. Bacteriol. 144, 592-602; (1983) J. Bacteriol. 153, 1238-1246) we demonstrated that an unusual class of sulfonolipids are major components of the cell envelope of gliding bacteria of the genus Cytophaga and of closely related genera. One of these lipids, to which we have assigned the trivial name capnine, was purified and was shown to be 2-amino-3-hydroxy-15-methylhexadecane-1-sulfonic acid (which might also be named as 1-deoxy-15-methylhexadecasphinganine-1-sulfonic acid). Though capnine accumulates as such in the cells of some Capnocytophaga spp., most organisms of the Cytophaga-like genera contain, instead, sulfonolipids that are less polar than capnine. These less polar lipids have been purified from a Capnocytophaga sp., a marine Cytophaga sp., Cytophaga johnsonae, and a Flexibacter sp. Acid methanolysis of the lipids yielded both aminosulfonates and a collection of fatty acid methyl esters. The infrared absorption spectra of the lipids indicated that the fatty acids were in amide (and not ester) linkage to the aminosulfonates. In every instance, analysis by mass spectrometry and other methods revealed that most, if not all, of the aminosulfonates obtained by methanolysis were structurally identical to capnine (though small amounts of variants of that compound may be present in some cases). The less polar sulfonolipids are, therefore, predominantly N-fatty acyl capnines, 1-deoxy-1-sulfonic acid analogs of ceramides. The fatty acid methyl esters obtained from the lipids were heterogeneous, but in all cases were rich in hydroxylated fatty acyl groups, which constituted 66 to 95% of the total.     

Capnocytophaga: New genus of Gram-negative gliding bacteria. II. Morphology and ultrastructure

Gram-negative, anaerobic gliding bacteria were isolated from normal supragingival plaque and from periodontal lesions. Isolates could be divided into two size classes: small 2.4–4.2 m×0.38–0.5 m and large 4.8–5.8 m×0.42–0.6 m cells. The outer membrane was either loose-fitting and wavy, or taut, and of variable thickness. An electron-dense fuzz was discernible on several of the isolates. The periplasmic region was of variable electron-density. The genus Capnocytophaga has been proposed for these organisms based on morphological and cultural characteristics.     

Capnocytophaga: New genus of Gram-negative gliding bacteria. III. Physiological characterization

Sixty-eight strains of capnophilic fusiform Gram-negative rods from the human oral cavity were subjected to extensive physiologic characterization, tested for susceptibility to various antibiotics, and the mol-percent guanine plus cytosine of each isolate determined. The characteristics of the isolates were compared with 10 fresh and 2 stock isolates of Fusobacterium nucleatum. The isolates clearly differed from the Fusobacterium species on the basis of molpercent guanine plus cytosine, end products, growth in a capnophilic environment and fermentation of carbohydrates.All of the gliding isolates required CO₂ and formed acetate and succinate, but not H₂S, indole or acetylmethylcarbinol. All fermented glucose, sucrose, maltose and mannose. The organisms may be differentiated on the basis of fermentation of additional carbohydrates, hydrolysis of polymers and reduction of nitrate. Three species are proposed: Capnocytophaga ochracea, Capnocytophaga sputigena and Capnocytophaga gingivalis. Ten isolates did not fit into the proposed species.     

Restriction endonucleases in Clostridium pasteurianum ATCC 6013 and C. thermohydrosulfuricum DSM 568

A small collection of clostridia was surveyed for type II restriction endonucleases. Enzymes were detected in two organisms. Clostridium pasteurianum ATCC 6013 contains an isoschizomer of ThaI (FnuDII) [5'-CGCG-3'] and preliminary evidence suggests that cleavage generates blunt-ended fragments. Clostridium thermohydrosulfuricum DSM 568 contains an isoschizomer of MboI (Sau3A) [5'-GATC-3'] that is inactive on dam methylated substrates. The DNA of this latter organism shows dam methylation.     

Restriction endonucleases: one of these things is not like the others

The crystal structure of the restriction endonuclease BglII in complex with its DNA target site has been determined. The DNA binding mode and chemistry of catalysis are observed to differ from BamHI which cleaves a similar target site. These observations indicate that more divergence has occurred within this family of proteins than originally thought.     

Biosynthesis of a sulfonolipid in gliding bacteria

Gliding bacteria of the genus Cytophaga synthesize sulfonolipids (1,2) that contain capnine (1-deoxy-15-methylhexadecasphinganine-1-sulfonic acid). Studies of the incorporation of radiolabeled compounds by C. johnsonae show that cysteate is utilized preferentially to both cystine and inorganic sulfate as a precursor of capnine sulfur and to both cystine and serine as a precursor of carbons 1 and 2 of capnine. The results are consistent with a pathway in which capnine is formed by condensation of cysteate with a fatty acyl CoA. Cystine, added as the sole sulfur source in the presence of glucose, provides the sulfur but not the carbon for capnine. Hence, these cells form cysteate not by direct oxidation of cystine (or cysteine), but by transfer of its sulfur to a different carbon compound.     

Type II restriction endonucleases cleave single-stranded DNAs in general.

Restriction endonucleases (13 out of 18 species used for the test) were certified to cleave single-stranded(ss)DNA. Such enzymes as AvaII, HaeII, DdeI, AluI, Sau3AI, AccII,TthHB8I and HapII were newly reported to cleave ssDNA. A model to account for the cleavage of ssDNA by restriction enzymes was proposed with supportive data. The essential part of the model was that restriction enzymes preferentially cleave transiently formed secondary structures (called canonical structures) in ssDNA composed of two recognition sequences with two fold rotational symmetry. This means that a restriction enzyme can cleave ssDNAs in general so far as the DNAs have the sequences of restriction sites for the enzyme, and that the rate of cleavage depends on the stabilities of canonical structures.