Characterization of pEC156, a ColE1-type plasmid from Escherichia coli E1585-68 that carries genes of the EcoVIII restriction-modification system.

The complete 4312-bp sequence of the pEC156 plasmid from Escherichia coli E1585-68, which carries genes encoding the EcoVIII restriction-modification (R-M) system, an isoschizomer of HindIII from Haemophilus influenzae, has been determined. Two clustered and convergently oriented open reading frames, large enough to encode genes of the EcoVIII R-M system, were found. The transcriptional start points were mapped by the primer extension method. The relative molecular masses of the EcoVIII endonuclease and EcoVIII methyltransferase deduced from the nucleotide sequence are 35,554 and 33,910, respectively. Nucleotide sequence analysis of pEC156 suggests that this plasmid is a ColE1-type replicon. It consists of an origin of replication and two untranslated genes encoding RNA I and RNA II, both involved in the regulation of plasmid DNA replication. The replication region also contains the gene encoding a 64-aa Rom-like protein. Inactivation of the putative rom gene by insertion of a kanamycin-resistance cassette resulted in 4.5-fold increase in pEC156-derived plasmid copy number in E. coli cells. All of these elements (RNA I, RNA II, and rom) reveal a high level of similarity to ColE1 homologs. The replication of all ColE1-type plasmids is dependent on the activity of E. coli DNA polymerase I. It was shown that a pEC156 derivative (pIB8) carrying an antibiotic resistance gene indeed failed to replicate in an E. coli polA12(ts) mutant at 43 degrees C, and its copy number was reduced in the E. coli pcnB80 mutant. These results prove that pEC156 is a ColE1-type replicon.

Isolation and characterization of type IIS restriction endonuclease from Neisseria cuniculi ATCC 14688.

Neisseria cuniculi produces the restriction enzyme NcuI which is an isoschizomer of MboII. We have demonstrated that NcuI recognizes a pentanucleotide sequence (5'-GAAGA-3'/3'-CTTCT-5'), and cleaves the DNA 8 and 7 nucleotides downstream from the recognition site leaving a single 3'-protruding nucleotide. We have purified this enzyme to electrophoretic homogeneity using a four-step chromatographic procedure. NcuI endonuclease is a monomeric protein with a M(r)=48,000+/-1000 under denaturing conditions. The properties of NcuI are consistent with those for MboII, the position of the cleavage site being identical and the pH profile and divalent cation requirements being similar. Moreover, NcuI cross-reacts strongly with anti-MboII serum suggesting the presence of similar antigenic determinants. We have determined the sequence of 20 N-terminal amino acids for NcuI and concluded that this sequence is identical to the N-terminal portion of the MboII enzyme.

Rapid preparation of denatured double-stranded DNA templates for sequencing.

This article proposes protocol for rapid preparation (ds) DNA templates for sequencing based on double-stranded DNA denaturation and its recovery by extraction with Wizard DNA purification resin (Promega). This method is an alternative to commonly used procedure employing denatured-DNA recovery by ethanol precipitation.

The FokI methyltransferase from Flavobacterium okeanokoites. Purification and characterization of the enzyme and its truncated derivatives.

The gene encoding the FokI methyltransferase from Flavobacterium okeanokoites was cloned into an Escherichia coli vector. The transcriptional start sites were mapped as well as putative -10 and -35 regions of the fokIM promoter. Enzyme overproduction was ensured by cloning the fokIM gene under the phi 10 promoter of phase T7. M.FokI was purified using a two-step chromatography procedure. M.FokI is a monomeric protein with a M(r) = 76,000 +/- 1,500 under denaturing conditions. It contains 21 Arg residues, and at least one of which is required for activity as shown by inhibition using 2,3-butanedione. Deletion mutants in the N- and C-terminus of M.FokI were isolated and characterized. The N-terminal derivative (M.FokIN) methylates the adenine residue within the sequence 5'-GGATG-3', whereas the C-terminal derivative (M.FokIC) modifies the adenine residue within the sequence 5'-CATCC-3'. Substrate-protection studies, utilizing chemical modification combined with data on the effect of divalent cations and pH on methylation activity, proved the existence of two catalytic centers within the FokI methyltransferase molecule. M.FokI and its truncated derivatives require S-adenosyl-L-methionine as the methyl-group donor, and they are strongly inhibited by divalent cations (Mg2+, Ca2+, Ba2+, Mn2+, and Zn2+) and S-adenosyl-L-homocysteine. The Km values for the methyl donor, S-adenosyl-L-methionine are 0.6 microM (M.FokI), 0.4 microM (M.FokIN), and 0.9 microM (M.FokIC) while the Km values for substrate lambda DNA are 1.2 nM (M.FokI), 1.4 nM (M.FokIN), and 1.3 nM (M.FokIC).

Identification, characterization and purification of the lantibiotic staphylococcin T, a natural gallidermin variant.

Staphylococcin T (StT), an antibacterial agent produced by a Staphylococcus cohnii T strain, was purified to homogeneity by ammonium sulphate precipitation, gel filtration, cation exchange and fast performance liquid chromatography (FPLC). The final yield was about 20%, and over a 1000-fold increase in the specific activity was obtained. Mass determination (2166 Da), amino acid sequencing (Ile-Ala-Xaa-Lys-Phe-Leu-Xaa-Xaa-Pro-Gly-Xaa-Ala-Lys-block) and DNA sequencing demonstrated that StT is identical to gallidermin, a lanthionine-containing antimicrobial peptide. StT has a broad spectrum of bactericidal activity against Gram-positive and some Gram-negative bacteria. StT appears to damage cell membrane, and as a result causes an efflux of ions and an immediate block in macromolecular synthesis. Moreover, electron microscopic observations reveal morphological changes, with a loss of ribosomes and condensation of the nucleoid DNA. These changes are followed by a dissolution of the cell contents resulting in a bacterial ghost composed of seemingly intact cell walls with remnants of the cytoplasmic membrane and internal structure. Since StT exhibits antimicrobial activity especially against the Staphylococcus species, this compound may be of use in the treatment of staphylococcal infections.

Genomic DNA sequencing by SPEL-6 primer walking using hexamer ligation.

DNA sequencing by SPEL-6 (Sequential Primer Elongation by Ligation of 6-mers) primer walking is based on the rapid assembly of true primers by ligation of several (three to 10) contiguous hexamers complementary to a DNA template saturated with Escherichia coli single-stranded DNA-binding protein. To prove the usefulness and to check the reliability of this method, a 3-kb DNA fragment carrying the genes encoding the EcoVIII restriction-modification (RM) system was sequenced with low redundancy (2.8). The use of both single-stranded (ss) and double-stranded (ds) DNA templates was compared. For this project, 27 primers were assembled by hexamer ligation to form 18-30-nt strings of three to five hexamers. Each primer was designed based on nucleotide sequence determined in a previous run, and was produced in a matter of minutes. The overall length of the easily readable sequencing ladders was about 300-450nt. We found that strong secondary structures in the ss DNA tend to interfere with its template function for the primer assembly by hexamer ligation, especially when they overlap the 3'-end of such a primer. This was easily overcome either by avoiding such hairpin regions or by using longer strings of hexamers, since we show that their ligation is highly cooperative, and ligation efficiency increases with the length of the string (). Some general rules for successful primer assembly and prospects for using the SPEL-6 method for large-scale, fully automated fluorescent sequencing of large genomes are discussed.

Co-operativity of hexamer ligation.

The SPEL-6 (sequential rimer elongation by ligation of 6-mers) procedure is based on the assembly of DNA primers by ligation of three or more hexamers taken from a library of 4096 hexamers. In this way, the synthesized primers enable DNA sequencing by primer walking. Ligation by both T4 DNA ligase and Rhodothermus marinus thermophilic DNA ligase is highly cooperative. Sequencing ladders obtained with 18-60-nucleotide (nt) primers (produced by ligation of three to ten hexamers using T4 DNA ligase) were all of high quality, with no spurious bands. R. marinus DNA ligase requires at least seven hexamers for successful primer synthesis. Long primers (up to 60 nt), which are easy to obtain, especially by automated ligation, offer a definite advantage in DNA priming in regions with pronounced secondary structure. Moreover, the SPEL-6 procedure for DNA sequencing reduces the sequencing effort manifold. An additional application of hexamer ligation is the detection of point mutants, as described here.

Automated four-color DNA sequencing using primers assembled by hexamer ligation.

A procedure based on the assembly of sequencing primers by hexamer ligation and then using them in automated DNA sequencing is described. This method is based on a four-color fluorescent terminator chemistry. Sequencing ladders were analyzed using an ABI 373 DNA sequencer (Applied Biosystems, Foster City, CA, USA). The best results were obtained for primers assembled by ligation of four to ten hexamers. The accuracy of the method was estimated to be 99.5% up to 400 nt of the read sequence, and somewhat lower at 400-600 nt.

Rapid removal of unincorporated label and proteins from DNA sequencing reactions.

This article presents a simple and rapid method for removal of unincorporated label and proteins from DNA sequencing reactions by using Wizard purification resin. This method can be successfully applied for preparation of end-labeled oligonucleotides free of unincorporated label, which is important in experiments (including DNA sequencing) when the level of background should be as low as possible. Also, this method is effective in removal of proteins from DNA sequencing reactions.

Interaction of the MboII restriction endonuclease with DNA.

The binding of the MboII restriction endonuclease (R.MboII; ENase) to DNA containing its recognition site was investigated using a mobility shift assay. R.MboII forms specific, stable and immunodetectable complexes with its canonical target sequence. The association constant (Ka) of R.MboII was calculated to be 2.8 x 10(9)/M, and is about 10(4)-fold higher than the Ka value for non-specific binding. Based on results obtained after sedimentation of the R.MboII-DNA complex in a glycerol gradient and measurement of the retardation of the complexes in polyacrylamide gels, we conclude that specific binding to the canonical sequence involves a monomer of R.MboII. DNase I footprinting has shown that the enzyme covers 16 nucleotides of DNA on the 5'-GAAGA-3' strand.

Assembly of 18-nucleotide primers by ligation of three hexamers: sequencing of large genomes by primer walking.

A novel method (SPEL-6) for sequencing large genomes permits 10-min synthesis of 18-mer primers and their immediate use in DNA sequencing by primer walking. Primers are ligated from three contiguous hexamers complementary to the single-stranded or denatured DNA (150:1 ratio), which is used both as the template for ligation and as the DNA to be sequenced. The complete library consists of 4096 hexamers, but sequencing remains very efficient with a fourfold smaller library composed of 1024 degenerate hexamers containing all four nucleotides at their position 3. The SSB protein (which binds to single-stranded DNA) greatly enhances the quality of sequencing ladders. The SPEL-6 method eliminates the need for subcloning, permits direct sequencing of large DNA fragments (of 50 kb or larger), is ideally suited for automation, and should accelerate the sequencing of large genomes by more than one order of magnitude.

Atypical DNA-binding properties of class-IIS restriction endonucleases: evidence for recognition of the cognate sequence by a FokI monomer.

The DNA-binding properties of the FokI restriction endonuclease were studied using the gel-mobility-shift assay. Specific recognition of the cognate sequence and cleavage of DNA are distinguishable functions and can be separated. FokI binds to its recognition site predominantly as a monomer. At high concentrations, FokI exhibits a cooperative recognition sequence-dependent aggregation. In 20 mM KCl/10 mM Tris.HCl buffer, the binding constant of FokI to its cognate site is equal 6.0-7.9 x 10(8)/mol and is lower than the values for most gene-regulatory proteins. FokI binding is 600-1500 times weaker to non-cognate double-stranded DNA than to the GGATG site, and 30,000 times weaker to single-stranded DNA or tRNA. The method of Bading [Nucleic Acids Res. 16 (1988) 5241-5248], used for determining the stoichiometry of protein bound to DNA by gel-mobility-shift assay, is extended.

Purification and properties of the MboII, a class-IIS restriction endonuclease.

After five purification steps a homogeneous preparation of endonuclease MboII was obtained, and several properties of the enzyme were determined. MboII is a monomer, with Mr under native and denaturing conditions being 47-49 x 10(3) Da. Endonuclease MboII is a basic protein (pI 8.3) which remains active when Mg2+ is replaced by Mn2+, Co2+, Ca2+, or Fe2+. MboII exhibits a star activity in the presence of some of the following reagents or ions: DMSO, glycerol, ethanol (and Co2+ or Mn2+ at pH 6). MboII does not bend DNA and is heat sensitive, losing activity after 15 min at 50 degrees C.

Purification and characterization of the FokI restriction endonuclease.

The restriction endonuclease FokI from Flavobacterium okeanokoites was purified to homogeneity. Based on gel filtration, sedimentation and sodium dodecyl sulfate-polyacrylamide-gel electrophoresis, the following properties of the enzyme were determined: FokI exists in one active monomeric form, and has an Mr of 64-65.4 x 10(3).FokI is a strongly basic protein with an isoelectric point of 9.4. The enzyme exhibits restriction activity in the pH range 5.0 to 10.5 (maximum level at pH 7.0-8.5) and its divalent cation requirement is satisfied not only by Mg2+, but also by Co2+, Mn2+, Ni2+, Cd2+, Zn2+ and Fe2+.