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.

Expression plasmid with a very tight two-step control: Int/att-mediated gene inversion with respect to the stationary promoter.

A very tightly controlled expression vector was constructed, which was originally designed as to be able to use any promoter, constitutive or regulated. Moreover, in vector pNH46T1, the repressible P(tac)/P(lac) promoters were used to transcribe genes cloned in the proximal multiple cloning site (MCS), which was flanked by convergent attB and attP sites. The gene of interest was cloned into MCS in the OFF orientation, i.e. facing the promoter(s). In such OFF orientation, the cloned gene could not be expressed, and only its anti-sense mRNA could be produced. Four strong rrnBT1 terminators, in a tandem arrangement and proximal to the N-terminal end of the cloned non-inverted gene, were protecting it from any inadvertent transcription originating in the vector. Moreover, the P(tac)/P(lac) promoters/operators are controlled by the LacI(q)ts and LacI(+) repressor(s) that further reduce the basal gene expression in the uninduced state. When induced, the total vector population is converted to the ON orientation by expression of the Int function that inverts the attB and attP-flanked MCS including the cloned gene. This places the gene under direct control of the P(tac)/P(lac) promoters, and thus results in very high expression. An additional feature is the anti-termination system that consists of the promoter-proximal nutL site and the inducible gene N, whose role in the ON state is to overcome the rrnBT1 terminators and any other adventitiously cloned terminators.

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).

Targeting and retrofitting pre-existing libraries of transposon insertions with FRT and oriV elements for in-vivo generation of large quantities of any genomic fragment.

A procedure is described that converts the pre-existing transposon insertion libraries to a collection of 'pop-out' strains, each allowing generation of 20- to 100-kb genomic fragments directly from the genome. The procedure consists of two steps: (1) single transposon insertions are targeted and retrofitted with excision and amplification elements (FRT and oriV), by homologous recombination with an FRT-oriV-carrying plasmid; and (2) two retrofitted neighbouring transposons are brought together by P1 transduction. From each strain, a 20- to 100-kb genomic fragment, bound by a pair of retrofitted transposons, could be excised and amplified upon supplying in trans the excision (Flp) and replication (TrfA) functions. To enhance the efficiency of crossing-in the FRT-oriV cassette, we transiently increased the copy number of our retrofitting plasmids using a temperature-sensitive TrfA-supplying helper plasmid. Using FRT-oriV and helper plasmids, we retrofitted four Tn10KmR and three Tn10CmR insertions. Subsequently, the FRT-oriV retrofitted insertions were crossed with each other in pairs (KmRxCmR), using P1 phage transductions. The resulting CmRFRT-[28-65-kb]-KmRFRT strains were transformed with a plasmid expressing FLP and trfA genes from the tightly controlled Ptet promoter. Induction of this tightly repressed promoter by autoclaved chlortetracycline (cTc) resulted in the efficient excision and amplification of genomic fragments located between FRT sites, but only in productive strains, i.e. having two parallel FRTs. We have shown that genomic fragments of 28-, 40-, 50- and 65-kb were efficiently excised and amplified. Furthermore, we could convert non-productive strains (having FRTs in non-parallel orientation), to productive combination of parallel FRTs, because one of the FRT elements was flanked by two convergent loxP sites, and thus could be inverted by the Cre function delivered either by the P1 phage or by a specially constructed temperature-sensitive Plac-cre plasmid. Although several microbial genomes were recently sequenced, the described method will help in supplying large quantities of any genomic fragment (prepared without the conventional cloning and its artifacts) for refined sequence comparison among strains and species, and for further analysis of uncharacterized ORFs, various mutations, and regulatory elements or functions. The excised and circularized DNA fragments (plasmids) could be propagated like any other large plasmids but only in hosts that could supply the appropriate Rep function. Our original 'pop-out' method [Pósfai et al. (1994) Nucleic Acids Res. 22, 2392-2398] was already employed for sequencing of the E. coli genome [Blattner et al. (1997) Science 277, 1453-1462]. Moreover, the Flp-mediated recombination between two FRT elements resulted in bacterial strains with large deletions (for parallel FRT orientations) or with large inversions (for inverted FRT orientations).

Tightly controlled two-stage expression vectors employing the Flp/FRT-mediated inversion of cloned genes.

We have developed a tightly controlled, two-stage expression system. It is based on a single plasmid that carries the TetR repressor/Ptet promoter/Otet operator for the first-stage control, and the Flp recombinase/ FRT sites for the second-stage control. The gene to be expressed (GENE) is cloned in an inverted orientation (with respect to the stationary promoter) into a multiple-cloning site (MCS) located between two convergent FRT1 and FRT2 sites. In the OFF stage, no inadvertent transcription can enter the 5' end of cloned GENE because of four rrnBT1 terminators, located just outside the FRT1-MCS-FRT2 cassette and because the FRT2 construct was deprived of any promoter function. When using the lacZ reporter, it was shown that in their OFF stage our two-stage expression plasmids exhibit a significantly lower basal expression than the repressed single-stage tetR/PtetOtet-lacZ vectors. To enter the ON stage, the tetR/PtetOtet module is induced by adding autoclaved chlortetracycline (cTc), leading to synthesis of the Flp recombinase, which in turn, inverts the FRT1-MCS-FRT2 module together with the cloned GENE. This results in the massive GENE expression from one (pInvMS) or two (pImpMS) stationary promoters.

Two forms of replication initiator protein: positive and negative controls.

The pir gene of plasmid R6K encodes the protein, pi, a replication and transcription factor. Two translational options for the pir gene give rise to two forms of pi protein: a 35.0-kDa form (pi35.0) and a shortened 30.5-kDa form (pi30.5). Although both proteins bind to a series of 22-bp direct repeats essential for plasmid R6K replication, only pi35.0 can bind to a site in the (A.T)-rich segment of its gamma ori and activate the gamma ori in vivo and in vitro. However, unlike pi35.0, pi30.5can inhibit in vivo and in vitro replication (activated by pi35.0). We propose that the two forms of pi might have distinct functions in replication. We show that although both forms of pi produce dimers, the nature of these dimers is not identical. The N-terminal 37 amino acid residues appear to control the formation of the more stable pi35.0 dimers, whereas another, apparently weaker interface holds together dimers of pi30.5. We speculate that the leucine zipper-like motif, absent in pi30.5, controls very specific functions of pi protein.

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.

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.