We developed a new simple high-throughput plasmid DNA extraction procedure, based on a modified alkaline lysis method, using only one 96-well microtiter glassfilter plate. In this method, cell harvesting, lysis by alkaline and plasmid purification are performed on only one microtiter glassfilter plate. After washing out RNAs or other contaminants, plasmid DNA is eluted by low-ion strength solut...

Among 15 strains of methanogens, one plasmid, pMP1, was identified in the new coccoid isolate PL-12/M. It could not be detected in the cleared lysate, but it was detected in the viscous pellet. The plasmid had a molecular weight of ca. 4.6 x 10(6). A restriction enzyme cleavage map of the cloned plasmid was derived.

The purification and characterization of a new restriction endonuclease, BclI from the extreme thermophile Bacillus caldolyticus is reported. This enzyme recognizes the sequence : formula: (see text) and cleaves at the positions indicated by the arrows.

Many DNA regulatory factors require communication between distantly separated DNA sites for their activity. The type IIF restriction enzyme SfiI is often used as a model system of site communication. Here, we used fast-scanning atomic force microscopy to monitor the DNA cleavage process with SfiI and the changes in the single SfiI-DNA complex in the presence of either Mg²⁺ or Ca²⁺ at a scan rat...

Cattle chromosomes were digested with two restriction endonucleases, MspI and HaeIII. The banding pattern induced by each enzyme allowed the identification and pairing of all individual chromosomes and consequently the elaboration of the karyotype. This method is rapid and technically easy, and proved to be of great utility in cattle cytogenetic studies.

The inverted repeats of Tn5, which have identical restriction endonuclease cleavage patterns, have different functional properties. They differ with respect to RNA polymerase binding, full promotion of neomycin resistance, the polypeptides coded for by the repeats and their function in the transposition process. There is a week RNA polymerase binding site present in one repeat and not in the ot...

Modern cloning methods are independent from restriction enzyme recognition sites. However, nearly all current cloning methods still require the introduction of overlaps by PCR, which can introduce undesired mutations. Here, we investigated whether overlaps needed for DNA assembly can be synthesized in situ and we tested if the de novo synthesis of sequences can be simultaneously combined with t...

We have mapped a region of homology within a 2.2 kilobase segment of plasmids of incompatibility group F I, WHICH IS CONTAINED IN AN EcoRI restriction enzyme fragment bearing genes for autonomous replication; The coordinates of the 2.2 kilobase segment are 46.4F-48.6F on the physical map of the F plasmid. The orgin of vegetative replication has been mapped at 42.5F, at which site F has no seque...

Virulent typical strains (Shikan, Morioka, Shizuoka) and Pasteur vaccine strains (no. 1, no. 2-H, no. 2-17JB) of Bacillus anthracis harboured two plasmid species with molecular masses of 110 MDal and 60 MDal. All of the 110 MDal plasmids isolated from the various strains showed indistinguishable patterns of digestion with restriction endonucleases. All the 60 MDal plasmids were also indistingui...

Although DNA-recognition sequences are among the most important characteristics of restriction enzymes and their corresponding methylases, determination of the recognition sequence of a Type-I restriction enzyme is a complicated procedure. To facilitate this process we have previously developed plasmid R-M tests and the computer program RM search. To specifically identify Type-I isoschizomers, ...