We have determined that the type II restriction enzyme Ava II, isolated from Anabaena variabilis, recognizes and cuts the sequence (formula: see article). The eight Ava II sites of pBR322 have been mapped, as well as a unique site for Ava I.

The distribution of cytosine methylation in 6.2 Mb of the mouse genome was tested using cohybridization of genomic representations from a methylation-sensitive restriction enzyme and its methylation-insensitive isoschizomer. This assay, termed HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR), allows both intragenomic profiling and intergenomic comparisons of cytosine methylation. ...

We have studied the resistance of cytosine methylated DNA to digestion by the restriction endonuclease HinfI, using a simple PCR procedure to synthesize DNA of known sequence in which every cytosine is methylated at the 5 position. We find that HinfI cannot digest cytosine methylated DNA at the concentrations normally used in restriction digests. Complete digestion is possible using a vast exce...

Sau3AI is a type II restriction endonuclease that recognizes the palindromic sequence 5'-GATC-3' and cleaves 5' to G residue on each strand. The E64A mutant full length protein was cloned and expressed in Escherichia coli. The purified (His) (6)-tagged protein has monomer and dimer fraction and was crystallized by the hanging-drop vapor-diffusion technique. The dimer protein crystals can diffra...

Pairs of Au nanoparticles have recently been proposed as "plasmon rulers" based on the dependence of their light scattering on the interparticle distance. Preliminary work has suggested that plasmon rulers can be used to measure and monitor dynamic distance changes over the 1- to 100-nm length scale in biology. Here, we substantiate that plasmon rulers can be used to measure dynamical biophysic...

INTRODUCTION Incubation of a blunt-end ligation reaction in the presence of an excess amount of an appropriate restriction enzyme can dramatically increase the yield of recombinant plasmids. The role of the restriction enzyme is to cleave circular and linear concatemers at restriction sites that are re-formed when linear, blunt-ended plasmid molecules ligate to themselves. In almost all cases, ...

Prokaryotic restriction-modification (R-M) systems defend the host cell from the invasion of a foreign DNA. They comprise two enzymatic activities: specific DNA cleavage activity and DNA methylation activity preventing cleavage. Typically, these activities are provided by two separate enzymes: a DNA methyltransferase (MTase) and a restriction endonuclease (RE). In the absence of a corresponding...

Restriction landmark genome scanning (RLGS) is a 2-DE of genomic DNA, which visualizes thousands of loci. In a conventional RLGS method for methylation analysis, we have used a methylation sensitive restriction enzyme, NotI as a landmark. However, it was unable to discriminate methylation polymorphism from sequence polymorphism. Here, we report an improved RLGS method to detect methylated sites...

A real-time assay for CpG-specific cytosine-C5 methyltransferase activity has been developed. The assay applies a break light oligonucleotide in which the methylation of an unmethylated 5'-CG-3' site is enzymatically coupled to the development of a fluorescent signal. This sensitive assay can measure rates of DNA methylation down to 0.34 +/- 0.06 fmol/s. The assay is reproducible, with a coeffi...

We have developed a method for genomic representation using Type IIB restriction endonucleases. Representation by concatenation of restriction digests, or RECORD, is an approach to sample the fragments generated by cleavage with these enzymes. Here, we show that the RECORD libraries may be used for digital karyotyping and for pathogen identification by computational subtraction.