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...

A full length, double-stranded ovalbumin complementary DNA (cDNA) was synthesized in vitro and used to assay the action of 35 restriction endonucleases upon this DNA sequence. Some 22 enzymes, Bal I, BamHl, Blu I and its isoschizomerXho I, Ppu 1,Xba I, Tag I, Sal I, Bgl I, Bgl II, Kpn I, Sst II, Hue II, Hpa I, Hap II, Hha I, Hind11 and its isoschizomer HincII, Hind111 and its isoschizomer Hsu I...

The effects of nucleotide analogue substitution on the cleavage efficiencies of type II restriction endonucleases have been investigated. Six restriction endonucleases (EcoRV, SpeI, XbaI, XhoI, PstI and SphI) were investigated respectively regarding their cleavage when substrates were substituted by 2'-O-methyl nucleotide (2'-OMeN) and phosphorothioate (PS). Substitutions were made in the recog...

Adenovirus type 3 (Ad3) isolates, isolated from 45 patients with acute conjunctivitis during the year 1990 in Japan, were studied by DNA restriction enzyme analysis with restriction endonucleases recognizing 6-bp sequences (BamHI, SmaI, HindIII, BglII) and endonucleases recognizing 5- or 4-bp sequences (HinfI and TaqI). All 45 isolates of Ad3 were identified as the genome type Ad3f by six endon...

Creating endonucleases with novel sequence specificities provides more possibilities to manipulate DNA. We have created a chimeric endonuclease (CH-endonuclease) consisting of the DNA cleavage domain of BmrI restriction endonuclease and C.BclI, a controller protein of the BclI restriction-modification system. The purified chimeric endonuclease, BmrI198-C.BclI, cleaves DNA at specific sites in t...

Hamilton Othanel Smith was born in 1931 in New York City. In 1937, he and his family moved to Champaign-Urbana, Illinois, because his father had joined the faculty of the department of education at the University of Illinois. As a boy, Smith was interested in chemistry, electricity, and electronics, and he spent many hours with his brother in their basement laboratory, which was stocked with su...

Introduction Some 3.5 years ago the study of heredity took a chemical turn when Avery and his colleagues discovered that deoxyribonucleic acid-DNA-is the " transforming principle " that converts bacteria from one genotype to another. Watson and Crick's structural model of DNA then provided the basis for investigating its role as the hereditary material. There followed rapid advances in the bioc...

The known nucleoside triphosphate-dependent restriction enzymes are hetero-oligomeric proteins that behave as molecular machines in response to their target sequences. They translocate DNA in a process dependent on the hydrolysis of a nucleoside triphosphate. For the ATP-dependent type I and type III restriction and modification systems, the collision of translocating complexes triggers hydroly...

It is well known that type II restriction enzyme activities and specificities can be modulated by altering solution conditions. The addition of co-solvents such as dimethyl sulfoxide (DMSO), alcohols and polyols can promote star activity, which is the cleavage of non-cognate sequences. While neutral detergents are often used to control protein aggregation, little is known about the effect of ne...

A computer program has been developed which aids in the determination of restriction enzyme recognition sequences. This is achieved by cleaving DNAs of known sequence with a restriction endonuclease and comparing the fragmentation pattern with a computer-generated set of patterns. The feasibility of this approach has been tested using fragmentation patterns of 0X174 DNA produced by enzymes of b...