Restriction EcoRI

Blasiak J, Kowalik J. 1998. Interaction between organophosphoms compounds and DNA assayed by the restriction endonuclease EcoRI. Acta Univ Lodz Folia Biochim Biophys 13 31-67. 

Restriction enzymes are named after the bacterium from which they are isolated. For example, EcoRI is from Escherichia coli, and BamEII is from Bacillus amyloliquefaciens (Table 40-2). The first three letters in the restriction enzyme name consist of the first letter of the genus (E) and the first two letters of the species (co). These may be followed by a strain designation (R) and a roman numeral (I) to indicate the order of discov-ery (eg, EcoRI, EcoRIE). Each enzyme recognizes and cleaves a specific double-stranded DNA sequence that is 4—7 bp long. These DNA cuts result in blunt ends (eg,... 

Cutting with restriction endonucleases is very useful for moving specific pieces of DNA around from place to place. It s also a useful way to name pieces of DNA. For example, a piece of DNA that is cut from a bigger piece of DNA is often named by size and given a surname that corresponds to the two restriction enzymes that did the cutting—the 0.3-kb EcoRI-BamHI fragment. Restriction enzymes themselves are named for the bacterial strains from which they were initially isolated. 

The localization of bNOS to the human genome was accomplished by Kishimoto and coworkers70. These investigators used a rat cerebellar cDNA to obtain a human cDNA from Clontech. This cDNA was hybridized to Southern blots containing DNA from a battery of human-rodent somatic cell DNA. Since the blots had been shown to be selective, the authors showed that the cDNA hybridized to chromosome 12. By using restriction nucleases EcoRI and Hind in the assignment was made to 12 ql4-qter. One or two copies were indicated in vivo but reducing the hybrid conditions showed more bands. It is necessary to conduct further studies to see whether the other cDNA are derived from this or another clone. 

This Is the DNA sequence recognized by the restriction endonuclease EcoRI. Rgure 1-6-1. EcoRI Recognition Sequence... 

Partial random DNA template (108 bp) (synthesized by Biosource International, Foster City, CA). 5 -ACC-CAG-TCC-ACA-AGC-TTG-TAG-TAC-TNN-NNN-NNN-NNN-NNN-NNN-NNN-NNN-NNN-NNN-NNN-NNN-NNN-NNC-CCT-AGA-TGG-CAG-TTG-AAT-TCT-CCC-TAT-AGT-GAG-TCG-TAT-TAC-3 (N=A, C, G, or T are incorporated with equal probabilities). Restriction sites (Hindlll and EcoRI) are underlined. 

Kits and enzymes Superscript reverse transcriptase (Invitrogen), Maxiscript and Megascript in vitro transcription kits (Ambion, Austin, TX). Taq-DNA polymerase, T4-polynucleotide kinase, EcoRI and Hindlll restriction enzymes (Invitrogen). 

Cut the constant regions using the restriction enzymes EcoRI and Hindlll. 

Figure 1.2 The restriction enzyme ECORI, illustrated here, is one of hundreds isolated from bacteria and used by biotechnologists to cut DNA molecules so that they can be joined with other DNA molecules. This allows scientists to insert a desired genetic sequence into a strand of DNA.

Restriction endonucleases (EcoRV, EcoRI, Sncl, Kpnl, BamHl, and Xbal). Store at -20°C. 

The EcoRI endonuclease cleaves this hexanucleotide sequence on each strand between the G and the first A residue (reading 5 -to-30 this point of cleavage is marked by arrows in the sequences in Table 3.1. Notice that the points of cleavage are offset, or staggered, for EcoRI. Not all restriction endonucleases make staggered cuts in DNA some cleave the DNA without any offset, to produce blunt-ended cuts. 

Recognition sequence of restriction endonuclease EcoRI shows two-fold rotational symmetry. 

To handle the mass of existing data, powerful computer programs have been developed and various graphical procedures have also been developed to help the human mind comprehend the results.654 655 One important problem is to define and locate what are called consensus sequences. The problem is best illustrated by examples.654 The cleavage site for the EcoRI restriction endonuclease is GAATTC. There is no ambiguity. In a DNA of random sequence this would be expected to occur by chance in about (1 / 4)6 nucleotides (4 kb). On the other hand, the Hmll restriction endonuclease cleaves within the consensus sequence GTYRAC where Y = C or T and R = A or G. 

In contrast to DNAse I, the restriction endonucleases, which are discussed in Chapter 5, Section H,2 and in Chapter 26, have precise substrate sequence specificities. Three of the best known restriction endonucleases are called EcoRI,832 - 834 an enzyme which binds... 

Restriction endonuclease EcoRI is able to cut a chain in dsDNA which has a chiral phophorothioate group at the specific cleavage site.838 Tire reaction occurs with inversion of configuration at phosphorus, suggesting direct in-line attack by a hydroxyl ion generated from H20. 

Although they often share little sequence similarity and have quite different specificiities, many restriction enzymes have similar three-dimensional structures as well as mechanisms of action. This is true for the EcoRI, BamHl (Fig. 26-5),83/90 EcoRV,91/91a and C/r 101 enzymes,84 and presumably many others. The specifically shaped and tightly packed active sites in the enzyme-substrate complexes ensure specificity. For example, the EcoRV endonuclease cleaves DNA at its recognition site at least a million times faster than at any other DNA sequence.91 As mentioned in Chapter 12, restriction endonucleases require a metal ion, preferably Mg2+, and probably act via a hydroxyl ion generated from Mg2+-OH2 at the active site. Three conserved active site residues, Asp 91, Glu 111, and Lys 113, in the EcoRI endonuclease interact with the DNA near the cleavage site. Lys 113 is replaced by Glu 113 in the BamHl enzyme.83 90... 

The cloning and manipulation of genes usually depends upon the precise cutting of DNA into discrete fragments by restriction endonucleases. Many restriction enzymes generate cohesive ends (sticky ends). Thus, EcoRI produces DNA fragments with the single-stranded "tails" shown here at the 5 -ends of the cut duplexes ... 

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