Restriction enzymes EcoRI

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.

Both linear and supercoiled double-stranded DNA can be used as substrate for site-selective scission. Typical methods for the scission of linear double-stranded DNA are given in Sections 7.6.2.1 and 7.6.2.2. The substrate is obtained by treating supercoiled PBR322 plasmid DNA by a restriction enzyme EcoRI. Here, the plasmid DNA is cut at one-site, providing a linear DNA (this DNA is known as form III). Section 7.6.2.3 describes the method for site-selective hydrolysis of supercoiled DNA. Figure 7.9(a)... 

Figure 3-30. Action of restriction enzymes. EcoRI cleaves this palindrome. Two fragments are produced that contain complementary single-stranded regions (sticky ends).

Rossi, S., Buera, M.P., Moreno, S., and Chirife, J. Stabilization of the restriction enzyme EcoRI dried with trehalose and other selected glass-forming solutes, Biotechnol. Prog., 13, 609,1997. 

A FIGURE 9-10 Cleavage of DNA by the restriction enzyme EcoRI. This restriction enzyme from coli makes staggered cuts at the specific 6-bp inverted repeat (palindromic) sequence shown, yielding fragments with single-stranded, complementary "sticky" ends. Many other restriction enzymes also produce fragments with sticky ends. 

Metal ions also participate in the functioning of other nucleases, although the structural details of their participation are not nearly as established as those for staphylococcal nuclease. DNAse I also requires Ca for its catalytic activity.SI endonuclease, mung bean nuclease, and Physarum polycephalem nuclease require zinc ion either as cofactors or intrinsically for nuclease activity, and the restriction enzyme EcoRI may also require intrinsically bound zinc ion. In terms of how the zinc ion might function in these enzymes, one can look both to staphylococcal nuclease and to bacterial alkaline phosphatase for some... 

Restriction enzymes EcoRI, Xbal, Spel, and Pstl (Promega, Madison, Wl). 

A reactant commonly produces an intermediate, a species that does not appear in the overall chemical equation for the reaction but which has been invoked in the mechanism. Biochemical processes are often elaborate versions of this simple model. For instance, the restriction enzyme EcoRI catalyzes the cleavage of DNA at a specific sequence of nucleotides (at GAATTC, making the cut between G and A on both strands). The reaction sequence it brings about is... 

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. 

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). 

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 ... 

Restriction enzymes—Htndlll, Bbsl, and EcoRI T4 DNA ligase DH5a cells... 

Extract the recombinant plasmids (pBSARs) (17) and double digest 5 pE (about 1 pg) of each of them with 1-5 U of EcoRI and Hindlll restriction enzymes (see Note 5). 

Nomenclature Restriction enzymes are isolated from bacteria, where they play a role in protecting the host cell against virus infection. Over 100 restriction enzymes have now been isolated and have been named according to the bacterial species from which they were isolated. The first three letters of the enzyme name are the first letter of the genus name and the first two letters of the species name. Since each bacterium may contain several different restriction enzymes, a roman numeral is also used to identify each enzyme. EcoRI, for example, was the first enzyme isolated from Escherichia cgli. 

As mentioned previously, bacteria defend themselves from their own restriction enzymes by methylating their DNA. For example, the EcoRI methylase adds a single methyl group to the amino group of... 

These are enzymes which occur in bacteria and cut DNA at specific sequences (e.g. the enzyme EcoRI, derived from E. coli (thus the derivation of the name), cuts the DNA chain between guanine and adenine in the sequence guanine-adenine-adenine-thymine-thymine-cytosine). Each time a particular DNA is cleaved by a restriction enzyme, precisely the same set of fragments is generated. The enzyme DNA ligase effectively works in a reverse manner to that of restriction enzymes in that it splices pieces of DNA back together again. 

Resuspend the DNA pellet in each of these three tubes with 8 pi of TE buffer. Label the tubes with your name and either H (iiimWW), E (EcoRI), or B (BamD ), depending on which restriction enzyme was used to neat each of the plasmid samples. 

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