Restriction Endo-nucleases

Flgiire 1 Restriction Map of the cloned 3.1 kbp tDNA /rom X. laevis. The map shows the sites of cleavage by Hind III, Hind II Hpa I, Hac II, EcoR I, and Hpa II. Hpa II Fragments are identified A, B, etc. Fragments AandT hybridize tRNAWet, the genes for which are shown by thicker lines. 

Plasmids for use as DNA vectors have been selected for the following properties  

Bukhari, J. A. Shapiro, and S. L. Adhya, DNA Insertion Elements, Plasmids and Episomes , Cold Spring Harbor Laboratory, 1977. 

Considerable controversy surrounds the safety of inserting foreign DNA into organisms that are potential human pathogens. Most research so far has employed Escherichia coli, an enteric bacterium and occasional pathogen. Currently, the potential risks of these techniques are negated as much as possible by the use of vectors which are unlikely to be transmitted in the wild, by rigorous containment and by the use of Escherichia coli Ku strain which is also unlikely to be able to survive in the wild. 

In the future the use of vectors to clone foreign DNA is certain to be extended to other host/vector partners. The value of less pathogenic bacterial hosts such as Bacillus subtilis has recently been reviewed. Plant viruses as vectors for higher plants hold much promise for the insertion of characters such as disease resistance and nitrogen fixation into plants of agricultural importance.  

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Restriction endonucleases are at the core of recombinant DNA technology. The specificity of some of these enzymes is described in Chapter 13. Fortunately the restriction endo-nucleases cleave DNA at... 

Restriction Endo nuclease Location on Map (g-k ) Recognition Sequence Content — Recognition Sequence + ... 

Suitable vectors have been derived from the bacteriophage M13 which, except in its replicating form (RF), contains single-stranded circular DNA. The derivative M13 mpl, produced by insertion of part of the Escherichia coli lac operon, was mutated to give a single site for the restriction endo nuclease EcoRl within the gene for /8-D-galactosidase. This enzyme produces a blue colour when the hosts of this... 

The application of directed evolution approaches for the change or extension of the specificity of a restriction enzyme is hampered by the fact that an in vivo selection assay is not available and that examination of endonuclease activity in vitro usually requires purification of the enzymes to avoid background activity by other nucleases prevalent in all cells. This means that an altered or extended specificity can only be observed with sufficiently purified protein preparations, thereby unfortunately separating genotype and phenotype. As neither a reliable in vivo test nor the secretion of restriction endo-... 

It has been shown that cis - DDP, at low platinum/nucleotide ratios selectively inhibits the activity of several restriction endo - and exo nucleases when their cutting sites are adjacent to (d6)n (d )n sequences with n > 2 This demonstrates the sequence specificity of the binding of cis - DDP to DNA (17, 18, 19). GAG and GCG sequences also appear to be selectively involved in base - pair substitution mutagenesis and this suggests the possibility of platinum chelation by two guanines separated by a third base (20). 

The binding properties of 2 -0,-4 -C-ethylene bridged nucleic acids (ENA) (65) have been examined and compared with LNA. NMR demonstrated that ENA adopts the iV-conformation, as does LNA. In hybridisation studies, ENA showed an enhanced affinity towards RNA, but was slightly less stable than the corresponding LNA analogues. However, it was reported that ENA exhibits superior nuclease resistance to LNA. The bicyclic uridine nucleoside (15,5S,6S)-6-hydroxy-5-hydroxymethyl-l-(uracil-l-yl)-3,8-dioxabicyclo[3.2.1]octane (66) has been incorporated into ODNs where it was expected to restrict the sugar moiety into an OA -endo conformation. It was found that the presence of the analogue caused some destabilisation in duplexes with either DNA or RNA, thus a locked nucleic acid does not always lead to duplex stability. 

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