Plasmids endonuclease cleavage sites

The MCS is a stretch of unique restriction endonuclease cleavage sites between the promoter and the poly (A) signal in expression plasmid vectors, and is used as a common area to insert a cDNA or gene of interest. By using the MCS, the genes of interest can be inserted and assayed with ease. Sticky-end and blunt-end vector strands are available at different restriction sites. Although the MCS is very convenient, it should be noted that these sites usually remain in the mRNA transcribed from the expression unit of the vector and may reduce its stability or the efficiency of translation. Furthermore, one must be careful not to produce an ATG codon at the junctional sequence between the MCS and the insert, because it may work as a false start codon in the resulting mRNA and interfere with the production of the correct product. 

Improvement (i) came with the development of phage (e.g. M13 and its constructs such as M13mpl9), plasmid (e.g. the pUC series) and phage-mid (e.g. pBluescript II KS+/-) DNAs with high copy numt is, resistance to specific antibiotics, multiple restriction endonuclease cleavage sites and known coding sequences into which the DNA to be sequenced could be inserted with precision. 

The plasmid should have only one cleavage site for a specific restriction endonuclease. This provides only two ends to which the foreign DNA can be attached. Ideally, the single restriction site should be within a gene, so that insertion of the foreign DNA will inactivate the gene (called insertional marker inactivation). 

The colicin El plasmid is a 4.43 MDa circular double stranded DNA molecule consisting of 6646 base pairs.1 Only one site is susceptible to cleavage by the restriction endonuclease ECoRl (Chapter 26) This feature has led to its widespread use in cloning of genes. 

The type II restriction endonucleases, which are the ones most widely used in molecular biology, are relatively small 50- to 100-kDa monomeric or dimeric proteins. About 2400 different enzymes with 188 different specificities had been isolated by 1995.83/84 The sites of attack, in most instances, are nucleotide sequences with a twofold axis of local symmetry.85 For example, the following sites of cleavage have been identified for two restriction endonucleases encoded by the DNA of R-factor plasmids of E. coli and for a restriction enzyme from Hemophilus influenzae. In the diagrams i are sites of cleavage, are sites of methyla-tion, and are local twofold axes (centers of palindromes) N can be any nucleotide with a proper base pairing partner. 

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