Reduced plasmid stability

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. 

While the in vitro studies on assembly have provided relatively little information, in vivo data can give us some suggestions on possible pathway of assembly. For a long time it has been known that in rho yeast cells, where cytochrome b is not produced, cytochrome c, is still accumulated in the inner membrane. This agrees with the plasmid studies of overproduction. On the other hand the cytoplasmically synthesized subunits of cytochrome c oxidase accumulate in much lower quantities in the absence of subunits I, II and III, which are mitochondrial products. It is unlikely that this diminished accumulation is due to substantially reduced gene expression. This may indicate that certain subunits are stabilized by their counterparts. 

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