Luan Swanepoel

What are the features of recombinant plasmids that enable it to be expressed in different organisms?
Recombinant plasmids, such as those found in E. coli, can be genetically modified to have specific features that enable them to be expressed in different organisms like prokaryotes and eukaryotes. One of the reasons why scientists transfer recombinant plasmids to human cells is because post-translational modification of peptides or proteins, needed in humans, does not occur in bacteria (Salehzadeh et al. 2012). For example, Li et al. (2009) successfully inserted the human calcitonin (hCT) gene into a pCDNA3.0 vector and expressed it in human fibroblast cells. The problem facing integration of a recombinant plasmid into a mammalian cell is its origin of replication; Pearson et al. (1991) states chromosomal DNA in mammalian cells initiates transcription at specific origins of replication different in base-pair sequence of those found in plasmid DNA. However, plasmids can be genetically modified to contain origins of replication similar to humans and research has proved some plasmids can replicate in non-bacterial cells without a change in origin of replication. A commonly used origin of replication, as explained by Piechaczek et al. (1998), in plasmids is the SV40 promoter sequence. This 314bp sequence requires only 1 viral protein to initiate transcription and high-level expression; other components can be supplied by mammalian cells. Other viruses, like the Cytomegalovirus (CMV) promoter is not recognised by mammalian cell lines (Tegtmeyer and Ozer 1971). However, the large T-antigen produced by the SV40 promoter has seen to cause mutations in animal models. But insertion of a chromosomal S/MARs sequence into a SV40 based vector has caused replication without mutations. Recombinant plasmids have also been used in plants but different bacteria are used. The Agrobacterium tumefaciens bacteria, carrying the Ti plasmid, naturally infect plants and inserts TDNA into plant chromosomes, causing mutations and disease. However, this plasmid is too large to be used as a vector (and has lack of restriction sites) so E. coli's plasmid is used as an intermediate vector and the Ti segment (containing the gene insertion) is inserted into a pBR322-based plasmid. The result is plant cells which can be infected with foreign DNA and then grown into a plant (Griffiths et al. 1999). However, for a plasmid to be used as a vector in any type of organism it must have a multiple cloning site that is able to be recognised and cleaved by restriction enzymes - for the insertion of foreign DNA using restriction enzymes. For example a commonly used cloning vector, pUC19, is able to be cleaved at a polylinker region within the LacZ gene (Cis-regulatory element) using restriction enzymes HindIII and XbaI. Another important factor required by a recombinant plasmid to be expressed in various mammalian organisms is a Kozak sequence a ribosome will not initiate translation without it. Kozak (1987) state at least six nucleotides preceding the AUG initiator condor are required for efficient translation in mammalian cell lines. The optimal mRNA sequence recognised by ribosomes in eukaryotes has been defined as 5 - CCA/GCCAUGG 3 so ideally a recombinant plasmid should contain this base pair sequence. For optimal expression in bacteria, the ribosomal binding site is different it is called a Shine-Dalgarno sequence (5 AGGAGGCAGCUAUG 3). Plasmid DNA can also feature sequences which code for polyadenylation in RNA which is required for gene expression in many species (Proudfoot et al. 2002). Overall, it is clear recombinant plasmids are highly versatile and can be expressed in multiple organisms. However, the challenges of ensuring plasmid DNA replicates at the same rate as mammalian cell division is a challenge as mammalian cells are very specific in their requirements. Other issues like size of the plasmid, multiple cloning sites and risk of mutation further complicate the use of inserting plasmid DNA into non-bacterial tissue. There are millions of different bacteria in nature to be analysed perhaps more viable recombinant plasmids can be engineered in the future.

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