The use of E. coli for general cloning and manipulation of DNA is well established; however, numerous developments have been made for cloning in eukaryotic cells. Plasmids used for cloning DNA in eukaryotic cells require a eukaryotic origin of replication and marker genes that will be expressed by eukaryotic cells. At present, the two most important applications of plasmids to eukaryotic cells are for cloning in yeast and in plants.

Although yeast has a natural plasmid, called the 2μ circle, this is too large for use in cloning. Plasmids such as the yeast episomal plasmid ( YEp) have been created by genetic manipulation using replication origins from the 2μ circle, and by incorporating a gene that will complement a defective gene in the host yeast cell. If, for example, a strain of yeast is used that has a defective gene for the biosynthesis of an amino acid, an active copy of that gene on a yeast plasmid can be used as a selectable marker for the presence of that plasmid. Yeast, like bacteria, can be grown rapidly, and it is therefore well suited for use in cloning. Of particular use has been the creation of shuttle vectors , which have origins of replication for yeast and bacteria such as E. coli. This means that constructs may be prepared rapidly in the bacteria and delivered into yeast for expression studies.

The bacterium Agrobacterium tumefaciens infects plants that have been damaged near soil level, and this infection is often followed by the formation of plant tumours in the vicinity of the infected region. It is now known that A. tumefaciens contains a plasmid called the Ti plasmid , part of which is transferred into the nuclei of plant cells that are infected by the bacterium. Once in the nucleus, this DNA is maintained through integration with the chromosomal DNA. The integrated DNA carries genes for the synthesis of opines (which are metabolised by the bacteria but not by the plants) and for tumour induction (hence ‘Ti’). DNA inserted into the correct region of the Ti plasmid will be transferred to infected plant cells, and in this way it has been possible to clone and express foreign genes in plants (Figure 1). This is an essential prerequisite for the genetic engineering of crops.

Fig1. Scheme for cloning in plant cells using the Ti plasmid.

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Screening Gene Libraries

Hybridisation and Gene Probes

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Cloning Vectors : Plasmids

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Chromosome Painting and DNA Sequencing Reveal the Evolution of Chromosomes