The Application Of Recombinant Dna Techniques

The transfer of DNA between different species of bacteria has been achieved experimentally using both in vivo and in vitro techniques (Atherton et al., 1979). Thus, genetic material derived from one species may be incorporated into another where it may be expressed. In vivo techniques make use of phage particles which will pick up genetic information from the chromosome of one bacterial species, infect another bacterial species and in so doing introduce the genetic information from the first host. The information from the first host may then be expressed in the second host. Whereas, the in vivo techniques depend on vectors collecting information from one cell and incorporating it into another, the in vitro techniques involve the insertion of the information into the vector by in vitro manipulation followed by the insertion of the carrier and its associated 'extra' DNA into the recipient cell. Because the DNA is incorporated into the vector by in vitro methods the source of the DNA is not limited to that of the host organism of the vector. Thus, DNA from human or animal cells may be introduced into the recipient cell. Atherton et al. (1979) listed the basic requirements for the in vitro transfer and expression of foreign DNA in a host micro-organism as follows:

(i) A 'vector' DNA molecule (plasmid or phage) capable of entering the host cell and replicating within it. Ideally the vector should be small, easily prepared and must contain at least one site where integration of foreign DNA will not destroy an essential function.

(ii) A method of splicing foreign genetic information into the vector.

(iii) A method of introducing the vector/foreign DNA recombinants into the host cell and selecting for their presence. Commonly used simple characteristics include drug resistance, immunity, plaque formation, or an inserted gene recognizable by its ability to complement a known auxotroph.

(iv) A method of assaying for the 'foreign' gene product of choice from the population of recombinants created.

The initial work focused on E. coli but subsequently techniques have been developed for the insertion of foreign DNA into a range of bacteria, yeasts, filamentous fungi and animal cells. The range of vectors has been discussed by Gingold (1993) for bacteria, Curran and Bugeja (1993) for yeasts, Hopwood et al. (1985a) for streptomycetes, Elander (1989) for filamentous fungi and Murray (1993) for animal cells. The insertion of information into the vector molecule is achieved by the action of restriction endonucleases and DNA ligase. Site-specific endonucleases produce specific DNA fragments which may be joined to another similarly treated DNA molecule using DNA ligase. The modified vector is then normally introduced into the recipient cell by transformation. Because the transformation process is an inefficient one, selectable genes must be incorporated into the vector DNA so that the transformed cells may be cultured preferentially from the mixture of transformed and parental cells. This is normally accomplished by the use of drug-resistant markers so that those cells containing the vector will be capable of growth in the presence of a certain antimicrobial agent. The process is shown diagrammatically in Fig. 3.29.

Once the desired gene has been introduced into the recipient cell the problem of expression of the gene arises. This is particularly difficult when a eukaryotic gene is introduced into a bacterium. In the late 1970s a large number of mammalian genes were successfully introduced into bacterial cells, but there was little evidence of any gene expression (Atherton et al, 1979). The problem of eukaryotic gene expression in prokary-otic cells is due to the different structure of eukaryote genes which contain non-coding segments of DNA. Thus, the production of a eukaryotic product by a prokaryotic cell necessitates the incorporation of the genes coding for the product in a form that may be translated by the recipient cell. Two approaches have been adopted to construct eukaryotic genes in a prokaryotic form: the first is to synthesize DNA corresponding to the primary structure of the protein product of the gene, although this method is suitable only for the construction of genes coding for small peptides of known structure. Itakura et al. (1977) synthesized the gene coding for the human hormone, somatostatin, and succeeded in incorporating it in E. coli where it was expressed. The alternative technique is to synthesize DNA from the messenger RNA, corresponding to the gene, using the enzyme, reverse transcriptase. Eukaryote messenger RNA is similar to bacterial RNA in that it does not contain non-coding segments so that DNA synthesized from an eukaryotic messenger RNA template should be in a form which is transcribable by a

Plasmid DNA EcoRI site

Drug-resistance gene

Treat with restriction endonuclease Eco RI

Foreign DNA -Eco RI site-,

Treat with restriction endonuclease Eco RI

Drug-resistance gene

Hybrid molecule
Transformation j of E. coli

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