By David M. Glover (auth.)
1 Introductory remarks.- 2 The enzymology of in vitro DNA recombination.- three Plasmid vectors.- four Bacteriophage ? vectors.- five Expression of cloned DNAs in E. coli.- 6 tools for the actual characterisation of cloned segments of chromosomal DNA from better eukaryotes.- 7 techniques for learning expression in eukaryotic structures.
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Extra info for Genetic Engineering Cloning DNA
1 Directed synthesis offused polypeptides. Oftwo major approaches used to achieve expression ofvertebrate polypeptides in E. coli, one involves the construction of a hybrid gene with the N terminal region of a bacterial protein joined to the C terminus of a vertebrate protein. In the other approach the vertebrate gene is placed downstream from prokaryotic controlling elements within the plasmid. As an example of the first approach, I will discuss in some detail the expression of a chemically synthesised gene for the hormone somatostatin , a tetradecapeptide which has the physiological role of inhibiting the secretion of growth hormone, glucagon and insulin.
Coli is likely to be of considerable importance in antigen production for the development ofvaccines. Hepatitis B viral DNA has recently been cloned in pBR322 [ 13 J. The virus is widespread in man and produces several types of chronic liver disorders. The limited amount of virus available from infected patients and the inability to grow the virus in cultured cells has seriously bindered its molecular characterisation and the development ofvaccines. arge single-stranded gap which must be repaired with an endogenaus viral polymerase before the molecule can be subject to restriction-endonuclease digestion and molecular cloning.
2. Biological containment In order to enhance biological Containment amber mutations have been introduced into a number of vectors. Three of the Charon phages have, for example, been certified for use in an EK2 vector-host system; that is to say they permit the lowering of physical containment requirements by one category. These phage are Charons 3A, 4A and 16A which differ from Charon 3, 4 and 16, from which they are derived, by the introduction of amber mutations in genes A and B. Infections resulting from these phage will therefore produce mature phage particles only in a suppressor strain of E.
Genetic Engineering Cloning DNA by David M. Glover (auth.)