Approaches for studying expression in eukaryotic systems

Abstract

DNA sequencing of cloned eukaryotic genes has revealed several interesting common features: a potential eukaryotic promoter sequence TATAAA [1], found approximately 30 residues up-stream from eukaryotic genes transcribed by RNA polymerase II; preferred bases at the boundaries between the coding parts of genes and their intervening sequences, which are postulated to be recognition sites for splicing enzymes [2]; regions of sequence homology which could present sites important in recombinational events [3] (see, for example, the discussion of immunoglobulin genes in Chapter 6). In none of these cases is there yet any proof of the functional importance of the precise nucleotide sequences. A general approach to solve these problems is to generate mutations in vitro at specific sites in cloned DNA segments and then to study the effects of the mutation when the DNA segments are reintroduced into eukaryotic cells or an in vitro system for eukaryotic gene expression. Since such an approach is in its infancy, this chapter will serve simply to draw attention first to techniques for in vitro mutagenesis, and second to some eukaryotic systems which are available to study the expression of cloned eukaryotic genes.