Use of Gene Fusions to Study Biological Problems

Abstract

A fundamental practical problem in molecular biology is the detection and quantitation of particular proteins of interest. In those cases in which biological or immunological assays exist, problems of how the expression of particular genes is regulated, how proteins find their correct location inside or outside of a cell, or what features govern protein structure and function in vivo are within the realm of accessibility. In many cases, however, reliable assays are not available and the alternative method of gene fusion is appropriate. The principle of the approach is to fuse either a promoter region or a gene whose product is under study to another gene encoding a product which can be assayed conveniently, for example the lacZ gene of E. coli which encodes β-galactosidase. In promoter fusions, the promoter region will direct synthesis of wild-type β-galactosidase with transcription initiating in the promoter region, and translation starting at the amino-terminal AUG encoded by lacZ (Figure 1). In gene fusions, a hybrid protein will be encoded containing sequences from the protein under study at the amino terminus, and an active β-galactosidase moiety at the carboxy-terminus (Figure 1). These fusions are active because the amino-terminal sequences of β-galactosidase may be replaced with a wide variety of sequences without a loss in its enzymatic activity. The β-galactosidase moiety, in such fusions, serves as a tag which can be used to monitor amounts or cellular location of the protein to which it is fused. In promoter or gene fusions, the myriad of genetic methods developed to study the lac operon can now be adapted to a genetic analysis of the promoter or gene fused to lacZ. For studies of fusions in bacteria, this adaptation is complete, while studies of fusions in eukaryotic cells such as yeast can draw from only a subset of the prokaryotic methods.