Genetic Fusions of the lac Operon: A New Approach to the Study of Biological Processes

Abstract

INTRODUCTION The lactose operon represents one of the best worked-out systems of gene expression and regulation. The extent to which the details of lac operon control have been elaborated depended, in good part, on aspects of lactose metabolism which make it a particularly easy system to analyze. These include a sensitive and rapid assay for β -galactosidase, a number of different indicator media for distinguishing phenotypes of mutant strains, and an ability to select both forward and reverse mutations in the operon and in the regulatory gene (see Beckwith 1970 for a review of most of these; see also Hopkins 1974). The most important aspect of lactose metabolism which distinguishes it from most other biochemical pathways and contributes to its ease of analysis is the nature of lactose itself. Since lactose is a disaccharide, glucose-1-4- β -D-galactose, substituted derivatives with various moieties replacing the glucose can be synthesized which will have affinity for one or another component of the system. For instance, one of the indicator media used includes 5-bromo-4-chloro-3-indolyl- β -D-galactoside (XG), which is a substrate for β -galactosidase. When hydrolyzed, this compound forms a blue dye, thus providing a sensitive test for the presence of β -galactosidase activity. Since XG is not an inducer of the lac operon, it can be used to distinguish inducible from constitutive strains, as well as to discriminate between strains which produce high and low levels of β -galactosidase, in general, and to detect plaque-forming transducing phages which carry the lacZ gene. The technical principles and approaches used in studying the...