<P> A conceptual breakthrough of Jacob and Monod was to recognize the distinction between regulatory substances and sites where they act to change gene expression . A former soldier, Jacob used the analogy of a bomber that would release its lethal cargo upon receipt of a special radio transmission or signal . A working system requires both a ground transmitter and a receiver in the airplane . Now, suppose that the usual transmitter is broken . This system can be made to work by introduction of a second, functional transmitter . In contrast, he said, consider a bomber with a defective receiver . The behavior of this bomber cannot be changed by introduction of a second, functional aeroplane . </P> <P> To analyze regulatory mutants of the lac operon, Jacob developed a system by which a second copy of the lac genes (lacI with its promoter, and lacZYA with promoter and operator) could be introduced into a single cell . A culture of such bacteria, which are diploid for the lac genes but otherwise normal, is then tested for the regulatory phenotype . In particular, it is determined whether LacZ and LacY are made even in the absence of IPTG (due to the lactose repressor produced by the mutant gene being non-functional). This experiment, in which genes or gene clusters are tested pairwise, is called a complementation test . </P> <P> This test is illustrated in the figure (lacA is omitted for simplicity). First, certain haploid states are shown (i.e. the cell carries only a single copy of the lac genes). Panel (a) shows repression, (b) shows induction by IPTG, and (c) and (d) show the effect of a mutation to the lacI gene or to the operator, respectively . In panel (e) the complementation test for repressor is shown . If one copy of the lac genes carries a mutation in lacI, but the second copy is wild type for lacI, the resulting phenotype is normal--- but lacZ is expressed when exposed to inducer IPTG . Mutations affecting repressor are said to be recessive to wild type (and that wild type is dominant), and this is explained by the fact that repressor is a small protein which can diffuse in the cell . The copy of the lac operon adjacent to the defective lacI gene is effectively shut off by protein produced from the second copy of lacI . </P> <P> If the same experiment is carried out using an operator mutation, a different result is obtained (panel (f)). The phenotype of a cell carrying one mutant and one wild type operator site is that LacZ and LacY are produced even in the absence of the inducer IPTG; because the damaged operator site, does not permit binding of the repressor to inhibit transcription of the structural genes . The operator mutation is dominant . When the operator site where repressor must bind is damaged by mutation, the presence of a second functional site in the same cell makes no difference to expression of genes controlled by the mutant site . </P>

Structural and regulatory genes of the lac operon