Mutants that make more lac repressor.

The gene that makes the lac repressor functions at an extremely low rate. It synthesizes only a few thousandths of a per cent of the total protein of an E. coli cell: about 5-10 molecules each generation.1 In order to study this molecule more easily, we have sought to enhance the amount present in a cell. Two approaches have been successful. The repressor gene, the i gene, itself can be mutated to a form that produces more repressor, or the number of copies of the gene can be drastically increased by incorporating the gene into a phage chromosome which will multiply in the cell. The combination of these two approaches yields a cell strain which can make 0.5 per cent of its protein lac repressor. The ij Mutant.-Several mutants that make about tenfold more repressor than the wild type have been found. These if mutants (q for quantity) were selected by forcing temperature-sensitive repressor mutants, strains constitutive at 430 and inducible at 300, to revert to an inducible phenotype at 430C. At the high temperature, the temperature-sensitive mutants make too little repressor, either because the protein itself is unstable (iTL) or because the final assembly of active repressor is blocked (iTSS).2 Although the reversion could simply correct the original defect, the temperature effects can also be overcome by producing more of the protein (or a more active protein: so that the small amount left at the high temperature will suffice). However, the ability of the lac repressor to bind IPTG (isopropyl-l-thio-f-D-galactopyranoside) gives one a direct, quantitative measure of the number of molecules present.1' By screening extracts from "revertant" strains, we could identify those mutants which, in contrast to the wild type, gave an easily detectable binding in the crude extract. The revertants were isolated using TONPG (o-nitrophenyl-1-thio-f3-D-galactopyranoside), which will inhibit the growth of lac constitutive cells that have a functioning lac permease. y(permease-) mutants will arise and can be coullterselected against by growth on lactose in the -presence of IPTG. Both iTSS and iTL strains have yielded overproducing derivatives. Table 1 shows the IPTG-binding data for some of these strains; the data suggest that there is about ten times more repressor in the ij strains. One of these mutants, a derivative of an iTSS strain, has been studied in detail, and was used to produce pure repressor for physical characterization (studies that will be reported elsewhere). The affinity of this ij repressor for IPTG has been checked and is the same as the wild type; therefore, the increase in the binding truly means an increase in amount of repressor. Dominance and Complementation.-To study further the properties of this i" mutant, we constructed heterozygous diploids with a variety of i and o (operator)