Molec. gen. Genet. 166, 229-231 (1978) © by Springer-Verlag 1978 Short Communication Binding of lac Repressor to the Secondary lac Operator in Escherichia coil Alain Rambach and Mai Lebastard Groupe INSERM U. 163, Institut Pasteur; 28, Rue du Dr Roux, Paris 15e, France Summary. In the lac operon, the existence of a second- ary repressor binding site, inside Z gene, had been inferred from in vitro binding studies (Reznikoff et al., 1974; Gilbert et al., 1975). A serie of deletions have been constructed from a lac transducing 2 bacteriophage. Some of those deleted bacteriophages have still the property of dere- pressing a chromosomal lac operon, even though they do not contain any more the lac operator. This phe- nomenon is an indication that the secondary repressor binding site is also active in vivo. Lac repressor acts on lac DNA by binding to a spe- cific site: the lac operator. The lac operator was first defined as a genetic locus. Mutations at this locus suppress repressor binding and hence such mutants express lac activities constitutively (Jacob and Monod, 1961). The lac operator was later character- ized directly by in vitro binding of lac repressor to DNA fragments (Gilbert and Muller Hill, 1966) and the relevant DNA fragment has been sequenced (Gil- bert and Maxam, 1967). The lac operator is located just next to the structural lac Z gene which codes for the/%galactosidase. In addition to this first lac operator, a secondary lac operator has been described. The secondary lac operator is located inside the lac Z gene, while the first operator is outside the structural gene. The sec- ondary operator has been characterized by experi- ments involving in vitro binding of lac repressor to DNA containing various deletions of the lac region (Reznikoff et al., 1974). We present here data showing that the lac repressor binds in vivo to this secondary operator. To obtain in vivo information on the secondary lac operator we wanted to compare lac expression from transducing bacteriophages carrying various re- gions of the lac genes but not containing the first operator. We expected some of these bacteriophages to contain no operator and some to contain the sec- ond operator. We have constructed transducing 2 bac- teriophage carrying various distal portions of the Iac Z gene in the following manner. Barnes et al. (1974), have constructed a coli strain with a thermoinducible 2 bacteriophage inserted at a site very close to a lac operon. Among the phages produced after thermoin- duction of such lysogens, 1% express a lac + pheno- type, but contain distal portions of the lac Z gene. These were detected as giving white plaques on Mac Conkey Lactose plates at 30 ° C on a strain carry- ing a small deletion of the second half of the lac Z gene, Hfr 3000 U163, but giving lac + recombinants with the same Hfr 3000 U163 strain. Approximatively 600 bacteriophages giving white plaques were screened and 3 were found to recombine with Hfr 3000 U163. Since they recombine with the U163 mutants these bacteriophages must contain distal regions of the lac Z genes. They were called 2AR102, ,~AR103 and 2AR104. Genetic Mapping The extent of the lac region incorporated in these transducing bacteriophages has been determined by genetic recombination with a series of lac Z mutants. As controls we have also used two other bacterio- phages picked out of the 600 noted above : a bacterio- phage giving a red plaque on Mac Conkey Lactose, designated 2plac and a bacteriophage giving a white plaque but no lac ÷ recombinanats, designated 2AR105. Another control phage 2placsW4680S carry- ing a known deletion in the lac Z gene, was used in this study. The extents of the lac region in the strains 2AR102 to 105 are shown in Figure 1: 2AR105 contains no lac Z gene, 2AR103 contains 0026-8925/78/0166/0229/$01.00