Cell, Vol. 49, 603-612, June 5, 1967, Copyright 0 1987 by Cell Press kc Repressor Can Regulate Expression from a Hybrid SV40 Early Promoter Containing a lac Operator in Animal Cells Myles Brown,’ James Figge: Ulla Hansen,t Christopher Wright: Kuan-Teh Jeang,* George Khoury,S David Y. Livingston,’ and Thomas M. Roberts’ * Division of Neoplastic Disease Mechanisms Dana-Farber Cancer Institute Boston, Massachusetts 02115 t Laboratory of Eukaryotic Transcription Dana-Farber Cancer Institute Boston, Massachusetts 02115 *Laboratory of Molecular Virology National Cancer Institute Bethesda, Maryland 20892 Summary The E. coli lac operator and repressor were adapted for function in mammalian cells. Plasmids containing an SV40 early region (pSVlac0) or a chloramphenicol acetyl transferase gene (pSV/acOCAT) linked to a hy- brid SV40 early promoter bearing a lac operator were tested for function. Identical plasmids lacking an op- erator (pX-8 and pX-8CAT) were controls. In vitro, early transcription from pSVlac0, but not from pX-8, was in- hibited by lac repressor, and repression was overcome by IPTG. Repression of large T synthesis or CAT activ- ity occurred in vivo only when the respective operator- containing plasmid was cotransfected with a plasmid encoding lac repressor, or when the recipient cells stably synthesized lac repressor. IPTG substantially relieved repression in both cases. CAT enzyme repros- sion was paralleled by a decrease in CAT mRNA abun- dance. Thus regulatory elements of the lac operon function physiologically in mammalian cells. Introduction The ability to regulate, on command, the transcription of various genes in cultured mammalian cells has already provided new insights into the functions of the products of some of these genes. In an ideal experiment of this type, one might envision eliciting the full activation or repres- sion of a gene of interest simply by changing the level of a specific, non-toxic inducing agent in the culture medium. The existing systems in higher eukaryotes for the experimental regulation of cloned genes, such as heavy metal induction of the metallothionein promoter (Mayo et al., 1982; Brinster et al., 1982; Pavlakis and Hamer, 1983) and the glucocorticoid induction of the mouse mammary tumor virus promoter (Hynes et al., 1981; Lee et al., 1981), frequently suffer from the existence of a relatively high level of constitutive activity of these ele- ments and from a limited increase in their function upon inducer addition. Moreover, the inducing agents them- selves may influence metabolism. The situation is differ- ent in prokaryotes, where there are several well-defined transcription regulatory systems, each of which can effect the stringent regulation of expression of a relevant gene. One of the best understood is that governing expression of the E. coli lacoperon (Miller and Reznikoff, 1980). Here, genes encoding enzymes important for lactose metabo- lism (/acZyA) are under the negative control of a repressor tetramer consisting of identical 38.6 kd subunits encoded by the lad gene. This protein can bind specifically to both allolactose and to a unique operator sequence (/acO) positioned immediately downstream of the lac promoter (/a#). In vitro, under standard conditions (I = 0.05 M [pH 7.41, 24%), the equilibrium association constant govern- ing the binding of repressor to lac0, &, is 1013 M-l (Riggs et al., 1970). Binding of repressor to lac0 can completely inhibit transcription from /a# in vivo. Normally, the rate of lac transcription is regulated by the concentration of al- lolactose (or an analogue) in the cell. This compound and certain of its analogues, such as isopropyl P-D-thiogalac- toside (KG), can function as inducers of lac operon tran- scription and appear to act by binding to repressor and decreasing the affinity of the latter for lac0, thereby allow- ing increased transcription of the lac operon. In vitro, under standard conditions, the equilibrium association constant for the repressor-IPTG complex binding to /acO, Et& is approximately 3 x lOlo M-l, i.e., 300-fold lower than that of free repressor for lac0 (Barkley and Bour- geois, 1960). Brent and Ptashne (1984) have demonstrated the feasi- bility of transferring a system of gene regulation from prokaryotes to yeast utilizing the E. coli /exA operator and repressor. In an analogous manner, we and others (Hu and Davidson, 1987) have transferred the regulatory com- ponents of the lac operon from E. coli to mammalian cells. Here we show that lac repressor can inhibit transcription from a hybrid SV40 early promoter containing a synthetic lac operator analogue and that this repression is over- come in the presence of IPTG, both in vitro and in vivo. Results Structure and Function In Vltro of a Hybrid SV40 Promoter Containing a lac Operator The plasmid, pX-8 (Fromm and Berg, 1982), contains an intact SV40 early region coding unit coupled to a mutant early promoter. Present between the TATA box and the start sites of early region transcription is an 8 bp deletion at the Bgll site (Figure 1). This deletion lies within the ori- gin of replication and is also the site of insertion of an 8 bp Xhol linker. The pX-8 promoter region contains all neces- sary elements for early transcription, including a wild-type TATA box, an intact set of 21 bp repeats, and tandem 72 bp repeat enhancer elements. Even though its replication origin is no longer functional (Fromm and Berg, 1982), when transfected into rat F-111 cells, pX-8 DNA trans- formed as efficiently as a wild-type SV40 plasmid (Fromm and Berg, 1982). Therefore, its early promoter functions effectively. Moreover, when transfected into CVl cells, pX-