Suzanne Bourgeois

The lac repressor-operator interaction. 3. Kinetic studies.

Abstract A sensitive membrane filter assay for the lac repressor-operator complex has been used to study the kinetics of repressor and operator interaction. The rate of dissociation of the complex is very slow and follows first-order kinetics with a half-life of 19 minutes in our standard buffer (0.05 m ionic strength). The rate of dissociation (kb) is rather insensitive to temperature or pH, but becomes more rapid at high ionic strength. In 0.2 m ionic strength buffer the dissociation half-life is five to six minutes. The rate of association of repressor and operator is very fast and follows second-order kinetics with the rate constant for association (ka) being 7 × 109 m−1 sec−1 in our standard buffer. The rate of association is only slightly affected by temperature or pH, but becomes slower with increasing ionic strength. It is concluded that the association of repressor and operator is aided by electrostatic attraction between the phosphate groups of DNA and a positively charged binding site on the lac repressor. Over a wide range of ionic strength, the ratio k b k a has been found to agree well with the equilibrium constant. The binding of repressor to operator results in an unfavorable enthalpy change; the driving force for the binding reaction comes from a large entropy increase. Binding mechanisms are discussed.

lac repressor-operator interaction: I. Equilibrium studies☆

Abstract A membrane filter technique for detecting lac repressor-operator complexes is discussed and it is shown that this method assays for complexes present in solution prior to filtering. The method is sensitive (10−16 mole), accurate, and suitable for physical chemical experiments. Our results are consistent with the idea that only one repressor binds per operator. We report on the equilibrium constant (1 × 10−13 m in our standard buffer) and the effect of several reaction conditions on this parameter. In particular, the binding is very sensitive to ionic strength, the logarithm of the equilibrium constant being proportional to the square root of the ionic strength. Evidence for repressor subunits and a preferential loss of DNA binding activity compared to inducer (isopropyl-thiogalactoside) binding activity is presented. Methods for determining the concentration of repressor capable of binding to DNA are described. Actinomycin D inhibits the binding, suggesting that there is at least one guanine in or near the operator. We also report on a sensitive competition assay for unlabeled operator and show that the repressor does not bind to denatured DNA but will bind following renaturation of the DNA. Native Escherichia coli DNA without the lac region does not compete even when a 300-fold weight excess (10 μg/ml.) is used. The binding to the operator is thus extremely specific.

lac repressor—Operator interaction: II. Effect of galactosides and other ligands☆☆☆

Abstract A membrane filter technique was used to study the effect of galactosides and other low molecular weight compounds on the interaction of purified lac repressor and lac operator. Compounds that function in vivo as inducers of the lac operon are found to inhibit repressor-operator binding in vitro. Compounds that prevent induction in vivo are found to counteract the effect of inducers in vitro. The order of effectiveness of these compounds is the same in vitro as in vivo and agrees with their affinity for the lac repressor. Glucose interacts with the lac repressor and behaves as a weak anti-inducer. Cyclic adenosine monophosphate was found to have no effect on repressor-operator binding. The effect of galactosides on the rate of dissociation of repressor-operator complexes was also studied. The rate of dissociation was found to be increased by an inducer, isopropyl-β- d -thiogalactoside, and decreased by an anti-inducer, o-nitrophenyl-β- d -fucoside. This result has implications both in regard to allosteric models for conformational changes in proteins and in regard to the interpretation of both in vivo and in vitro induction curves.

lac repressor-operator interaction: VI. The natural inducer of the lac operon

Abstract Allolactose (1-6-O-β- d -galactopyranosyl- d -glucose) is the inducer of the lac operon when Escherichia coli are grown in the presence of lactose (1-4-O-β- d -galactopyranosyl- d -glucose). An in vitro assay for inducer was used to define the conditions of inducer production from lactose by β-galactosidase. The inducer can be made directly on the enzyme by a rearrangement reaction converting the 1 to 4 linkage of lactose to a 1 to 6 linkage. Allolactose was shown to be present in cell extracts by addition of purified repressor as carrier, isolation of the repressorligand complex and then chromatographic identification. The binding constant of allolactose for repressor and the effect of allolactose on the repressor-operator complex are the same as that of the synthetic inducer isopropyl-β- d -thiogalactoside. Furthermore, in vivo, synthetic allolactose is a more effective inducer than IPTG ‡ , probably because it is more efficiently concentrated by the permease.

FKBP51, a novel T-cell-specific immunophilin capable of calcineurin inhibition.

The immunosuppressive drugs FK506 and cyclosporin A block T-lymphocyte proliferation by inhibiting calcineurin, a critical signaling molecule for activation. Multiple intracellular receptors (immunophilins) for these drugs that specifically bind either FK506 and rapamycin (FK506-binding proteins [FKBPs]) or cyclosporin A (cyclophilins) have been identified. We report the cloning and characterization of a new 51-kDa member of the FKBP family from murine T cells. The novel immunophilin, FKBP51, is distinct from the previously isolated and sequenced 52-kDa murine FKBP, demonstrating 53% identity overall. Importantly, Western blot (immunoblot) analysis showed that unlike all other FKBPs characterized to date, FKBP51 expression was largely restricted to T cells. Drug binding to recombinant FKBP51 was demonstrated by inhibition of peptidyl prolyl isomerase activity. As judged from peptidyl prolyl isomerase activity, FKBP51 had a slightly higher affinity for rapamycin than for FK520, an FK506 analog. FKBP51, when complexed with FK520, was capable of inhibiting calcineurin phosphatase activity in an in vitro assay system. Inhibition of calcineurin phosphatase activity has been implicated both in the mechanism of immunosuppression and in the observed toxic side effects of FK506 in nonlymphoid cells. Identification of a new FKBP that can mediate calcineurin inhibition and is restricted in its expression to T cells suggests that new immunosuppressive drugs may be identified that, by virtue of their specific interaction with FKBP51, would be targeted in their site of action.

Diploid and haploid states of the glucocorticoid receptor gene of mouse lymphoid cell lines

Abstract A glucocorticoid-sensitive mouse thymoma line, W7, is compared to the mouse lymphoma line S49 which has been extensively used in studies of steroid action. Glucocorticoid-resistant variants are known to arise spontaneously at high rate from S49 (3.5 × 10 −6 per cell per generation) and at a frequency orders of magnitude lower in the case of W7 ( −9 ). The receptors of both cell lines have the same affinity for dexamethasone (K d = 1.3 ± 0.3 × 10 −8 M), but W7 cells contain twice the amount of glucocorticoid receptors present in S49 and are measurably more sensitive than S49 cells to dexamethasone. By selection for resistance to low concentrations of dexamethasone, derivatives of W7 have been isolated which are similar to S49 in that they have a higher resistance than the parental W7 line and approximately half the receptor content. Moreover, like S49, the partially resistant variants of W7 give rise to fully resistant derivatives at a high rate (2 × 10 −6 per cell per generation). These results suggest that a structural gene (r) coding for the receptor is present in two functional copies in W7 (r −,+ ), but in only one functional copy (r +/− ) in partially resistant derivatives of W7 and in S49. The gene dosage effect observed in these pseudodiploid lines indicates that the receptor gene, r, is autosomal, and that the inactivation of the r gene is a recessive genetic event. Consequences of the homozygous and heterozygous states of the receptor locus are discussed.

Regulation of fibronectin biosynthesis by glucocorticoids in human fibrosarcoma cells and normal fibroblasts

When treated with the synthetic glucocorticoid dexamethasone, HT1080 human fibrosarcoma cells show changes in morphology, adhesion, and the extracellular matrix. Dexamethasone treatment results in a tenfold increase in the rate of fibronectin biosynthesis in HT1080 cells and a twofold increase in untransformed, normal human fibroblasts. Maximal induction levels are attained within one cell generation, while decay of the response requires several cell cycles. Pulse-chase studies showed that most of the newly synthesized fibronectin is secreted into the medium. The glucocorticoid antagonist, RU-486, blocks the dexamethasone-induced changes but does not alter the basal rate of fibronectin production. Therefore, fibronectin biosynthesis appears to be controlled by two distinct mechanisms--one, regulating basal rates of fibronectin production, which is transformation-sensitive and glucocorticoid-independent; and another, which is mediated by the glucocorticoid receptor, resulting in elevated rates of fibronectin biosynthesis upon dexamethasone treatment both in normal fibroblasts and in HT1080 cells.

DNA binding properties of glucocorticosteroid receptors bound to the steroid antagonist RU-486.

RU‐486 is an anti‐fertility steroid which also has anti‐glucocorticosteroid effects. RU‐486 is shown to be a strong antagonist of the glucocorticosteroid‐induced cytolytic response of the murine thymoma lines W7TB and T1M1b, and of the induction of mouse mammary tumor virus (MMTV) mRNA in T1M1b cells. The glucocorticosteroid receptor of W7 cells has high affinity for RU‐486 (Kd = 3 X 10(‐9) M) but the complex formed has low nuclear transfer capacity. Binding of RU‐486, as compared with the glucocorticosteroid agonist triamcinolone acetonide, to mouse receptor results in a decreased affinity for DNA in general and a reduced specific recognition of a site in the promoter region of MMTV proviral DNA. The RU‐486 complex formed with rat liver receptor exhibits the same behavior; in addition, it is shown that only a fraction of these complexes are activated by temperature and these form highly salt‐sensitive interactions with DNA. These results indicate that the binding of RU‐486 to glucocorticosteroid receptors mimics pharmacologically the properties of a class of receptor variants (nt‐) which are non‐functional and have reduced nuclear transfer and altered DNA binding capacity. These results substantiate the importance of DNA binding in receptor function.

A low-copy-number vector utilizing β-galactosidase for the analysis of gene control elements

A low-copy-number vector, pFZY1, with the multiple restriction site linker of M13mp18 inserted upstream from a promoterless beta-galactosidase (beta Gal)-coding lacZ gene has been constructed to provide a convenient and accurate system to analyze regulatory elements in vivo. The plasmid contains the oriF replication origin without the par locus and is present in the cell in one to two copies per genome. It is retained in the host by the presence of ampicillin, and each inserted promoter yielded consistent values of beta Gal activity under all the conditions tested. A series of tetracycline resistance (TcR) promoter fragments and lac promoter fragments have been compared in pFZY1 and the high-copy-number pKO-vector series. The transcriptional activity measured for different fragments containing the same TcR promoter varied within a six-fold range among the several constructs tested. Regulation of the wild-type lac promoter and mutants in pFZY1 was similar to that observed for lac promoters in the chromosome while their regulation in pKO-1mp18 was significantly affected by the high copy number, as expected.

Isolation and characterization of glucocorticoid- and cyclic AMP-induced genes in T lymphocytes.

Glucocorticoids and cyclic AMP exert dramatic effects on the proliferation and viability of murine T lymphocytes through unknown mechanisms. To identify gene products which might be involved in glucocorticoid-induced responses in lymphoid cells, we constructed a lambda cDNA library prepared from murine thymoma WEHI-7TG cells treated for 5 h with glucocorticoids and forskolin. The library was screened with a subtracted cDNA probe enriched for sequences induced by the two drugs, and cDNA clones representing 11 different inducible genes were isolated. The pattern of expression in BALB/c mouse tissues was examined for each cDNA clone. We have identified two clones that hybridized to mRNAs detected exclusively in the thymus. Other clones were identified that demonstrated tissue-specific gene expression in heart, brain, brain and thymus, or lymphoid tissue (spleen and thymus). The kinetics of induction by dexamethasone and forskolin were examined for each gene. The majority of the cDNA clones hybridized to mRNAs that were regulated by glucocorticoids and forskolin, two were regulated only by glucocorticoids, and three hybridized to mRNAs that required both drugs for induction. Inhibition of protein synthesis by cycloheximide resulted in the induction of all mRNAs that were inducible by glucocorticoids. Preliminary sequence analysis of four of the 11 cDNAs suggests that two cDNAs represent previously undescribed genes while two others correspond to the mouse VL30 retrovirus-like element and the mouse homolog of chondroitin sulfate proteoglycan core protein.