Magnus Pfahl

Specific binding of the glucocorticoid-receptor complex to the mouse mammary tumor proviral promoter region

To elucidate the molecular mechanism by which steroid hormones exert their regulatory function, we investigated the interaction of a glucocorticoid-receptor complex with purified DNA fragments from cloned mouse mammary tumor (MMTV) proviral DNA. With a DNA-cellulose binding assay, rat and mouse glucocorticoid receptors were found to interact with a high affinity site or sites in or near the promoter region of the MMTV proviral DNA. The assay allowed the use of unpurified as well as purified receptor, and therefore made it possible to investigate the binding properties of mutant receptors. Two nuclear-transfer-deficient receptors have a decreased affinity for specific as well as unspecific DNA, but are still capable of distinguishing between the two types of DNA. The existence of specific DNA binding sites for steroid-receptor complexes is discussed in the context of a general model of steroid hormone action.

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.

General features of steroid resistance in lymphoid cell lines

Some general features of dexamethasone resistance in five murine lymphoid cell lines were investigated. To obtain large numbers of dexamethasone-resistant (Dexr) variants, a technique was developed by which mouse lymphoid cell lines can be grown with high efficiency on the surface of agar plates without a feeder layer. A total of 271 Dexr variants were investigated, and 90% of them turned out to lack detectable steroid receptor whereas 10% have receptor with, in most cases, a normal affinity for the steroid hormone. Most of this latter class of variants, however, have reduced amounts of receptor and the receptor of all of them displayed altered nuclear binding characteristics. None of the five investigated lymphoid cell lines yielded a Dexr variant with a normal receptor. These results confirm the idea that the high incidence of receptor variant may be due, at least in part, to the haploid state of a gene coding for the receptors. In cell fusion experiments it could be shown that Dexs is dominant over Dexr, but that a Dexr a-lele in a tetraploid cell can lead to an increased frequency of steroid resistance.

Interaction of glucocorticoid receptors from lymphoid cell lines with their nuclear acceptor sites

Procedures have been developed which provide simple means of determining binding constants of steroid receptors for glucocorticoids in mouse lymphoid cell lines and of characterizing the interaction of the steroid--receptor complex with the nucleus. An average of 70% of the steroid--receptor complexes is found associated with the nuclear fraction in three investigated cell lines, whereas 30% of the steroid--receptor complexes is found in the cytosol fraction. This distribution of the steroid-receptor complex within the cell is independent of whether steroid uptake of the cells is performed at low or at high steroid concentration. Part of the binding of the steroid receptor to the nuclear fraction is sensitive to high ionic strength and to high pH. A larger fraction of the steroid--receptor complex binding to the nuclear fraction is insensitive to high ionic strength and pH when the steroid uptake is performed at low steroid concentrations than when performed at high steroid concentrations. Steroid--receptor complex is released from the nuclear fraction by DNAase treatment but not by RNAase treatment. The possible correlation between the sensitivity to ionic strength and pH and the specificity of the binding is discussed.

lac repressor-operator interaction. Analysis of the X86 repressor mutant☆

Abstract In vitro measurements show that the X86 repressor, which has an increased affinity for the lac operator as compared to wild-type repressor, also has an increased affinity for non-operator sites on Escherichia coli DNA. The rate constant of association of repressor and operator is decreased by E. coli DNA fivefold more for X86 repressor than for wild-type repressor. Low inducer concentrations increase the rate of association of X86 repressor and operator in the presence of E. coli DNA. In a partial equilibrium situation where part of the X86 repressor is bound to the operator, and part to either non-operator sites on E. coli DNA or to an O c operator, the formation of complexes between X86 repressor and wild-type operator is favored by low inducer concentrations. Repression of the lac enzymes increases drastically in the X86 mutant in the absence of DNA synthesis in vivo . A new explanation for the in vivo characteristics of the X86 mutant is suggested.

Second and "third operator" of the lac operon: an investigation of their role in the regulatory mechanism.

Abstract The influence of the two operator-like regions lying within or near the lac regulatory region on the binding of lac repressor to lac operator has been investigated. λdlac phages deleted either for the “second operator” in the beginning of the Z gene or deleted for the “third operator” at the end of the I gene were constructed. In in vitro binding experiments it could be shown that the deletion of secondary repressor binding sites from the lac regulatory region does not significantly alter the stability of the repressor—operator complex. Measuring the rate constant of association of repressor with operator in the presence of a 150-fold excess of unspecific DNA, we observed a concentration-dependent effect of the unspecific DNA, although the ratio of operator to non-operator DNA was kept constant. A small effect of the secondary binding sites is seen on the rate of association of repressor with operator, indicating that the secondary binding sites might play a role in facilitating association of repressor with operator under in vivo conditions.

Analysis of steroid resistance in lymphoid cell hybrids

In an attempt to obtain a more detailed understanding of the action of the glucocorticoid-receptor complex in mouse lymphoid cell lines, steroid sensitivity has been investigated in hybrids. Hybrids between dexamethasone (dex)-sensitive and dexamethasone-resistant (Dexr) variants, and hybrids between different Dexr variants were investigated. In the case of Dexr x Dexs hybrids, the possibility of negative complementation was tested; in the case of Dexr x Dexr hybrids, positive complementation was investigated. Neither positive nor negative complementation could be detected; dex sensitivity was always dominant over dex resistance. However, hybrids which contain positive receptor allele(s) segregated out Dexr clones at higher frequencies than expected from studies of pseudodiploid cell lines. This study suggests that different mechanisms give rise to the dex-resistant phenotype in pseudodiploid lymphoid cell lines and in pseudotetraploid hybrids of these cell lines.

Characteristics of tight binding repressors of the lac operon

Abstract A number of tight binding lac repressor mutants (Itb) have been analyzed. The mutant repressors could be shown to have altered DNA binding properties, which can include an increased affinity for operator DNA, an increased affinity for non-specific DNA, or a decreased affinity for non-specific DNA. Inducer binding of the repressors is normal even though the induction profile of the Itb strains is similar to that of Is mutants. In general, the investigated Itb repressors can be divided into three groups, of which type I shows an increase in affinity for operator as well as for non-specific DNA. A typical example for this type is the well-characterized X86 repressor. Type II repressors show only an increase in affinity for operator DNA. This new type of mutant repressor is of particular interest, since its further analysis might provide information on which amino acid residues of the repressor are directly involved in the specific interaction between repressor and operator. Type III repressors do not show a considerable change in affinity for wild-type operator but might have an increased affinity for Oc operators. Some of these repressors also have a decreased affinity for non-specific DNA.

Mapping of I gene mutations which lead to repressors with increased affinity for lac operator

Abstract A genetic mapping system is used to locate mutations on the lac repressor gene ( I ) which lead to repressor proteins with an increased affinity for operator DNA. These tight binding repressors (I tb ) are of particular interest since their analysis should allow some conclusions on the mechanism of interaction between repressor and operator. I tb mutations were found to map in two regions of the I gene. One is near the amino-terminal end, a region which has been shown to be essential for the DNA binding properties of the repressor. The other region in which I tb mutations were mapped codes for approximately amino acids 255 to 295 of the repressor, a region which had so far not been considered to be essential for the DNA binding properties of the repressor protein.

Effect of DNA denaturants on the lac repressor-operator interaction.

The nitrocellulose filter assay was used to study the effect of the DNA denaturants glycerol and dimethylsulfoxide (Me2SO) on the lac repressor-operator interaction. Both glycerol and Me2SO decrease the rate of dissociation (kb) of the repressor-operator complex but do not significantly alter the rate of association of repressor and operator. In the presence of 10% Me2SO an almost 10-fold increase of affinity of repressor for operator is observed. A small increase in affinity of repressor for Escherichia coli DNA, chicken blood DNA, and poly(dA-dT) is also found. The results lead to the conclusion that lac repressor when interacting with the operator causes local destabilization of the DNA.