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Bovine neurophysins I and I1 have been obtained in a highly purified state using isoelectric focusing. The interactions of oxytocin and [8-lysine]vasopressin with these proteins have been investigated by thin-film equilibrium dialysis using highly radioactive hormones retaining their full biological activities. I n this report, we present the results of binding studies carried out using the equilibrium dialysis technique with highly purified neurophysins obtained by isoelectric focusing of crude protein material. The thermodynamic parameters and the stoichiometry of the reaction between the hormones and the proteins have been established. These findings are discussed in terms of the molecular mechanisms by which the neurophysins bind to the hormonal peptides.

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Using gel retardation and DNase I protection techniques, we have demonstrated that the Escherichia coli integration host factor (IHF) stabilizes the interaction between Mu repressor and its cognate operator-binding sites in vitro. These results are discussed in terms of a model in which IHF may commit the phage to the lytic or lysogenic pathway depending on the occupancy of the operator sites by the repressor.

The repressor of bacteriophage Mu, c, binds to three operator sites, O1, O2, and O3, overlapping two divergent promoters, which regulate the lytic and lysogenic pathways. Its binding to this operator region generates several complexes, which were analyzed by DNase I protection experiments. We demonstrate that c first binds to two 11-base pair partially repeated sequences in O2 that could represent "core" binding sites for the repressor. This initial interaction serves as an organizer of a more complex nucleoprotein structure in which O2, O1, and O3 become successively occupied. The quaternary structure of the repressor was also investigated. Size exclusion chromatography and protein-protein crosslinking experiments with chemicals that possess linking arms of various lengths indicate that the repressor oligomerizes in solution. A model is proposed describing the successive interactions of c with the operator sites O2, O1, and O3 leading to the elaboration of a higher order structure in which the early lytic functions are repressed.The repressor, c, of the temperate bacteriophage Mu controls the choice between lysogeny and lytic development. The decision is exercised at the level of transcription by binding of c to the early operator region located about 1 kilobase from the left end of the phage genome ( Fig. 1). This operator is contained within a 200-bp 1 region composed of two divergent promoters (Pe and Pc) and three operator sequences, O1, O2, and O3. These have been defined by DNase I protection of the region by the repressor (Krause et al., 1983(Krause et al., , 1986Alazard et al., 1992;Goosen et al., 1984). Binding of c to O1 and O2 appears to occur simultaneously at low repressor concentrations and prevents the initiation of transcription at the early promoter Pe. Binding to O3, which requires higher concentrations of c, inhibits transcription of the c gene from the Pc promoter (Goosen et al., 1984;Alazard et al., 1992). A binding site for integration host factor (IHF) is located between O1 and O2. This DNA-binding protein bends DNA and promotes the formation of looped structures (Craig and Nash, 1984;Gamas et al., 1987;Prentki et al., 1987;Stenzel et al., 1987;Robertson et al., 1988;Thompson et al., 1988). IHF plays a complex role in the regulation of the early functions of Mu. It has been demonstrated that it stimulates transcription from Pe, favoring lytic growth (Krause et al., 1986), possibly by direct interaction between bound IHF and RNA polymerase (Goosen and . On the other hand, IHF stabilizes the interaction among the repressor and O1 and O2 and allows the establishment and the maintenance of lysogeny (Vogel et al., 1991;Alazard et al., 1992;Gama et al., 1992;Bétermier et al., 1995).Previous results (Alazard et al., 1992) show that the interaction of c with a linear operator fragment leads to the formation of at least five high molecular weight DNA-protein complexes, which can be separated using gel retardation techniques. The complexes with lower apparent molecular weight can be chased successiv...

We show, using gel retardation, that crude Escherichia coli cell extracts contain a protein which binds specifically to DNA fragments carrying either end of the phage Mu genome. We have identified this protein as Fis, a factor involved in several site-specific recombinational switches. Furthermore, we show that induction of a Mucts62 prophage in a fis lysogen occurs at a lower temperature than that of a wild-type strain, and that spontaneous induction of Mucts62 is increased in the fis mutant. DNasel footprinting using either crude extracts or purified Fis indicate that binding on the left end of Mu occurs at a site which overlaps a weak transposase binding site. Thus, Fis may modulate Mu growth by influencing the binding of transposase, or other proteins, to the transposase binding site(s), in a way similar to its influence on Xis binding in phage lambda.

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