Walter Wehrli

Root induction on several Solanaceae species by Agrobacterium rhizogenes and the determination of root tropane alkaloid content.

Using Agrobacterium rhizogenes, roots were induced on explants of 24 different Solanaceae species and established as in vitro cultures. Some of the root clones produced tropane alkaloids at levels similar to roots of the corresponding intact plants and maintained these levels over several passages.

The use of dextran-coated charcoal for kinetic measurements: Interaction between rifampicin and DNA-dependent RNA polymerase ofEscherichia coli

Abstract Rifampicin inhibits bacterial DNA-dependent RNA polymerase by forming a 1:1 complex with the enzyme. In previous studies the extent of this binding was determined by chromatographic isolation of the complex on Sephadex columns with 14 C-labeled rifampicin as tracer. To measure the kinetics of the enzyme-antibiotic interaction, however, this procedure is too slow and laborious. In this paper we describe the use of dextran-coated charcoal, which proved to be ideal for isolating the complex rapidly and quantitatively. The free [ 14 C]rifampicin is adsorbed on the charcoal and can be removed by centrifugation, whereas the [ 14 C]rifampicin-enzyme complex remains in the supernatant and can be easily measured. An account is given of the application of this procedure in measuring the dissociation and association kinetics of the RNA polymerase-rifampicin complex.

A convenient method for permeabilizing the fungus Cephalosporium acremonium.

Abstract After ether treatment the fungus Cephalosporium acremonium is permeable to enzyme substrates which cannot enter the intact cells. Two enzyme systems, hexokinase/glucose 6-phosphate dehydrogenase and RNA polymerases, have been assayed in ether-treated cells. The first two enzymes can only be measured upon addition of ATP and NADP + and are remarkably stable. The DNA transcription requires the four nucleoside triphosphates as substrates and can be stopped by the addition of actinomycin D.

Interaction between Rifampicin and DNA-dependent RNA Polymerase of E. coli

INTRODUCTION The antibiotic rifampicin specifically inhibits bacterial RNA polymerase by blocking the initiation, but not the elongation, of RNA chains (Sippel and Hartmann 1968). As shown earlier, rifampicin forms a tight 1:1 complex with the enzyme (Wehrli et al. 1968a; Wehrli and Staehelin 1970; Eilen and Krakow 1973). Therefore very low concentrations of antibiotic are required to inhibit the enzyme. Mammalian RNA polymerases, as well as other nucleic acid polymerases of both bacterial and mammalian origin, are not affected at these low levels of rifampicin (Wehrli et al. 1968b; Wehrli and Staehelin 1975). Thus rifampicin is a very specific and powerful inhibitor and has been extensively used in the study of RNA biosynthesis and metabolism. Although it has been suggested that the strength of rifampicin binding to RNA polymerase is correlated to the extent of enzyme inhibition (Wehrli and Staehelin 1971), no direct quantitative measurements of this binding have been made. In this paper we describe a new method that allows both the rate of association and the rate of dissociation of rifampicin and RNA polymerase to be measured directly. The application of this method makes it possible to quantify the kinetic interaction of rifampicin not only with the free holo- or core enzyme under a variety of physical conditions, but also with RNA polymerase engaged in the various steps of transcription. MATERIALS AND METHODS [ 14 C] Rifampicin (11.6 μ Ci/ μ mole) was prepared as previously described (Keberle, Meyer-Brunot and Schmid 1966). The preparation of dextran-coated charcoal, the isolation of the rifampicin–RNA...