Yoshinobu Sugino

Purification and characterization of the N gene product of bacteriophage lambda

The N protein (pN) specified by bacteriophage lambda is an antitermination factor and is required for phage development. pN can be assayed by making use of the observation that the in vitro synthesis of trp mRNA in a reaction programmed with DNA template from lambda trp transducing phage bearing N- and fed- mutations is pN dependent (Ishii et al., 1980). The assay has been used to purify pN. We have observed that pN forms a complex with E. coli protein(s) and is dissociated in the presence of urea. The complex is not formed in host bacteria bearing the nusA-nusB- mutations. pN is a basic protein and heat-stable. Using these characteristics, we have purified pN to virtual homogeneity as judged by polyacrylamide gel electrophoresis in the presence of SDS. pN is a monomeric protein and its mol. wt. is approx. 14 000. The antiterminating activity of pN appears to be enhanced by complex formation with host-encoded protein(s) depending on the nusA and/or nusB gene function.

A biochemical assay for the transcription-antitermination function of the coliphage λ N gene product

Abstract An in vitro assay has been developed for the antiterminating activity of the N gene product (pN) of bacterio-phage λ, based on the N-dependent stimulation of trp mRNA synthesis from the DNA templates of λtrp transducing phages. Using this assay system, we show (a) that the stimulation of trp mRNA synthesis by pN requires the cis -dominant nut L site on the λ chromosome and (b) that pN can participate in vitro in the formation of functional antiterminating transcription complexes.

Mutants of Escherichia coli sensitive to methylene blue and acridines

Enteric bacteria, and Escherichia coli in particular, can grow in the presence of comparatively high concentrations of methylene blue. For example, EMB sugar medium which is commonly used for test of sugar fermentation contains 65 jug. of methylene blue per millilitre. Mutants oi Escherichia coli K12 have been isolated which are sensitive to this dye and inhibited by low concentrations. These mutants were found to be sensitive also to acridine dyes, such as acridine orange. Genetic studies of this mutation were carried out using crossing experiments, F-duction and transduction with phage Pike. Part of this work was reported previously (Sugino, 1963).