Open AccessBiosynthesis of the Crystal Protein of Bacillus thuringiensis var. tolworth
Author(s) -
Gould Hannah J.,
Loviny Thérèse F. L.,
Vasu SabinSorin,
Herbert Barry N.
Publication year - 1973
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1973.tb03005.x
Subject(s) - immunoprecipitation , rna , bacillus thuringiensis , messenger rna , biochemistry , protein biosynthesis , biology , microbiology and biotechnology , antiserum , biosynthesis , gel electrophoresis , gene , antigen , bacteria , genetics
In the accompanying paper, we present evidence to show that the biosynthesis of the two characteristic polypeptide constituents “A” and “B” of the crystal protein toxin of Bacillus thuringiensis var. tolworth , is restricted to a short interval, from 16–18 h, in the mid‐stationary phase of the growth cycle of 24 h in culture. Here we examine the question of the kinetics of biosynthesis of the corresponding crystal protein messenger RNA. We have taken the direct approach of assaying a series of RNAs isolated from B. thuringiensis cultures at varying times of culture for messenger activity in a heterologous Escherichia coli , subcellular system. The product of RNA‐stimulated protein synthesis in vitro was characterized by immunoprecipitation with antiserum against purified crystal protein and by polyacrylamide‐gel electrophoresis of the antigen‐antibody precipitates dissolved in the detergent sodium dodecylsulfate. RNA from B. thuringiensis , but not that of Bacillus cerens , stimulates the synthesis of protein which specifically precipitates with crystal protein antiserum. The time of extraction of RNA from B. thuringiensis does not appear to be critical in determining its messenger activity: all samples taken in the interval of 11–20 h contained messenger RNA for crystal protein by the criterion of specific immunoprecipitation. In each case a significant proportion of the synthesized protein migrated in the region of the smaller‐molecular‐weight component A (55000) of the crystal protein in the electrophoretic separation. However, hardly any synthesized protein co‐migrated with the larger‐molecular‐weight component B (120000) of this protein. Since all the labelled protein precipitated by the crystal protein antiserum was larger than about 15000 molecular weight we conclude that mainly large fragments of the crystal protein were synthesized in the E. coli system. We failed in our attempts to isolate polysomes as a source of messenger RNA from stationary‐phase cells. Thus it was not possible to determine by experiments similar to those described above whether the crystal protein messenge is stored in ribosomes or in some other fraction of the cell when it is not being translated.