1887

Abstract

Summary

Insecticidal crystal proteins, or protoxins, of are composed of two domains, an amino-terminal half essential for toxicity, and a carboxy-terminal half with an as yet unassigned function. To define the boundary of the two domains, sequential termination codons were introduced from the 3′-end of the DNA sequence encoding the toxic domain of the 1155-residue gene product. The mutated and the intact genes were placed under the control of the inducible promoter PrecA, and toxicity of the cell extracts was determined using silkworm larvae. Under non-induced conditions, in which the gene products accumulated to a limited degree, mutations encoding 606 amino acid residues or more were toxic, whereas those encoding 605 residues or less were non-toxic. Comparison of the toxicities and the levels of the toxic proteins suggested that the mutant proteins had comparable activity to that of the intact protoxin. Furthermore, the non-toxic protein seemed to be unstable inthe extracts. To investigate the roles of the non-toxic domain, the mutant proteins were overproduced in both and . The intact and the mutated genes carrying natural promoters were introduced into acrystalliferous . Upon induction of PrecA in , and upon sporulation in , there was a large accumulation of gene products which formed inclusion bodies. The inclusion bodies of the intact protoxin were active, whereas those of the mutant proteins were inactive. Inclusion bodies of the intact protein could be solubilized in alkali, whereas the mutant inclusion bodies were insoluble. Since solubilization under alkaline conditions in the insect midgut is considered to be the first step of toxic action, the non-toxic domain is required to direct the synthesis of inclusion bodies as an active soluble form.

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1995-03-01
2021-10-21
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