Bacillus thuringiensis is characterized by the production of a large, intracellular inclusion during sporulation. Each of these inclusions is comprised of one or a few classes of large polypeptides (protoxins) that are toxic for specific species of insect larvae. There is a spectrum of these toxins produced by subspecies of B. thuringiensis each with a unique pattern of toxicity for certain insect larvae. Included among these are many that cause major damage to crops and trees (Lepidoptera) as well as others that serve as carriers of human diseases, i.e. mosquitoes and flies. In some subspecies, more than one inclusion is formed and each contains a unique toxin. These polypeptides are produced during a portion of the sporulation process and comprise a substantial part of the protein synthetic activity during the period of formation. Several of these protoxin genes have been cloned from B. thuringiensis plasmids into B. subtilis E. coli shuttle vector and express sufficiently well in both organisms to permit determination of lethal doses for select neonate larvae. The basis for toxin specificity will be determined by comparing relative LC50's for parental B. thuringiensis strains, cloned protoxin genes (alone or in combination) and of B. cereus transcipients containing selected arrays of B. thuringiensis plasmids. B. thuringiensis subspecies and cloned genes will be chosen to provide a comparison of those with overlapping as well as non-overlapping specificities. Various regions of the cloned protoxin genes will be subcloned to examine toxicity levels as well as alterations in specificity. The regulation of protoxin synthesis in at least one subspecies is dependent on the activities of two cryptic plasmids. One plasmid affects the temperature of protoxin synthesis, the other the amount produced. Bioassay systems will be developed employing cloned protoxin genes plus cloned fragments of these regulatory plasmids in order to define the portion of each plasmid involved in regulation, whether there is a gene product and whether the regulatory function is at the level of transcription or elsewhere.
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