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A strain of Bacillus thuringiensis (Bt) subsp. galleriae highly toxic to the cupreous chafer, Anomala cuprea, was isolated from a soil sample collected in Japan. The strain, SDS-502, produces a crystal consisting of a 130-kDa protein. The gene encoding the protein was cloned in Escherichia coli using antiserum directed to the protein. The gene was expressed in E. coli, producing a 130-kDa protein toxic to A. cuprea. Sequencing of the cloned gene indicated a typical Bt cry gene with substantial homology to cry8 genes. Based on the peptide sequence comparison, the gene found in SDS-502 was designated as cry8Da (AB089299). The cry8Da gene was highly expressed in SDS-502 in a laboratory scale bioreactor. The fermentation product of SDS-502 was formulated for soil application and tested in a peanut field for chafer control along with a chemical insecticide reference, a fenthion organophosphate granular formulation. Judging from the amount of undamaged nuts harvested from plots of different treatments, plots treated with SDS-502 had significantly better insect control than the untreated plots. The chemical insecticide plots showed no significant difference in nut damage from the Bt-treated or control plots. [Copyright &y& Elsevier]

Highlights: [•] The Cry8Da binding protein was purified by column chromatography. [•] Three internal amino acid sequences of the Cry8Da binding protein were determined. [•] The internal amino acid sequences shared homology with Coleopteran β-glucosidases. [•] The fraction containing the 150kDa Cry8Da binding protein had β-glucosidase activity. [•] Partial β-glucosidase genes were amplified by PCR using degenerate primers. [ABSTRACT FROM AUTHOR]

Abstract: Most δ-endotoxins produced by Bacillus thuringiensis require proteolytic processing in order to become active. The in vitro and in vivo activation processes of Cry39A, a δ-endotoxin that is highly toxic to Anopheles stephensi, were investigated. Cry39A with a molecular mass of 72kDa was processed in vitro into a 60kDa fragment by trypsin and gut extract from A. stephensi larvae. N-terminal amino acid sequencing of the 60kDa fragment revealed that trypsin and the protease(s) in the gut extract cleaved Cry39A between Arg61 and Gly62. In contrast, 40 and 25kDa polypeptides were generated in vivo by intramolecular cleavage of the 60kDa fragment in A. stephensi larvae. Further, a co-precipitation assay was used to investigate the binding property of the activated Cry39A to A. stephensi BBMV. Cry39A bound to A. stephensi BBMV specifically and did not compete with the Cry4Aa toxin. This indicated that the binding molecule(s) for Cry39A might differ from those for Cry4A. In addition, Cry39A preferentially bound to the Triton X-100-insoluble membrane fraction. [Copyright &y& Elsevier]

The Bombyx mori nucleopolyhedrovirus (BmNPV) ie - 1 promoter exhibits strong transcriptional activity and is used in transient foreign gene expression systems in insect cells. In a reporter assay experiment using the BmNPV ie - 1 promoter, we found that it exhibited activity even in non-host mammalian BHK cells, plant BY-2 cells, and also bacterial Escherichia coli cells. An analysis using a deletion series of the BmNPV ie - 1 promoter demonstrated that the core promoter region of this promoter was sufficient to display promoter activity in BHK cells, BY-2 cells, and E . coli cells, whereas upstream elements were required for higher activity in insect cells. Furthermore, we found that the BmNPV ie - 1 promoter exhibited sufficient activity for a β-galactosidase assay in E . coli cells. The results obtained here suggest that the BmNPV ie - 1 promoter has potential as a universal promoter for transient expression systems in insect, mammalian, plant, and bacterial cells. [ABSTRACT FROM AUTHOR]

Highlights: [•] The baculovirus vector infiltrates the cells of economic important fishes. [•] Drosophila Mos1 transposase expressed in fish cells maintains its ability to localize to the nucleus. [•] The baculoviral vector carrying Mos1 is a useful tool to stably transform fish cells. [ABSTRACT FROM AUTHOR]

Abstract: Bacillus thuringiensis (Bt) Cry8D insecticidal proteins are unique among Cry8 family proteins in terms of its insecticidal activity against adult Scarab beetles, such as Japanese beetle (Popillia japonica Newman). From the sequence homology with other Bt Cry proteins especially those active against beetles, such as Cry3Aa whose 3D structure is available, the structure of the Cry8D protein has been predicted to be a typical three-domain Cry protein type. In addition, the activation process of Cry8D in gut juice of susceptible insects is presumed to be similar to that of Cry3A (). In this study, the activation process of Cry8Da in insect gut juice was closely examined. Japanese beetle gut juice proteases digested the 130kDa Cry8Da protein to produce a 64kDa protein. This 64kDa protein was active against both adult and larval Japanese beetle and considered to be an activated toxin. N-terminal sequencing of this 64kDa protein revealed that the Cry8Da leader sequence consisting of 63 amino acid residues from M1 to F63 was removed. As in the case of Cry3Aa, the proteases further digested the 64kDa protein to two 8kDa and 54kDa fragments. N-terminal amino acid analysis of these smaller fragments indicated that the proteases digested the loop between Alpha Helix (Alpha for short) 3 and Alpha 4. This means that the 8kDa fragment consists of Alpha 1–3 of Domain I and that the 54kDa fragment contains the remaining Domain I and full Domain II and Domain III. Size exclusion chromatography and anion exchange chromatography could not separate these 64, 54 and 8kDa proteins suggesting that the 54kDa and 8kDa fragments are still forming the toxin complex equivalent to the 64kDa protein by size and ionic charge. The sequencing and chromatography results suggest that the gut juice proteases merely nicked the loop between Alpha 3 and Alpha 4. This nicking process appeared to be essential for receptor binding of the Cry8Da toxin. BBMV binding assay revealed that the Cry8Da toxin bound to BBMV preparations from both adult and larval Japanese beetle only after the loop was nicked. Only the 54kDa fragment bound to the BBMV preparations but not the 64kDa protein. Ligand blot showed that the protease activated Cry8Da toxin, presumably the 54kDa fragment, bound to specific BBMV proteins, one or more of those would be receptor(s). The sizes and binding affinities of these Cry8Da-bound proteins of Japanese beetle BBMV differed between larvae and adults. [Copyright &y& Elsevier]

Abstract: A novel cry gene, cry8Db, highly toxic to scarab beetles such as the Japanese beetle, Popillia japonica Newman, was cloned from an isolate of Bacillus thuringiensis (Bt), BBT2-5. The cry8Db gene has 3525bp nucleotides and codes for a protein of 1174 amino acid residues. The protein, Cry8Db, has typical Bt characteristics such as the 8-block, conserved sequences and the three-domain 3D toxin structure as defined with Cry3Aa. When the amino acid sequence of Cry8Db was compared with that of Cry8Da whose gene was cloned and characterized in our laboratory earlier, substantial sequence diversities were found in their Domain III. The cry8Db gene was expressed in an acrystalliferous B. thuringiensis strain, BT51. BT51 expressing cry8Db formed a spherical crystal like the natural crystal of BBT2-5. The Cry8Db protein was assayed along with the other scarab active Cry8Da and Cry8Ca against the Japanese beetle. While Cry8Da and Cry8Db had toxicity against both adults and larvae of the Japanese beetle, Cry8Ca was toxic to only larvae. Cry8Ca showed no toxicity against the adult beetle up to 30μg per 1cm2 of leaf discs on which the protein was applied. The activation process of Cry8Db by adult and larval gut juice was compared in vitro with the processes of Cry8Da and Cry8Ca. All three proteins, Cry8Db, Cry8Da and Cry8Ca, produced a toxic core of approximately 70kDa equally indicating that the activation process does not inactivate the adult activity of Cry8Ca. We concluded that the adult activity of Cry8D proteins is encoded in Domain II. Further tests against other beetle species showed a significant difference between Cry8D’s and Cry8Ca but no difference between Cry8Da and Cry8Db. Comparison of 3D structural models of Cry8Ca, Cry8Da and Cry8Db, which were constructed by using Cry3Bb as the structural template, indicated significant structural differences, especially between Cry8Ca and Cry8D proteins, in three major surface-exposed loops of Domain II that may be involved in determining the adult beetle activity. [Copyright &y& Elsevier]