A ca. 10-kilobase (kb) HindHII fragment of plasmid DNA from Bacillus thuringiensis subsp. israelensis was cloned into plasmid pUC9 and transformed into Escherichia coli. Extracts of the recombinant strain contained a 27-kilodalton (kDa) peptide that reacted with antibodies to a 27-kDa peptide isolated from crystals produced by B. thuringiensis subsp. israelensis. Extracts of the recombinant strain were hemolytic and toxic to Aedes aegypti larvae. Full expression of the 27-kDa peptide required the presence of a ca. 0.8-kb region of DNA located 4 kb upstream from the structural gene; the 0.8-kb region could be present in cis or trans relative to the gene and apparently acted post-transcriptionally. Analysis of maxicelis showed that the 10-kb insert also coded for peptides of 67, 20, and 16 kDa; data obtained with different subclones suggest that the 20-kDa peptide is encoded in the 0.8-kb DNA region.During sporulation, Bacillus thuringiensis subsp. israelensis (hereafter referred to as Bti) produces irregular, multicomponent parasporal crystals (6,15,29). The crystals are lethal to larvae of mosquitoes and black flies (11); solubilized preparations of crystals lyse erythrocytes and cultured mosquito cells (26). Electrophoretic analyses of solubilized, denatured crystals show several peptides ranging in size from 27 to 130 kilodaltons (kDa) (29). The relationship of these peptides to each other is not known, and there are conflicting reports concerning the identity of the toxic and cytolytic peptides. A number of investigators (1,9,15,16,35,38) have presented evidence that toxicity alone or that toxicity and hemolytic and cytolytic activities are associated with a 27-kDa peptide or its 25-to 26-kDa derivative. A gene coding for a peptide with a deduced molecular mass of 27,340 daltons has been cloned in Escherichia coli (32, 35) and sequenced (32, 34). reported that extracts of recombinant E. coli strains containing the 27-kDa gene are toxic and hemolytic, whereas Waalwijk et al. (32) found that extracts of recombinant strains are hemolytic but not mosquitocidal. The latter investigators reported that mosquitocidal activity is associated with peptides of 130 and 230 kDa (31). Others (14,19) have attributed the mosquitocidal activity to a ca. 66-kDa peptide and the hemolytic activity to the 27-kDa peptide. Walfield et al. (33) and Thorne et al. (27) have cloned and sequenced a gene coding for a 72-kDa peptide and have reported that the apparent processed product of this gene, a peptide of 58 kDa, is highly toxic but not hemolytic. Yet other investigators (7) propose that toxicity resides in a 31-to 35-kDa peptide or that toxicity involves the synergistic action of two peptides (15, 37).The present communication reports studies made with a recombinant E. coli strain containing the genes coding for the 27-kDa crystal protein peptide of Bti. Extracts of the recombinant strain are toxic to mosquito larvae and are hemolytic in vitro. We also report the unexpected observation that the presence of the 27-kDa peptide, bu...
Cells cope with radiation damage through several mechanisms: (1) increased DNA repair activity, (2) scavenging and inactivation of radiation-induced radical molecules, and (3) entry into a G0-like quiescent state. We have investigated a chromosomal rearrangement to elucidate further the molecular and genetic mechanisms underlying these phenomena. A mutant of Escherichia coli JM83 (phi 80dlacZ delta M15) was isolated that demonstrated significantly increased resistance to both ionizing and ultraviolet radiation. Surviving fractions of mutant and wild-type cells were measured following exposure to standardized doses of radiation. Increased radioresistance was directly related to a chromosomal alteration near the bacteriophage phi 80 attachment site (attB), as initially detected by the LacZ- phenotype of the isolate. Southern hybridization of chromosomal DNA from the mutant and wild-type E. coli JM83 strains indicated that a deletion had occurred. We propose that the deletion near the attB locus produces the radioresistant phenotype of the E. coli JM83 LacZ- mutant, perhaps through the alteration or inactivation of a gene or its controlling element(s).
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