A pair of adjacent genes, cry5Ad and orf2-5Ad, encode the typical N- and C-terminal regions of a Cry5Adelta-endotoxin as two separate proteins in Bacillus thuringiensis strain L366.
ABSTRACT A new DNA sequence cry5Ad/orf2-5Ad (GenBank accession number EF219060) was isolated from Bacillus thuringiensis strain L366. This DNA sequence contains two ORFs: cry5Ad (a previously unreported member of the cry5A gene family) and orf2-5Ad. cry5Ad is unique among cry5A genes in that it encodes only the N-terminal region of a typical Cry5Adelta-endotoxin. The cry5Ad sequence includes homology blocks 1-5, which are present in most B. thuringiensisdelta-endotoxins. The usual C-terminal region of a Cry5Adelta-endotoxin (including homology blocks 6-8) is encoded by orf2-5Ad. Both proteins encoded by cry5Ad and orf2-5Ad were found in IPTG-induced Escherichia coli, after a copy of cry5Ad/orf2-5Ad was cloned into the pQE32 expression vector and transformed into pREP4 E. coli cells. Both proteins were also found in parasporal crystal inclusions of B. thuringiensis L366. Sequencing of cDNA derived from transformed E. coli cells showed that the two ORFs are transcribed as a single mRNA. Extracts prepared from the recombinant E. coli expressing Cry5Ad and Orf2-5Ad were not toxic to nematode larvae (Haemonchus contortus), indicating that these two proteins are most likely not responsible for the nematocidal activity seen previously in the B. thuringiensis strain L366.
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ABSTRACT: Two novel crystal protein genes, cry30Ba and cry44Aa, were cloned from Bacillus thuringiensis subsp. entomocidus INA288 and expressed in an acrystalliferous strain. Cry44Aa crystals were highly toxic to second-instar Culex pipiens pallens (50% mortality concentration [LC50] = 6 ng/ml) and Aedes aegypti (LC50 = 12 ng/ml); however, Cry30Ba crystals were not toxic.Applied and Environmental Microbiology 09/2006; 72(8):5673-6. · 3.68 Impact Factor
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ABSTRACT: Avermectin (AVM) inhibition of the development of the free-living stages of Haemonchus contortus has been quantified in an assay in which nematode eggs are placed on an agar matrix containing serial dilutions of a drug in the wells of a microtitre plate. Development is allowed to proceed for 6 days by which time larvae in control wells (no drug) have reached the infective, third (L3) stage. At high concentrations (> 30 nM) ivermectin (IVM) paralyses L1 larvae soon after hatching, however, much lower concentrations (approximately 1 nM) are sufficient to inhibit development to the L3 stage which suggests that effects of the drug other than those relating to gross motor activity are responsible for the latter effect. The larval stages of IVM-susceptible H. contortus isolates from both Australia and South Africa, including isolates known to be resistant to levamisole or rafoxanide and/or the benzimidazoles, were equally sensitive to inhibition by AVMs. In contrast, 6 isolates of H. contortus resistant to IVM in vivo showed a reduced sensitivity to AVM inhibition of development. The order of potency of a limited range of AVMs as inhibitors of larval development was consistent with in vivo efficacy. Resistance ratios for IVM-resistant isolates were dependent on AVM structure, with AVM B2 the most sensitive probe for IVM resistance in the isolates tested.International Journal for Parasitology 04/1995; 25(4):463-70. · 3.64 Impact Factor
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ABSTRACT: A study was carried out in order to compare two techniques for culture of strongyle larvae: the usual fecal culture and a new one where eggs were extracted from feces and larvae were grown in a more defined medium (agar plate fortified with Earle's medium and yeast extract). The efficiency of "on-agar" cultures was better than that of the fecal cultures with a difference of 26% for Trichostrongylus colubriformis and 32% for Ostertagia circumcincta. The variability observed between the number of larvae collected from each culture was low (8% on average) in comparison with that of fecal cultures (15% on average) thus demonstrating a better reproducibility. On the other hand growth of the larvae was similar to that obtained in fecal medium. This new technique avoids the difficulty of controlling the conditions found in fecal cultures (particularly moisture) and could become a more accurate method for diagnosis than the conventional methods. A morphometric study of infective larvae derived from the two methods of culture was carried out. Despite the more defined conditions of development in the "on-agar" cultures, it was not possible to obtain sufficiently homogeneous populations with regard to their measurements so as to distinguish larvae merely with the help of these criteria.Canadian journal of comparative medicine. Revue canadienne de medecine comparee 02/1984; 48(1):63-71.