thuringiensis HD-1), hybridization experiments were performed using fragments of the DAPT in vitro three most abundant IS elements (IS231C, IS232A and ISBth166) of different families as probes (Fig. 1). There are no sites for Bst1107I and EcoRI within these elements. The result showed that
the most multitudinous IS231C had slightly different hybridization profiles between these two isolates, which indicated that it did not cause large-scale rearrangements. The observed variations in band patterns for IS232A and ISBth166 may result from homologous recombination between copies of these elements on chromosome or plasmids or may just be a result of their transposition to different locations. The increased number of identical copies within the YBT-1520 genome, numerous insertion events within functional genes and the identical positioning in different isolates suggest the recent expansion of IS231C. To explore the mobility of these three IS elements, we examined the changes in their Southern blot pattern during 30 repeated passages (bacterial generations) of YBT-1520 in vitro, which showed completely identical patterns, suggesting that these elements are relatively stable (data not shown). As two molecular markers of Btk were found in ISBth166 and PCR experiments indicated its existence in non-kurstaki
strains, the distribution of ISBth166 among B. thuringiensis NU7441 clinical trial serovars was further examined by Southern 17-DMAG (Alvespimycin) HCl blot analysis (Fig. 2). The restriction enzyme EcoRI was selected for B. thuringiensi genomic DNA digestions because
of the clear banding patterns. The resulting fragments were separated by agarose gel electrophoresis and probed with ISBth166 (see Materials and methods). The hybridization patterns showed that the five kurstaki strains hybridized strongly with the ISBth166 probe, while the other strains showed weak or null hybridization, suggesting that ISBth166 is widely distributed among kurstaki strains. The weak hybridization signal among some non-kurstaki strains may indicate the presence of ISs distantly related to ISBth166 or may reflect a lower copy number of the target sequence. The hybridization profiles of kurstaki strains showed a slight variation in the number and position of bands, while the strong hybridization signals may indicate the plasmid-borne elements that had more than one copy. Strains HD-231 and HD-232 showed the same hybridization patterns. The hybridization pattern of YBT-1520 was most similar to that of HD-263, with only one additional band. This result yielded useful indications for further experimental work to reveal the possible correlation between Lepidopteran larvae toxicity and the presence of ISBth166-related elements and the evolutionary relationships among the ISBth166 variants. In this study, 68 intact copies of 14 distinct IS elements were identified in the B.