Smith G P, Ellar D J, Keeler S J, Seip C E
Department of Biochemistry, University of Cambridge, United Kingdom.
Plasmid. 1994 Jul;32(1):10-8. doi: 10.1006/plas.1994.1039.
A 5.8-kb DNA fragment encoding the cryIC gene from Bacillus thuringiensis (Bt) subsp. aizawai HD229 was subcloned into the pMex7 vector for expression in Escherichia coli. In addition to the 135-kDa CryIC delta-endotoxin, this DNA fragment also encoded a 30-kDa polypeptide whose open reading frame (orfX) was located less than 200 bp upstream of cryIC. Nucleotide sequencing showed that orfX was truncated at the 5' end, and full sequence was obtained from a second overlapping clone. Sequence analysis showed that orfX could encode a polypeptide closely related to the putative transposase from IS150. OrfX was flanked by a 17-bp imperfect inverted repeat, defining the length of the element as 998 bp. Southern blot analysis revealed that the novel insertion sequence was present in a single copy and located in an identical position immediately upstream of cryIC in plasmid DNA from both Bt subsp. aizawai and entomocidus.
将来自苏云金芽孢杆菌（Bt）日本亚种HD229的编码cryIC基因的5.8 kb DNA片段亚克隆到pMex7载体中，以便在大肠杆菌中表达。除了135 kDa的CryICδ-内毒素外，该DNA片段还编码一种30 kDa的多肽，其开放阅读框（orfX）位于cryIC上游不到200 bp处。核苷酸测序表明，orfX在5'端被截断，通过第二个重叠克隆获得了完整序列。序列分析表明，orfX可以编码一种与来自IS150的假定转座酶密切相关的多肽。OrfX两侧是一个17 bp的不完全反向重复序列，确定该元件的长度为998 bp。Southern杂交分析表明，该新型插入序列以单拷贝形式存在，并且在来自Bt日本亚种和杀昆虫亚种的质粒DNA中位于cryIC紧上游的相同位置。
