Wang Z H, Fallon A M
Department of Entomology, University of Minnesota, St Paul 55108, USA.
Insect Mol Biol. 1998 Nov;7(4):317-25. doi: 10.1046/j.1365-2583.1998.740317.x.
When maintained under continuous selection with the folate inhibitor, methotrexate, cultured Aedes albopicfus mosquito cells amplify an 200 kb region of DNA containing the dihydrofolate reductase gene. To determine whether the amplicon contained additional coding regions, Southern blots of cosmid clones containing amplicon DNA were probed separately with reverse-transcribed mRNA from methotrexate-sensitive and methotrexate-resistant cells. Cosmid pWED118 contained five EcoRI fragments (A, B, C, F, G) ranging in size from 2 to 5 kb that hybridized with cDNA from resistant cells. Of these, fragments B and F also hybridized to probe representing mRNA from sensitive cells, and all but fragment G hybridized to repetitive DNA from wild-type cells. Fragment G, which appeared to encode a low copy number gene in wild-type cells that subsequently became part of the dihydrofolate reductase amplicon in methotrexate-resistant cells, hybridized strongly to a 7 kb band and more weakly to bands measuring 9 and 3 kb on Northern blots containing RNA from resistant cells. Fragment G contained a 1203 bp open reading frame, encoding 401 amino acids homologous to synaptic vesicle protein SV2, a member of a transmembrane transporter family expressed in neural and endocrine cells. The region of homology included the six N-terminal transmembrane domains, an internal cytoplasmic loop, a seventh transmembrane domain, and most of an intravesicular loop. This partial sequence, which appears to correspond to a truncated gene generated during formation of the dihydrofolate reductase amplicon, provides a useful basis for more extensive characterization of an important gene family that may be the target of novel insecticides.
在叶酸抑制剂甲氨蝶呤的持续选择压力下培养时，埃及伊蚊细胞会扩增一个包含二氢叶酸还原酶基因的200 kb DNA区域。为了确定扩增子是否包含其他编码区域，分别用来自对甲氨蝶呤敏感和耐药细胞的逆转录mRNA探测含有扩增子DNA的黏粒克隆的Southern杂交印迹。黏粒pWED118含有5个大小在2至5 kb之间的EcoRI片段（A、B、C、F、G），它们与耐药细胞的cDNA杂交。其中，片段B和F也与代表敏感细胞mRNA的探针杂交，除片段G外，所有片段都与野生型细胞的重复DNA杂交。片段G在野生型细胞中似乎编码一个低拷贝数基因，该基因在对甲氨蝶呤耐药的细胞中随后成为二氢叶酸还原酶扩增子的一部分，它在含有耐药细胞RNA的Northern杂交印迹上与一条7 kb的条带强烈杂交，与9 kb和3 kb的条带杂交较弱。片段G包含一个1203 bp的开放阅读框，编码401个氨基酸，与突触小泡蛋白SV2同源，SV2是在神经和内分泌细胞中表达的跨膜转运蛋白家族的成员。同源区域包括六个N端跨膜结构域、一个内部细胞质环、一个第七个跨膜结构域以及大部分囊泡内环。这个部分序列似乎对应于在二氢叶酸还原酶扩增子形成过程中产生的一个截短基因，为更广泛地表征一个可能是新型杀虫剂靶标的重要基因家族提供了有用的基础。
