Ranson H, Cornel A J, Fournier D, Vaughan A, Collins F H, Hemingway J
Department of Pure and Applied Biology, University of Wales, Cardiff CF1 3TL, Wales, United Kingdom.
J Biol Chem. 1997 Feb 28;272(9):5464-8. doi: 10.1074/jbc.272.9.5464.
1,1,1-Trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) resistance in both adults and larvae of Anopheles gambiae is mediated by stage-specific glutathione S-transferases (GSTs). On the basis of their biochemical characteristics the larval resistance-associated GSTs are likely to be insect class I GSTs. Aggst1-2, a class I GST gene, which is expressed in larvae, has been cloned from the malaria vector A. gambiae. The gene was inserted into a bacterial expression system, and the detection of 1-chloro-2,4-dinitrobenzene (CDNB) conjugating activity in Eschericia coli expressing the recombinant enzyme confirmed that aggst1-2 encodes a catalytically active GST. The gene encodes a 209 amino acid protein with 46% sequence similarity to a Drosophila melanogaster class I GST (GST-D1), 44% similarity with a Musca domestica class I GST (MdGST-1), but only low levels of homology with class II insect GSTs, including the adult specific AgGST2-1 from A. gambiae. Southern analysis of genomic DNA indicated that A. gambiae has multiple class I GSTs. In situ hybridization of class I genomic and cDNA clones to polytene chromosomes identified a single region of complementarity on chromosome 2R division 18B, suggesting that these class I GSTs in A. gambiae are arranged sequentially in the genome. Three positive overlapping recombinant clones were identified from an A. gambiae genomic library. Mapping and partial sequencing of these clones suggests that there are several GSTs and truncated GST pseudogenes within the 30kb of DNA that these clones span.
冈比亚按蚊成虫和幼虫对1,1,1-三氯-2,2-双（对氯苯基）乙烷（滴滴涕）的抗性是由阶段特异性谷胱甘肽S-转移酶（GSTs）介导的。根据其生化特性，幼虫抗性相关的GSTs可能属于昆虫I类GSTs。已从疟疾媒介冈比亚按蚊中克隆出一个在幼虫中表达的I类GST基因Aggst1-2。该基因被插入细菌表达系统，在表达重组酶的大肠杆菌中检测到1-氯-2,4-二硝基苯（CDNB）结合活性，证实Aggst1-2编码一种具有催化活性的GST。该基因编码一个209个氨基酸的蛋白质，与黑腹果蝇I类GST（GST-D1）的序列相似性为46%，与家蝇I类GST（MdGST-1）的相似性为44%，但与II类昆虫GSTs的同源性较低，包括冈比亚按蚊成虫特异性的AgGST2-1。基因组DNA的Southern分析表明冈比亚按蚊有多个I类GSTs。I类基因组和cDNA克隆与多线染色体的原位杂交在2R分区18B上确定了一个单一的互补区域，表明冈比亚按蚊中的这些I类GSTs在基因组中是顺序排列的。从冈比亚按蚊基因组文库中鉴定出三个阳性重叠重组克隆。这些克隆的定位和部分测序表明，在这些克隆跨越的30kb DNA内有几个GSTs和截短的GST假基因。
