Shotkoski F A, Fallon A M
Department of Entomology, University of Minnesota, St Paul 55108, USA.
Insect Mol Biol. 1993;2(3):155-61. doi: 10.1111/j.1365-2583.1993.tb00135.x.
The dihydrofolate reductase amplicon in methotrexate-resistant mosquito cells provides an amplified gene in insects that can be compared directly to the corresponding amplified locus in mammalian cells. A cloned Aedes albopictus dihydrofolate reductase gene was used as a probe to examine the structure of dihydrofolate reductase alleles in sensitive and resistant cells. In wild type cells, two distinct alleles could be distinguished by restriction fragment length polymorphisms, one of which was amplified in methotrexate-resistant cells. Subsequent to amplification, an additional polymorphism at a ten base-pair XmnI recognition site indicated that the amplified mosquito gene is subject to genetic changes similar to those that have been described in amplified genes from mammalian cells. Pulsed-field gel electrophoresis was used to determine that the minimum size of the mosquito dihydrofolate reductase amplicon was 140 kilobases; ethidium bromide staining patterns suggested a size of at least 233 kilobases. Dihydrofolate reductase probes hybridized to distinct locations in five of the thirteen chromosomes in Mtx-5011-128 cells, indicating that the amplified DNA probably occurs as tandem direct or inverted repeats.
耐甲氨蝶呤蚊虫细胞中的二氢叶酸还原酶扩增子在昆虫中提供了一个可与哺乳动物细胞中相应扩增位点直接比较的扩增基因。一个克隆的白纹伊蚊二氢叶酸还原酶基因被用作探针，以检测敏感细胞和耐药细胞中二氢叶酸还原酶等位基因的结构。在野生型细胞中，通过限制性片段长度多态性可区分出两个不同的等位基因，其中一个在耐甲氨蝶呤细胞中被扩增。扩增后，在一个十个碱基对的XmnI识别位点出现了另一种多态性，表明扩增的蚊虫基因经历了与哺乳动物细胞扩增基因中所描述的类似的遗传变化。脉冲场凝胶电泳用于确定蚊虫二氢叶酸还原酶扩增子的最小大小为140千碱基；溴化乙锭染色模式表明其大小至少为233千碱基。二氢叶酸还原酶探针与Mtx-5011-128细胞13条染色体中的5条染色体上的不同位置杂交，表明扩增的DNA可能以串联直接重复或反向重复的形式出现。
