Dicker A P, Volkenandt M, Schweitzer B I, Banerjee D, Bertino J R
Gornell University Graduate School of Medical Sciences, New York, New York.
J Biol Chem. 1990 May 15;265(14):8317-21.
A methotrexate-resistant Chinese hamster ovary cell line (Pro-3 MtxRIII), resistant due to a low-level amplified, altered target enzyme, dihydrofolate reductase (DHFR), has been characterized on the molecular level. The cDNA and coding regions of all six DHFR exons were amplified in vitro using Taq polymerase and directly sequenced. Analysis of the Pro-3 MtxRIII DHFR cDNA demonstrated a C----T base transition at nucleotide 67 that results in the substitution of phenylalanine for leucine at residue 22 and the loss of a BsaI site in the Pro-3 MtxRIII cDNA. This mutation results in a decreased binding of methotrexate to the altered enzyme. Molecular modeling of Leu22----Phe supports the concept of the importance of Leu22 in the active site of the enzyme and indicates that replacement with phenylalanine will decrease the binding of methotrexate.
一种对甲氨蝶呤耐药的中国仓鼠卵巢细胞系（Pro-3 MtxRIII），其耐药是由于靶酶二氢叶酸还原酶（DHFR）低水平扩增且发生改变所致，已在分子水平上进行了表征。使用Taq聚合酶在体外扩增了所有六个DHFR外显子的cDNA和编码区，并直接进行了测序。对Pro-3 MtxRIII DHFR cDNA的分析表明，在核苷酸67处发生了C→T碱基转换，导致第22位残基处的亮氨酸被苯丙氨酸取代，并且Pro-3 MtxRIII cDNA中一个BsaI位点缺失。这种突变导致甲氨蝶呤与改变后的酶的结合减少。Leu22→Phe的分子建模支持了Leu22在酶活性位点中的重要性这一概念，并表明用苯丙氨酸取代会降低甲氨蝶呤的结合。
