Morris J A, McIvor R S
Institute of Human Genetics, University of Minnesota, Minneapolis 55455.
Biochem Pharmacol. 1994 Mar 29;47(7):1207-20. doi: 10.1016/0006-2952(94)90393-x.
Naturally occurring amino acid substitutions conferring resistance to methotrexate (MTX) have been reported previously at codon positions 22 (leu-->arg, phe) and 31 (phe-->ser, trp) of mammalian dihydrofolate reductases (DHFR). To explore the character of other substitutions, a polymerase chain reaction (PCR)-assisted saturation mutagenesis protocol was devised to introduce all possible codon sequences at positions 22 and 31 of the murine DHFR coding sequence in an expressible simian virus 40 (SV40)-regulated transcription unit. Nucleotide sequencing confirmed the presence of all four nucleotides at each of the three codon positions in the mutagenized material. Transfection of these "codon libraries" into DHFR-deficient Chinese hamster ovary cells resulted in an increased frequency of MTX-resistant colony formation in comparison with wild-type DHFR transfected cells. DHFR variants contained in different clones were characterized by PCR amplification and DNA sequencing, identifying six different amino acid substitutions at position 22 and seven substitutions at position 31. DHFR variants were extracted for determination of MTX inhibition character and catalytic activity, normalizing for the amount of DHFR protein by western blot analysis. A wide range of MTX sensitivities and catalytic activities were observed which is consistent with the role of these side chains in DHFR catalytic function. We observed that codon 22 variants were generally more resistant to MTX, but codon 31 variants retained substantially more catalytic activity (about 2.5-fold) at a given level of MTX resistance. This heterogeneity in catalytic and inhibition character has important implications for the function of different DHFR variants as mediators of drug resistance.
先前已有报道，哺乳动物二氢叶酸还原酶（DHFR）的第22位密码子（亮氨酸→精氨酸、苯丙氨酸）和第31位密码子（苯丙氨酸→丝氨酸、色氨酸）发生天然存在的氨基酸替换可赋予对甲氨蝶呤（MTX）的抗性。为了探究其他替换的特性，设计了一种聚合酶链反应（PCR）辅助的饱和诱变方案，以在可表达的猿猴病毒40（SV40）调控的转录单元中，在小鼠DHFR编码序列的第22位和第31位引入所有可能的密码子序列。核苷酸测序证实了诱变材料中三个密码子位置的每一个都存在所有四种核苷酸。将这些“密码子文库”转染到缺乏DHFR的中国仓鼠卵巢细胞中，与转染野生型DHFR的细胞相比，MTX抗性集落形成的频率增加。通过PCR扩增和DNA测序对不同克隆中包含的DHFR变体进行表征，确定了第22位有六种不同的氨基酸替换，第31位有七种替换。提取DHFR变体以测定MTX抑制特性和催化活性，并通过蛋白质印迹分析对DHFR蛋白的量进行标准化。观察到广泛的MTX敏感性和催化活性，这与这些侧链在DHFR催化功能中的作用一致。我们观察到，第22位密码子变体通常对MTX更具抗性，但在给定的MTX抗性水平下，第31位密码子变体保留了更多的催化活性（约2.5倍）。这种催化和抑制特性的异质性对于不同DHFR变体作为耐药性介质的功能具有重要意义。
