Muralidhar G, Utz E D, Elliott M S, Katze J R, Trewyn R W
Department of Physiological Chemistry, Ohio State University, Columbus 43210.
Anal Biochem. 1988 Jun;171(2):346-51. doi: 10.1016/0003-2697(88)90496-4.
Altered queuine modification of tRNA has been associated with cellular development, differentiation, and neoplastic transformation. Present methods of evaluating agents for their ability to induce queuine hypomodification of tRNA are tedious, time-consuming, and not readily amenable to examining cell-type or tissue specificity. Therefore, a rapid, small-scale assay was developed to identify agents that alter queuine modification of tRNA in cultured cells. Monolayer cultures (2cm2) of Chinese hamster embryo cells depleted of queuine for 24 h were evaluated for their ability to incorporate [3H]dihydroqueuine into acid precipitable material (tRNA) in the presence and absence of potential inhibitors. Known inhibitors of the queuine modification enzyme tRNA-guanine ribosyltransferase (e.g., 7-methylguanine, 6-thio-guanine, and 8-azaguanine) were very effective in blocking incorporation of the radiolabel, and the dose-dependent results exhibited small standard deviations in independent experiments. The data indicate that the method is rapid, reliable, and potentially useful with a variety of cell types.
tRNA中喹啉修饰的改变与细胞发育、分化和肿瘤转化有关。目前评估试剂诱导tRNA喹啉低修饰能力的方法繁琐、耗时，且不易用于检测细胞类型或组织特异性。因此，开发了一种快速、小规模的检测方法来鉴定改变培养细胞中tRNA喹啉修饰的试剂。对缺乏喹啉24小时的中国仓鼠胚胎细胞单层培养物（2cm2），在有和没有潜在抑制剂的情况下，评估其将[3H]二氢喹啉掺入酸沉淀物质（tRNA）的能力。喹啉修饰酶tRNA-鸟嘌呤核糖基转移酶的已知抑制剂（如7-甲基鸟嘌呤、6-硫代鸟嘌呤和8-氮杂鸟嘌呤）在阻断放射性标记掺入方面非常有效，并且剂量依赖性结果在独立实验中显示出小的标准差。数据表明该方法快速、可靠，并且可能适用于多种细胞类型。
