Will C L, Dolnick B J
Mol Pharmacol. 1986 Jun;29(6):643-8.
Several fragments of the human dihydrofolate reductase gene (tetrahydrofolate dehydrogenase, 5,6,7,8-tetrahydrofolate NADP+ oxidoreductase, EC 1.5.1.3) were isolated from gene-amplified KB7B cells and characterized. Recombinant plasmids containing intron sequences were constructed. Probes prepared from these plasmids were tested for dihydrofolate reductase precursor mRNA specificity via solution hybridization studies and Northern blot analysis. One probe, p0.69EH, was shown to be specific for dihydrofolate reductase RNA by its greatly enhanced level of hybridization with total RNA from dihydrofolate reductase gene-amplified versus non-amplified cells. In addition, solution hybridization studies with various classes of RNA and Northern blot analysis revealed that p0.69EH hybridizes predominantly with polyadenylated, high molecular weight, nuclear RNA species. Subsequent solution hybridization studies revealed a disproportionate 5-fluorouracil-induced increase in dihydrofolate reductase intron-containing RNA over dihydrofolate reductase mRNA. These results suggest that 5-fluorouracil incorporation into RNA may inhibit the conversion of precursor mRNA to mature mRNA.
从基因扩增的KB7B细胞中分离并鉴定了人类二氢叶酸还原酶基因（四氢叶酸脱氢酶，5,6,7,8 - 四氢叶酸NADP +氧化还原酶，EC 1.5.1.3）的几个片段。构建了含有内含子序列的重组质粒。通过溶液杂交研究和Northern印迹分析，测试了从这些质粒制备的探针的二氢叶酸还原酶前体mRNA特异性。一个探针p0.69EH通过与来自二氢叶酸还原酶基因扩增细胞与未扩增细胞的总RNA杂交水平大大提高，显示出对二氢叶酸还原酶RNA具有特异性。此外，用各类RNA进行的溶液杂交研究和Northern印迹分析表明，p0.69EH主要与多聚腺苷酸化的、高分子量的核RNA种类杂交。随后的溶液杂交研究显示，5 - 氟尿嘧啶诱导的含二氢叶酸还原酶内含子的RNA相对于二氢叶酸还原酶mRNA的增加不成比例。这些结果表明，5 - 氟尿嘧啶掺入RNA可能抑制前体mRNA向成熟mRNA的转化。
