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首页> 外文期刊>Nucleic Acids Research >THE VIRAL THYMIDINE KINASE GENE AS A TOOL FOR THE STUDY OF MUTAGENESIS IN TRYPANOSOMA BRUCEI
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THE VIRAL THYMIDINE KINASE GENE AS A TOOL FOR THE STUDY OF MUTAGENESIS IN TRYPANOSOMA BRUCEI

机译：病毒胸腺激酶基因作为布鲁氏菌突变研究的工具

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摘要

We have tested the use of thymidine kinase as a negative selection system for Trypanosoma brucei. To this end we have targeted a construct containing a Herpes simplex virus thymidine kinase (TK) gene into the ribosomal DNA array of procyclic T.brucei, This resulted in TK activity 30-50-fold above background and in susceptibility to the nucleoside analogues ganciclovir, ethyl-deoxyuridine and 1-[2-deoxy,2-fluoro-8-D-arabinofuranosyl]-5-iodouracil, all of which have no effect on wild-type trypanosomes, TK+ trypanosomes, however, reverted to a ganciclovir resistant phenotype at a rate of 10(-6) per cell-generation. A similar reversion rate was observed using the Varicella-zoster virus TK gene, Loss of TK activity was not due to detectable DNA rearrangements or a decrease in TK mRNA, Sequence analysis of the revertant genes demonstrated, however, the occurrence of point mutations and frameshifts, One revertant line had a mutation in the thymidine binding site leading to the substitution of a conserved arginine by a glycine, Other mutations included single base insertion, single base deletion and the introduction of a premature termination codon by point mutation.
机译：我们已经测试了胸苷激酶作为布鲁氏锥虫阴性选择系统的用途。为此，我们已将含有单纯疱疹病毒胸苷激酶（TK）基因的构建体靶向到前环布鲁氏杆菌的核糖体DNA阵列中，这导致TK活性比背景高30-50倍，并且对核苷类似物更昔洛韦敏感，乙基脱氧尿苷和1- [2-脱氧，2-氟-8-D-阿拉伯呋喃糖基] -5-碘尿嘧啶，它们都对野生型锥虫，TK +锥虫无影响，但恢复为更昔洛韦耐药表型每个细胞世代的速率为10（-6）。使用水痘带状疱疹病毒TK基因观察到相似的回复率，TK活性的降低不是由于可检测到的DNA重排或TK mRNA的下降，回复基因的序列分析证明了，但是，点突变和移码的发生，一个回复系在胸苷结合位点发生突变，导致保守的精氨酸被甘氨酸取代。其他突变包括单碱基插入，单碱基缺失和通过点突变引入过早终止密码子。

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