Artificial mutants generated by the insertion of random oligonucleotides into the putative nucleoside binding site of the HSV-1 thymidine kinase gene.
Artificial mutants generated by the insertion of random oligonucleotides into the putative nucleoside binding site of the HSV-1 thymidine kinase gene.
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DOI：
10.1021/bi00115a004
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发表时间：
1991-12
期刊：
影响因子：
2.9
通讯作者：
D. Dube;J. D. Parker;D. C. French;D. Cahill;S. Dube;M. Horwitz;K. Munir;L. Loeb
中科院分区：
文献类型：
--
作者：
D. Dube;J. D. Parker;D. C. French;D. Cahill;S. Dube;M. Horwitz;K. Munir;L. Loeb
通过用随机核苷酸序列替换密码子166和167，我们获得了42个HSV-1胸苷激酶（ATP：胸苷5'-磷酸转移酶，EC 2.7.1.21）的活性人工突变体。密码子 166 和 167 位于 HSV-1 tk 基因中推定的核苷结合位点内。活性突变谱表明 Ile166 和 Ala167 都不是胸苷激酶活性所绝对必需的。这些氨基酸中的每一种都可以被其他 19 种氨基酸中的一些但不是全部取代。活性突变体可根据两个基础分为高活性或低活性：(1) 大肠杆菌 KY895（缺乏胸苷激酶活性的菌株）在胸苷存在下生长；(2) 当携带随机插入的质粒时，该菌株摄取胸苷。携带高活性质粒或野生型质粒的大肠杆菌 KY895 可在低量胸苷（低于 1 微克/mL）的情况下生长，但在高量胸苷的情况下无法生长。另一方面，携带低活性质粒的大肠杆菌 KY895 可以在培养基中高浓度胸苷（大于 50 微克/mL）下生长。高活性质粒还具有增强的 [3H]dT 摄取。未分级提取物中的体外胸苷激酶活性量与低胸苷浓度下的生长或胸苷摄取速率不相关。热失活研究表明，突变酶毫无例外地比野生型酶对温度更敏感。（摘要截断为 250 字）
We have obtained 42 active artificial mutants of HSV-1 thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) by replacing codons 166 and 167 with random nucleotide sequences. Codons 166 and 167 are within the putative nucleoside binding site in the HSV-1 tk gene. The spectrum of active mutations indicates that neither Ile166 nor Ala167 is absolutely required for thymidine kinase activity. Each of these amino acids can be replaced by some but not all of the 19 other amino acids. The active mutants can be classified as high activity or low activity on two bases: (1) growth of Escherichia coli KY895 (a strain lacking thymidine kinase activity) in the presence of thymidine and (2) uptake of thymidine by this strain, when harboring plasmids with the random insertions. E. coli KY895 harboring high-activity plasmids or wild-type plasmids can grow in the presence of low amounts of thymidine (less than 1 microgram/mL), but are unable to grow in the presence of high amounts of thymidine. On the other hand, E. coli KY895 harboring low-activity plasmids can grow at a high concentration of thymidine (greater than 50 microgram/mL) in the media. The high-activity plasmids also have an enhanced [3H]dT uptake. The amounts of thymidine kinase activity in vitro in unfractionated extracts do not correlate with either growth at low thymidine concentration or the rate of thymidine uptake. Heat inactivation studies indicate that the mutant enzymes are without exception more temperature-sensitive than the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)