Isolation of the thymidylate synthetase gene (TMP1) by complementation in Saccharomyces cerevisiae
Isolation of the thymidylate synthetase gene (TMP1) by complementation in Saccharomyces cerevisiae
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通过互补在酿酒酵母中分离胸苷酸合成酶基因 (TMP1)
DOI：
10.1128/mcb.2.4.437-442.1982
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发表时间：
1982
影响因子：
5.3
通讯作者：
R. Haynes
中科院分区：
文献类型：
--
作者：
G. R. Taylor;B. Barclay;R. Storms;J. Friesen;R. Haynes
通过酿酒酵母中的遗传互补从嵌合质粒库中分离出酵母胸苷酸合成酶（胸苷酸合酶；EC 2.1.1.45）的结构基因（TMP1）。用纯化质粒 (pTL1) 重新转化 dTMP 营养缺陷型 GY712 和温度敏感突变体 (cdc21)，以高频率产生 Tmp+ 转化体。此外，还测试了该质粒补充缺乏功能性胸苷酸合成酶的细菌 thyA 突变体的能力。虽然不可能直接选择Thy+转化体，但发现所有pTL1转化体在非选择性条件下生长几代后都具有Thy+表型。因此，酵母胸苷酸合成酶在大肠杆菌中具有生物活性。通过高压液相色谱法测定酵母细胞裂解物中的胸苷酸合成酶，以监测 [6-3H]dUMP 至 [6-3H]dTMP 的转化。在胸苷酸营养缺陷型 (tmp1-6) 的蛋白质提取物中，几乎检测不到 dUMP 到 dTMP 的酶促转化。然而，该菌株的 pTL1 转化体的裂解物具有与野生型菌株相当的胸苷酸合成酶活性。
The structural gene (TMP1) for yeast thymidylate synthetase (thymidylate synthase; EC 2.1.1.45) was isolated from a chimeric plasmid bank by genetic complementation in Saccharomyces cerevisiae. Retransformation of the dTMP auxotroph GY712 and a temperature-sensitive mutant (cdc21) with purified plasmid (pTL1) yielded Tmp+ transformants at high frequency. In addition, the plasmid was tested for the ability to complement a bacterial thyA mutant that lacks functional thymidylate synthetase. Although it was not possible to select Thy+ transformants directly, it was found that all pTL1 transformants were phenotypically Thy+ after several generations of growth in nonselective conditions. Thus, yeast thymidylate synthetase is biologically active in Escherichia coli. Thymidylate synthetase was assayed in yeast cell lysates by high-pressure liquid chromatography to monitor the conversion of [6-3H]dUMP to [6-3H]dTMP. In protein extracts from the thymidylate auxotroph (tmp1-6) enzymatic conversion of dUMP to dTMP was barely detectable. Lysates of pTL1 transformants of this strain, however, had thymidylate synthetase activity that was comparable to that of the wild-type strain.