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首页> 外文期刊>Molecular and Cellular Biology >Isolation of the thymidylate synthetase gene (TMP1) by complementation in Saccharomyces cerevisiae.
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Isolation of the thymidylate synthetase gene (TMP1) by complementation in Saccharomyces cerevisiae.

机译：通过酿酒酵母中的互补作用分离胸苷酸合成酶基因（TMP1）。

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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.
机译：酵母胸苷酸合成酶（胸苷酸合成酶; EC 2.1.1.45）的结构基因（TMP1）通过酿酒酵母中的基因互补从嵌合质粒库中分离出来。用纯化的质粒（pTL1）对dTMP营养缺陷型GY712和温度敏感突变体（cdc21）进行重新转化，可高频率产生Tmp +转化子。另外，测试了质粒与缺乏功能性胸苷酸合成酶的细菌thyA突变体互补的能力。尽管不可能直接选择Thy +转化子，但发现在非选择性条件下生长了几代后，所有pTL1转化子在表型上都是Thy +。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在酵母细胞裂解物中测定胸苷酸合成酶，以监测[6-3H] dUMP向[6-3H] dTMP的转化。在胸腺嘧啶营养缺陷型（tmp1-6）的蛋白质提取物中，几乎无法检测到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。

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