New vectors for simplified construction of BrdU-incorporating strains of Saccharomyces cerevisiae
New vectors for simplified construction of BrdU-incorporating strains of Saccharomyces cerevisiae
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DOI：
10.1002/yea.1406
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发表时间：
2006-10-01
期刊：
影响因子：
2.6
通讯作者：
Aparicio, Oscar M.
中科院分区：
文献类型：
--
作者：
Viggiani, Christopher J.;Aparicio, Oscar M.
胸腺嘧啶类似物BrdU是DNA复制或修复研究中分析核苷酸结合的有力工具。酿酒酵母缺乏胸腺嘧啶回收途径，这种途径能够使细胞有效地摄取胸腺嘧啶类似物并将其纳入DNA。最近在酵母体内通过高水平表达单纯疱疹病毒胸苷激酶（HSV-TK）或HSV-TK与人平衡核苷转运蛋白（hENT1）的联合表达重建了这一途径，从而能够分析BrdU在酵母中的掺入。虽然BrdU合并系统非常有价值，但利用这些系统的菌株的构建和使用可能因可用系统的特定要求而变得复杂。我们已经创建了一组载体，简化了brdu -合并（BrdU-Inc）芽殖酵母菌株的构建和使用。该集合中的每个载体都包含HSV-TK和hENT1，在组成性表达启动子的控制下，以及用于基因组整合的四种不同选择标记（HIS3, TRP1， LEU2或URA3）中的一种。有了这些BrdU- inc载体，一步整合一个拷贝产生酵母，有效地将BrdU加入到培养基中。这些载体简化了菌株的构建和维护，从而促进了BrdU在酵母中分析的常规使用。版权所有(c) 2006约翰威利父子有限公司
The thymidine analogue BrdU is a powerful tool for analysing nucleotide incorporation in studies of DNA replication or repair. S. cerevisiae lacks the thymidine salvage pathway that enables efficient cellular uptake and incorporation of thymidine analogues into DNA. Recent in vivo reconstitution of this pathway in yeast by high-level expression of Herpes simplex virus thymidine kinase (HSV-TK) or combined expression of HSV-TK and human equilibrative nucleoside transporter (hENT1) has enabled analysis of BrdU incorporation in yeast. While the BrdU incorporation systems are highly valuable, the construction and use of strains utilizing these systems can be complicated by specific requirements of the available systems. We have created a set of vectors that simplify the construction and use of BrdU-Incorporating (BrdU-Inc) strains of budding yeast. Each vector in the set contains HSV-TK and hENT1 under the control of promoters that express constitutively, and one of four different selectable markers (HIS3, TRP1, LEU2 or URA3) for genomic integration. With these BrdU-Inc vectors, one-step integration of a single copy produces yeast that efficiently incorporate BrdU upon its addition to the medium. These vectors ease strain construction and maintenance, thereby facilitating routine use of BrdU for analysis in yeast. Copyright (c) 2006 John Wiley & Sons, Ltd.