Monitoring Recombination During Meiosis in Budding Yeast.
Monitoring Recombination During Meiosis in Budding Yeast.
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DOI：
10.1016/bs.mie.2017.12.005
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发表时间：
2018
影响因子：
--
通讯作者：
Hunter N
中科院分区：
文献类型：
--
作者：
Owens S;Tang S;Hunter N
同源重组是有性生殖的基础，在减数分裂的第一次分裂中促进同源染色体的准确分离，并创造新的等位基因组合，促进进化。程序性DNA双链断裂（DSB）引发减数分裂重组后，同源配对和DNA链交换形成接合分子（JM）中间体，最终分解为交叉和非交叉修复产物。物理监测减数分裂重组的DNA步骤在酿酒酵母（芽殖酵母）培养物进行同步减数分裂提供了开创性的见解减数分裂重组的分子基础，并提供了一个强大的工具，解剖重组因子的分子作用。本章介绍了一套电泳和Southern杂交技术，用于检测和定量的DNA中间体的减数分裂重组在芽殖酵母重组热点。DSB和重组产物（交叉和非交叉）使用一维电泳进行解析，并通过亲本染色体之间的限制性位点多态性进行区分。使用pebraen交联来稳定支化JM，其通过天然/天然二维电泳从线性物质中分离。采用天然/变性二维电泳来确定JM的组分DNA链并测量DSB的加工。这些技术通常适用于任何基因座，其中重组频率高到足以通过Southern杂交检测中间体。
Homologous recombination is fundamental to sexual reproduction, facilitating accurate segregation of homologous chromosomes at the first division of meiosis, and creating novel allele combinations that fuel evolution. Following initiation of meiotic recombination by programmed DNA double-strand breaks (DSBs), homologous pairing and DNA strand exchange form joint molecule (JM) intermediates that are ultimately resolved into crossover and noncrossover repair products. Physical monitoring of the DNA steps of meiotic recombination in Saccharomyces cerevisiae (budding yeast) cultures undergoing synchronous meiosis has provided seminal insights into the molecular basis of meiotic recombination and affords a powerful tool for dissecting the molecular roles of recombination factors. This chapter describes a suit of electrophoretic and Southern hybridization techniques used to detect and quantify the DNA intermediates of meiotic recombination at recombination hotspots in budding yeast. DSBs and recombination products (crossovers and noncrossovers) are resolved using one-dimensional electrophoresis and distinguished by restriction site polymorphisms between the parental chromosomes. Psoralen cross-linking is used to stabilize branched JMs, which are resolved from linear species by native/native two-dimensional electrophoresis. Native/denaturing two-dimensional electrophoresis is employed to determine the component DNA strands of JMs and to measure the processing of DSBs. These techniques are generally applicable to any locus where the frequency of recombination is high enough to detect intermediates by Southern hybridization.
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影响因子：
2.9
作者：
BELL, L;BYERS, B
通讯作者：
BYERS, B
DOI：
10.1016/0921-8777(94)90018-3
发表时间：
1994-09-01
期刊：
MUTATION RESEARCH-DNA REPAIR
影响因子：
--
作者：
RUVEN, HJT;SEELEN, CMJ;VANZEELAND, AA
通讯作者：
VANZEELAND, AA
影响因子：
16
作者：
Allers, T;Lichten, M
通讯作者：
Lichten, M
影响因子：
2.6
作者：
Chia, Minghao;van Werven, Folkert J.
通讯作者：
van Werven, Folkert J.
影响因子：
64.5
作者：
Hunter, N;Kleckner, N
通讯作者：
Kleckner, N