Sequence Determinants of Meiotic Recombination Hotspots

描述(由申请人提供)：减数分裂重组是几乎所有有性生殖有机体共同的基本生物学过程。这一过程创造了新的等位基因组合，从而增加了物种内的遗传多样性；此外，在大多数情况下，这对适当的染色体分离至关重要，这可以防止非整倍体后代。我们的长期目标是了解减数分裂重组的机制和调控。减数分裂重组最引人注目但最不为人所知的方面之一是，它在整个基因组中不是以统一的频率发生的。相反，有一些离散的位置(热点)，重组事件优先发生在这些位置。最近，我们发现一个特定的核苷酸序列5‘-ATGACGTCA-3’与裂殖酵母重组热点的一个子集相关。这个热点序列基序可能只是决定减数分裂重组热点位置的一组不同的短核苷酸序列中的一个例子。这个建议的目标是：1)寻找更多序列定义的重组热点。为此，我们正在进行一种筛选，使我们能够快速筛选非常大量的独特序列，并识别热点基序。该屏幕应显示大多数或所有序列定义的热点。2)在确定热点序列基序后，我们将对这些序列进行调查，并确定它们是否是减数分裂DNA断裂的部位，这是重组热点的标志。在适当的情况下，这些基序将被突变，以确认DNA断裂和/或热点活动是由这些位置的特定序列造成的。3)由于重组热点经常是转录因子的结合部位，而且这些因子在生物体之间往往是保守的，我们将测试在S.pombe中发现的热点序列基序是否也在远缘芽生酵母Saccharmyces cerevisiae中活跃。在这两种生物中都活跃的序列可能在真核生物中广泛保守，包括人类。

DESCRIPTION (provided by applicant): Meiotic recombination is a fundamental biological process common to virtually all sexually reproducing organisms. This process creates new allelic combinations and thus, increases the genetic diversity within a species; furthermore, it is essential, in most cases, for the proper segregation of chromosomes, which ensures against aneuploid progeny. Our long term goal is to understand the mechanism and regulation of meiotic recombination. One of the most striking, but least-well understood aspects, of meiotic recombination is that it does not occur at uniform frequency throughout the genome. Rather there are discrete sites (hotspots) at which recombination events preferentially occur. Recently, we showed that a specific nucleotide sequence, 5'-ATGACGTCA-3,' is associated with a subset of recombination hotspots in the fission yeast Schizosaccharomyces pombe. This hotspot sequence motif is likely to be just one example among a diverse group of short nucleotide sequences that determine the location of meiotic recombination hotspots. The goals of this proposal are: 1) to find additional sequence-defined hotspots of recombination. To this end, we are performing a screen that will allow us to rapidly sift through very large numbers of unique sequences and identify hotspot motifs. This screen should reveal the majority, or perhaps all, sequence-defined hotspots. 2) After identification of hotspot sequence motifs, we will survey the S. pombe genome for those sequences and determine whether they are sites of meiotic DNA breakage, a hallmark of recombination hotspots. Where appropriate, these motifs will be mutated to confirm that DNA breakage and/or hotspot activity results from the specific sequence at those sites. 3) Since recombination hotspots are frequently binding sites for transcription factors, and these factors are frequently conserved between organisms, we will test whether hotspot sequence motifs discovered in S. pombe are also active in the distantly related budding yeast Saccharomyces cerevisiae. Sequences that are active in both organisms may be widely conserved among eukaryotes, including man.