SGER: A New Approach to the Study of Meiotic Chiasma Interference

这一建议为理解减数分裂交叉干扰的机制以及基因转换和交叉之间的联系提供了一种新的途径。干扰，即相邻染色体区域交叉的抑制，自本世纪初以来就已为人所知，但减数分裂期间的一次交叉对相邻交叉（跨越数百千碱基对的距离）产生抑制作用的机制尚不清楚。研究干扰的困难之一是，在任何给定的减数分裂中，引发减数分裂重组的双链断裂的位置是未知的，因此很难确定一个重组事件如何影响另一个。研究者在减数分裂特异性SPO13启动子的控制下表达了位点特异性HO内切酶基因，以便在明确的间隔内产生明确的双链断裂，从而研究基因转换、交叉和干扰之间的关系。初步研究证实，在减数分裂前DNA复制后，SPO13::HO的表达导致减数分裂基因转换。此外，研究者还表明，在所有正常减数分裂双链断裂缺失的情况下，rad50 spol3二倍体可以进行完全的减数分裂重组；因此，在没有任何其他初始病变的情况下检查交叉是可能的。这就有可能通过使用针对重组蛋白的抗体的间接免疫荧光来确认，与建立干扰有关的突触复合体的元素将在双链断裂的位置聚集。这个项目的几个方面是“高风险的”。首先，必须更精确地确定SP013:: ho诱导的双链断裂是否与正常减数分裂断裂同时发生。其次，必须确定在rad50 spol3背景下与基因转换相关的明显杂交是否实际上是相互交换的结果。最后，用细胞学方法检测突触复合体的单个区域可能比在正常减数分裂中观察许多这样的区域更困难。干扰，即相邻染色体交叉区域的交叉抑制，自本世纪初以来就已为人所知，但减数分裂期间的一次交叉对相邻交叉（跨越数百千碱基对的距离）产生抑制作用的机制尚不清楚。这项研究设计了一系列优雅的实验，以进一步了解这一基本过程。＊＊＊

9424330 Haber This proposal presents a new approach to understanding the mechanisms of meiotic crossover interference and the connection between gene conversions and crossing-over. Interference, the inhibition of crossing-over in chromosomal regions adjacent to another crossover, has been known since early in this century, but the mechanism by which one crossover during meiosis can exert an inhibiting effect on an adjacent crossover - over distances of hundreds of kilobase pairs - is not yet understood. One of the difficulties in studying interference is that the location of double-strand breaks that initiate meiotic recombination in any given meiosis is not known, so that it is difficult to determine how one recombination event influences another. The investigator has expressed the site-specific HO endonuclease gene under the control of a meiotic-specific SPO13 promoter in order to create defined double-strand breaks in well-defined intervals, so that the relationship between gene conversion, crossing-over and interference can be studied. Preliminary studies have confirmed that the expression of SPO13::HO leads to meiotic gene conversions after premeiotic DNA replication. Moreover the investigator has shown that complete meiotic recombination can be carried out in rad50 spol3 diploids in which all normal meiotic double-strand breaks are absent; hence, examining crossing-over in the absence of any other initiating lesions is possible. This should make it possible to confirm - by indirect immunofluorescence using antibodies directed against recombination proteins - that elements of the synaptonemal complex, implicated in the establishment of interference, will assemble at the site of a double-strand break. Several aspects of this project are "high risk." First, it must first be established more precisely if SP013::HO-induced double-strand breaks are produced at the same time as normal meiotic breaks. Second, it must be established if apparent crossing-over associated with gene conversion in a rad50 spol3 background is in fact the consequence of reciprocal exchange. Finally, the detection of a single region of synaptonemal complex by cytological means may prove to be more difficult than to see many such regions in a normal meiosis. %%% Interference, the inhibition of crossing-over in chromosomal regions adjacent to another cross-over, has been known since early in this century, but the mechanism by which one crossover during meiosis can exert an inhibiting effect on an adjacent crossover - over distances of hundreds of kilobase pairs - is not yet understood. The investigation has designed an elegant series of experiments to further our understanding of this fundamental process. ***