Meiotic Silencing by Unpaired DNA

病毒、反转录转座子和插入序列使所有生物体的基因组完整性处于危险之中。可以说，真菌比植物和动物面临更大的危险，因为它们是同心胞制的：一个宏观的“个体”可能包含数百万个细胞核，共享一个连续的细胞质，而细胞质没有隔膜或细胞膜分开。不出所料，真菌至少有三种复杂的反击机制：抑制、RIP和非配对DNA （MSUD）的减数分裂沉默。本课题的研究旨在了解MSUD的机制及其在一种实验生物神经孢子虫（Neurospora crassa）的整体生物学中的作用。早期的研究表明，在这个生物体非常短暂的二倍体阶段，可能是同系物在减数分裂的粗线期配对时，任何相当大的未配对DNA片段都被生物体“看到”为外来序列，这种序列不存在于双亲的同一位点，因此具有恶作剧的能力。产生双链RNA形式的信号。最终，这不仅沉默了未配对DNA的表达，也沉默了相同序列的任何其他DNA的表达，无论是成对的还是未成对的。选择和筛选在这种沉默过程中失败的抑制突变在获得洞察力方面已经是有益的；其中一种名为Sad-1 （ascus显性抑制因子），编码一种性相特异性RNA定向RNA聚合酶，其他Sad突变体将被寻找和研究。研究的主要重点将放在两个被认为与抑制有关的同源基因dicer-1和dicer-2的作用上。在许多生物体中，以及在真菌生长的营养阶段，这些名字贴切的基因被认为是将长双链RNA分子切成短双链片段，而短双链片段反过来又产生单链分子。然后，这些基因直接破坏与它们同源的mRNA，导致“沉默”。目前尚不清楚它们在MSUD中是否具有类似的作用，并且将花费相当大的努力来阐明这个问题。最后，很明显，功能性Sad基因对于完成减数分裂是必要的。这表明沉默的一些机制，可能涉及双链RNA及其切割，与减数分裂机制是共享的。寻找关键分子是这项研究的另一个目标。所有生物体都在“自私”的DNA序列或分子寄生虫的持续挑战下，这些寄生虫能够以牺牲宿主生物为代价进行自我繁殖。通常，寄主有复杂的反击机制，要么攻击寄生DNA并使其变异为无害的状态，要么阻止寄生DNA制造其增殖所必需的分子。了解这些沉默机制是如何工作的，不仅可以防止病毒的繁殖，还可以防止其他细胞基因产生不需要的产物，或过量产生良性产物。

Viruses, retrotransposons, and insertion sequences put the genomic integrity of all organisms at risk. Fungi are arguably under even greater jeopardy than plants and animals because they are organized coenocytically: a macroscopic "individual" may contain millions of nuclei sharing a continuous cytoplasm that is undivided by septa or cell membranes. Unsurprisingly, fungi have at least three sophisticated mechanisms for fighting back: quelling, RIP, and meiotic silencing by unpaired DNA (MSUD). The research that is the subject of this project is aimed at understanding the mechanism of MSUD and its role in the overall biology of one experimental organism, Neurospora crassa. Earlier work showed that during the very brief diploid phase of this organism, presumably when homologs are paired in the pachytene stage of meiosis, any sizeable stretch of unpaired DNA is "seen" by the organism as a foreign sequence, one that was not present in both parents at the same locus, and hence having the capacity for mischief. A signal in the form of double-stranded RNA is generated. Ultimately, this silences the expression not only of the unpaired DNA, but also any other DNA of the same sequence, whether paired or unpaired. Selecting and screening for suppressor mutations which fail in this silencing process has already been rewarding in terms of insight gained; one called Sad-1 (suppressor of ascus dominance) encodes a sexual-phase specific RNA-directed RNA polymerase, and other Sad mutants will be sought and studied. A major focus of the research will be on the role of two paralogous genes called dicer-1 and dicer-2 thought to be involved in quelling. In many organisms, and in the vegetative phase of fungal growth, these aptly-names genes are thought to dice up long double-stranded RNA molecules into short, double-stranded pieces which, in turn, give rise to single-stranded molecules. These then direct the destruction of mRNA homologous to them, resulting in "silencing". It is not clear that they have a similar role in MSUD, and a sizeable effort will be expended in trying to illuminate this issue. Finally, it has become clear that functional Sad genes are necessary for the completion of meiosis. This suggests that some of the machinery of silencing, presumably involving double-stranded RNA and its cleavage, is shared with the machinery of meiosis. A search for the key molecules is an additional aim of this research. All organisms are under continuous challenge by "selfish" DNA sequences or molecular parasites that are able to reproduce themselves at the expense of the host organism. Often the host has sophisticated machinery for fighting back, either by attacking and mutating the parasitic DNA to a harmless condition, or by preventing the parasitic DNA from making molecules necessary for its multiplication. An understanding of how these silencing mechanisms work would make it possible to prevent not only the reproduction of viruses, but also to prevent other cellular genes from making undesired products, or from making benign products in excessive amounts.