How do Spore killers kill spores? Elucidating the mechanism of meiotic drive by spore killing in Neurospora fungi.

这个项目研究了粗糙脉孢菌中的两个基因，一个杀死真菌孢子，另一个防止孢子杀死。虽然这两个基因可以被认为形成了一个毒药和解药系统，但它们的目的尚不清楚。它们对真菌有益还是有害？它们似乎形成了一种自私的伙伴关系，其杀戮和抵抗特性允许这两个基因传递给每一个有机体的后代，即使交配对中只有一个父母拥有这两个基因。这个项目的主要目标是确定这些毒药和解毒剂基因是如何实现这一非凡壮举的。此外，这两个基因似乎推动了它们所在的染色体的重大重组。因此，该项目的次要目标是调查自私基因是真核生物基因组重组的主要驱动因素的假设。主要目标将由位于伊利诺伊州中部的伊利诺伊州立大学的科学家、研究生和本科生组成的团队实现，而次要目标将由同一团队与瑞典的进化生物学家合作实现。此外，来自伊利诺伊州中部的一名GK-12 STEM教师将协助该项目。这不仅将为教师提供宝贵的研究经验，PI和教师还将为GK-12课堂设计基于探究的学习活动，涉及到对粗毛拟青霉和其他无害微生物的实验。本研究中研究的两个基因分别是粗毛念珠菌孢子杀伤基因rfk和抗性基因rsk。虽然RFK杀伤基因的边界尚未明确，但其杀伤功能已被追踪到一株名为Sk2的菌株第三条染色体上的1500个碱基DNA片段。rsk抗性基因位于同一染色体上。自然界中存在不同的rsk等位基因，并不是所有的rsk等位基因都对RFK具有抗性。此外，rsk还具有蛋白质编码基因的典型特征。例如，它具有清晰可识别的起始密码子、终止密码子和开放阅读框架。这与杀手基因形成鲜明对比，杀手基因没有明显的蛋白质编码特征，因此可能产生有毒的非编码RNA或有毒蛋白质。该项目的第一个目标是区分这两种可能性，并收集有关孢子杀灭机制的证据。首先，将对6个先前分离的RFK突变体进行测序，以产生扰乱杀戮的突变目录；其次，将使用RNA测序来定位功能RFK基因座的转录本，并确定与杀戮相关的整体转录变化；第三，将利用快速扩增cDNA端(RACE)技术克隆全长RFK转录本；第四，将使用定点突变来确定随机插入突变是否比随机点突变更具破坏性；第五，将修复一组六个点突变，当所有这些点突变都在同一个RFK等位基因内时，就会扰乱杀戮；第六，将从耐药和非耐药版本的RSK中产生蛋白质嵌合体，以确定抗性蛋白的哪些部分对功能至关重要。该项目的第二个目标是对一种名为Sk-3的不同菌株进行相同的一组实验。这项提案的两个目标加在一起，将界定杀死基因的边界，确定杀手基因产物是有毒的RNA还是蛋白质，提供扰乱孢子杀死的突变目录，确定与孢子杀死机制相关的转录变化，并确定与抗性所需的蛋白质的关键区域，所有这些都与Sk-2和Sk-3有关，这是自私基因功能和进化的两个模型。

This project investigates two genes in the fungus Neurospora crassa, one that kills fungal spores and another that prevents spore killing. While these two genes can be thought of as forming a poison and antidote system, their purpose is unclear. Are they beneficial for the fungus or are they detrimental? It seems they have formed a selfish partnership whose killing and resistance properties allow the two genes to be transmitted to every one of the organism's offspring, even when only one parent of a mating pair possesses the genes. The primary goal of this project is to determine how these genes, poison and antidote, achieve this remarkable feat. Additionally, the two genes appear to have driven a major reorganization of the chromosome in which they reside. Therefore, a secondary goal of the project is to investigate the hypothesis that selfish genes are major drivers of genome reorganization in eukaryotic organisms. The primary goal will be pursued by a team of scientists, graduate students, and undergraduate students at Illinois State University in Central Illinois, while the secondary goal will be pursued by the same team in collaboration with evolutionary biologists based in Sweden. In addition, a GK-12 STEM teacher from Central Illinois will assist with the project. Not only will this provide valuable research experience to the teacher, the PI and the teacher will design inquiry-based learning activities for GK-12 classrooms that involve experimentation with N. crassa and other harmless microorganisms. The two genes under investigation in this study are rfk, the N. crassa spore killing gene, and rsk, the resistance gene. Although the boundaries of the rfk killing gene have not been precisely defined, the killing function has been tracked to a 1500 base pair fragment of DNA on the third chromosome of a strain called Sk 2. The rsk resistance gene is found on the same chromosome. Different rsk alleles exist in nature, and not all of them provide resistance to rfk. Additionally, rsk possesses features typical of protein-coding genes. For example, it possesses a clearly identifiable start codon, stop codon, and open reading frame. This is in stark contrast to the killer gene, which does not have obvious protein-coding features, and thus could produce either a toxic non-coding RNA or a toxic protein. The first aim of this project is to differentiate between these two possibilities and gather evidence on the mechanism of spore killing. First, six previously isolated rfk mutants will be sequenced to produce a catalog of mutations that disrupt killing; second, RNA sequencing will be employed to map transcripts from a functional rfk locus and to determine the global transcriptional changes associated with killing; third, the full-length rfk transcript will be cloned with Rapid Amplification of cDNA End (RACE) technology; fourth, site-directed mutagenesis will be used to determine if random insertion mutations are more disruptive of killing than random point mutations; fifth, a group of six point mutations, which are known to disrupt killing when all are found within the same rfk allele, will be repaired to determine which combination of the six is critical for loss of killing; and sixth, protein chimeras will be produced from resistant and non-resistant versions of RSK to determine which parts of the resistance protein are critical for function. The second aim of this project is to perform the same set of experiments on a different strain called Sk-3. Together, the two aims of this proposal will define the borders of the killing gene, determine if the killer gene product is a toxic RNA or a protein, provide a catalog of mutations that disrupt spore killing, identify transcriptional changes associated with the spore killing mechanism, and identify critical regions of the protein required for resistance, all for Sk-2 and Sk-3, two models of selfish gene function and evolution.