Humanized yeast as a platform to study human gene function and evolution

蛋白质编码基因在人类和酵母之间在功能上仍然相同的惊人程度，突显了遥远生物在研究人类基因方面的力量。在前期工作中，我们已经人源化了&gt；400菌株。值得注意的是，47%的人类基因可以弥补因失去相应的酵母基因而导致的致命生长缺陷。因此，这些分析揭示了哪些保守的过程仍然可以互换，为可交换的子集创建了人类基因功能的直接分析。目标1.使萌芽酵母人性化。*在目标1中，我们将专注于系统化大约1,000个酵母基因，一次一个或作为一个完整的系统，在它们的天然位点上，从而保持天然的表达，建立研究简化有机体中基因功能和变异的试剂。具体地说，通过关注已经获得谱系特异性复制的人类基因，我们将测试哪些共同同源基因(Paralog)倾向于保留功能，为什么？完成后，这将是有史以来进行的最广泛、最严格和最系统的人源化测试，导致许多人源化酵母菌株用于后续研究(如目标2)，同时也可免费向科学界提供。*目标2.深度扫描突变以表征变异对人类基因功能的贡献。*在目标2中，我将用人类基因变异文库人源化酵母，并直接测量遗传变异对人类基因功能的影响。我们将专注于可替换和不可替换的人类基因，同时旨在了解基因变异对功能可替换性的贡献。在目标2a中，我们将测试不可替代的人类20S核心亚单位的变异是否可以在酵母中实现功能替代，从而确定允许替代的关键残基/相互作用。人性化范式允许我们测试完整的基本酵母和人类系统是否在第一个近似值上是可以互换的。因此，可以研究局部功能网络对遗传变异的影响。在目标2b中，我们将测试在大量酵母和人源化局部功能网络的背景下遗传变异对属于类固醇生物合成途径的人类基因的影响。目标3.人源化分裂酵母和萌芽酵母在4亿年前已经分化，虽然它们之间的许多细胞过程相似，但许多其他过程，如端粒保护素复合体，RNAi系统在人类中更相似。值得注意的是，这些关键细胞系统中的许多在萌芽酵母中是不可替代的。在目标3中，我们将对人类基因在分裂酵母中的可替换性进行系统的筛选，从而测试是否相似的同源基因的性质预测了可替换性(如在芽殖酵母中)，同时也创造了许多新的人源化酵母菌株。**

The remarkable extent to which protein-coding genes are still functionally equivalent between humans and yeast emphasizes the power of distant organisms for studying human genes. In preliminary work, we've already humanized >400 strains. Remarkably, 47% of the human genes can complement a lethal growth defect conferred by loss of the corresponding yeast gene. Thus, the assays reveal which conserved processes are still interchangeable, creating direct assays of human gene function for the swappable subset.******Aim 1. Humanize budding yeast. ***In Aim 1, We will focus on systematically humanizing approximately 1,000 yeast genes, one at a time or as systems in their entirety, at their native loci thus maintaining native expression, building reagents to study gene function and variation in a simplified organism. Specifically, by focusing on human genes that have acquired lineage specific duplication, we will test which of the co-orthologs' (paralogs') tends to retain function and why? Upon completion, this will be the most extensive, rigorous and systematic test of humanization ever performed resulting in many humanized yeast strains to be used in subsequent studies (as in Aim 2) while also being made freely available to the scientific community. ******Aim 2. Deep scanning mutagenesis to characterize the contribution of variation to human gene function.***In Aim 2, I will humanize yeast with libraries of human gene variants and directly measure the impact of genetic variation on human gene function. We will focus on both replaceable and non-replaceable human genes while aiming to understand the contribution of genetic variation to functional replaceability. In Aim 2a, we will test whether variations in the non-replaceable human 20S core subunits can allow functional replaceability in yeast, thus identifying key residues/interactions that allow replaceability. The humanization paradigm allows us to test whether complete essential yeast and human systems are, to a first approximation, interchangeable. Thus, allowing investigations in the effects of local functional network on genetic variation. In Aim 2b, we will test the effect of genetic variation in the context of largely yeast and humanized local functional network on human genes belonging to sterol biosynthesis pathway.******Aim 3. Humanize fission yeast***Fission yeast and budding yeast have diverged >400 million years ago and while many cellular processes are similar between them, many other processes like Telomere Shelterin complex, RNAi systems that are more alike in humans. Notably, many of these critical cell systems are non-replaceable in budding yeast. In Aim 3, we will perform systematic screen for human gene replaceability in fission yeast, thus, testing whether similar properties of orthologous genes predict replaceability (as in budding yeast) while also creating many new humanized yeast strains. **