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

人类和酵母之间的蛋白质编码基因在功能上仍然相当，这一显著程度强调了远距离生物研究人类基因的力量。在初步的工作中，我们已经使400个菌株人性化。值得注意的是，47%的人类基因可以补充由于相应酵母基因缺失而导致的致命生长缺陷。因此，分析揭示了哪些保守的过程仍然是可互换的，为可互换的子集创建了人类基因功能的直接分析。* * * * * *的目标1。使出芽酵母人性化。***在目标1中，我们将专注于系统地将大约1000个酵母基因人源化，一次一个或作为一个完整的系统，在它们的天然位点上保持天然表达，构建试剂来研究简化生物体中的基因功能和变异。具体来说，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“同源”倾向于保留功能，为什么？一旦完成，这将是迄今为止进行的最广泛、最严格和最系统的人源化测试，结果是许多人源化酵母菌株将用于后续研究（如目标2），同时也将免费提供给科学界。* * * * * *的目标2。深度扫描诱变表征变异对人类基因功能的贡献。***在Aim 2中，我将用人类基因变异文库对酵母进行人源化，并直接测量遗传变异对人类基因功能的影响。我们将重点关注可替代和不可替代的人类基因，同时旨在了解遗传变异对功能可替代性的贡献。在Aim 2a中，我们将测试不可替代的人类20S核心亚基的变化是否允许酵母的功能可替代性，从而确定允许可替代性的关键残基/相互作用。人性化范例使我们能够测试完整的基本酵母菌和人体系统是否可以初步近似地互换。因此，允许研究局部功能网络对遗传变异的影响。在Aim 2b中，我们将测试遗传变异在酵母和人源化局部功能网络的背景下对属于甾醇生物合成途径的人类基因的影响。* * * * * *的目标3。裂变酵母和出芽酵母在4亿年前就已经分化，虽然它们之间的许多细胞过程相似，但许多其他过程，如端粒庇护蛋白复合体，RNAi系统在人类中更相似。值得注意的是，这些关键的细胞系统在出芽酵母中是不可替代的。在Aim 3中，我们将对裂变酵母中人类基因的可替代性进行系统筛选，从而测试同源基因的相似特性是否可以预测可替代性（如出芽酵母），同时也可以创建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. **