PROJECT 4

项目4：体内蛋白质翻译后修饰的光学监测(索恩)(#27) 我们开发了利用荧光共振能量研究蛋白质修饰和相互作用的工具 转移(FRET)显微镜。主要的工作是检测wVo12中的蛋白质-蛋白质相互作用并使用这些 用于预测大型蛋白质复合体结构的数据。我们开发了各种方法来生产精密和 来自酵母的可重复的FRET数据，并使用它们推导出Septin复合体的低分辨率结构 (这是从酵母到哺乳动物的细胞分裂所必需的)。与Michael Laub(项目7)一起，我们发现了互动 在两组分信号蛋白之间，翻译后调控的主要参与者 细菌，并与多尔蒂和他的同事合作开发出高通量、定量、基于FRET的 鉴定蛋白质相互作用的方法13。私家侦探库尔特·索恩曾是鲍尔的研究员，现在是 加州大学旧金山分校的尼康成像中心。 新项目4：基因中的串联重复：适应的超变量模块？(Verstrepen牌)(#28-30) 我们测试了假设35，即在萌芽酵母中，重复序列是高度可变的遗传 模块，其可变性帮助生物体适应不断变化的环境。测序最多的基因组 包含串联重复序列；其中一些在编码序列中出现，其中重组可以改变数目 翻译的蛋白质中的多肽重复。我们已经开发了一种算法 (http://hulsweb1.cgr.harvard.edu/SERV/)用于查找和注释重复，预测它们是否在数量上变化， 将它们与其他信息(表达水平、与转录因子结合位点重叠、 核小体站点等)，并将该数据存储在可公开访问的数据库中 (http://hulsweb1.cgr.harvard.edu/TandemRepeat/)允许快速、自动地与现有 数据库；2)在启动子和开放阅读中产生不同重复数目的酵母菌株 选定基因的框架，并研究了这种变异的表型后果(正在进行)；3)研究 Tandem在拟南芥和水稻中重复(与C.Queitsch，新项目7)。我们用了两个 对基因组进行测序以找到所有包含可变重复次数的ORF，选择150个基因， 对来自不同地点的12个拟南芥分离物的变异进行了表征，发现了70多个 在不同分离株之间显示重复数差异的基因。凯文·弗斯特莱本成为鲍尔 2005年加入中巴，2006年加入中巴。

Project 4: Optical monitoring of post-translational modification of proteins in vivo (Thorn)(#27) We developed tools to study protein modification and interactions using fluorescence resonance energy transfer (FRET) microscopy. The main effort was to detect protein-protein interactions in wVo12 and use these data to predict the structure of large protein complexes. We developed methods to produce precise and reproducible FRET data from yeast, and used them to derive a low-resolution structure for the septin complex (which is required for cell division from yeast to mammals). With Michael Laub (project 7) we found interactions between two-component signaling proteins, the predominant participants in post-translational regulation in bacteria, and collaborated with Daugherty and his colleagues to develop high throughput, quantitative, FRETbased methods to identify protein interactions13. Kurt Thorn, the PI, was a Bauer Fellow and is now Director of the Nikon Imaging Center at UCSF. New project 4: Tandem repeats in genes: hyper-variable modules for adaptation? (Verstrepen) (#28-30) We tested the hypothesis35 that in budding yeast, tandemly repeated sequences are hyper-variable genetic modules, whose variability helps organisms adapt to changing environments. Most sequenced genomes contain tandem repeats; some of these occur in coding sequences, where recombination can alter the number of peptide repeats in translated proteins. We have, 1) developed an algorithm36 (http://hulsweb1.cgr.harvard.edu/SERV/) to find and annotate repeats, predict whether they vary in number, associate them with other information (expression level, overlap with transcription factor binding sites, nucleosome sites etc.), and deposit this data in a publicly accessible database (http://hulsweb1.cgr.harvard.edu/TandemRepeat/) that allows rapid and automated comparison to existing databases; 2) generated yeast strains with different numbers of repeats in the promoters and open reading frames of selected genes, and studied the phenotypic consequences of this variation (ongoing); 3) studied tandem repeats in Arabidopsis thaliana and Oryza sativa (with C. Queitsch, new project 7). We have used two sequenced genomes to find all ORFs containing variable numbers of repeats, selected 150 genes, characterized their variation across twelve A. thaliana isolates from different locations, and found more than 70 genes that show variation in repeat number between different isolates. Kevin Verstrepen became a Bauer Fellow in 2005 and joined the CMB in 2006.