Characterization of Small Open Reading Frames (sORFs) that Encode for Proteins

利用来自独立实验方法和计算分析的数据， 提出S.酿酒酵母 基因组，占约5%的注释的ORF。我们确定， sORF在其他真核生物中也有注释，包括人类，184个S. 酿酒酵母sORF与其他生物的ORF具有相似性。在 与Jef Boeke（约翰霍普金斯大学）、罗恩戴维斯 （斯坦福大学）和迈克尔斯奈德（耶鲁大学），我们构建了一个集合， 140个新鉴定的sORF的基因缺失突变体，并将它们整合到现有的 删除集合。我们的实验室对sORF缺失进行了全面分析 菌株，并鉴定了22个单倍体生长、高温生长 在不可发酵碳源存在下生长，或在DNA存在下生长 破坏和复制抑制剂。我们对sORF的有限分析是基于一个狭窄的 实验条件的范围和与目前可获得的序列的比较 数据库。我们建议随着数据库的扩展，将确定更多的sORF。在 继续我们正在进行的研究目标，我们发现，三个新确定的 在全球质谱研究中，sORF已被鉴定为动粒组分。 这一结果表明，这些sORF可能在动粒功能中起作用，并表明 额外的sORF在动粒功能和基因组稳定性中的潜在作用。 我们将分析sORF缺失菌株的染色体丢失和检查点缺陷， 功能，并进行二次遗传筛选，以进一步确定的分子作用， sORF。在努力定义sORF的其他作用时，我们有几个正在进行的合作， 检查sORF缺失菌株是否具有与细胞周期缺陷相关的表型 进展、核转运、基因沉默和转录调控。我们还 产生表位标记的sORF菌株和过表达sORF的质粒供公众使用。 我们对sORFs的研究将确定和建立sORFs在不同生物学过程中的作用。 途径。我们建议随着数据库的扩展，将确定更多的sORF。我们 将利用计算、生物化学和基因组方法来验证 sORF并了解其生物学功能。

Utilizing data from independent experimental approaches and computational analyses we proposed the existence for 299 sORFs in the S. cerevisiae genome, representing about 5% of annotated ORFs. We determined that a similar percentage of sORFs are annotated in other eukaryotes, including humans, and 184 of the S. cerevisiae sORFs exhibit similarity with ORFs in other organisms. In collaboration with the laboratories of Jef Boeke (The Johns Hopkins University), Ron Davis (Stanford University), and Michael Snyder (Yale University), we constructed a collection of gene-deletion mutants of 140 newly identified sORFs and integrated them into the existing deletion collection. Our laboratory undertook a comprehensive analysis of the sORF deletion strains and identified 22 sORFs required for haploid growth, growth at high temperature, growth in the presence of a non-fermentable carbon source, or growth in the presence of DNA damage and replication arrest agents. Our limited analysis of the sORFs was based on a narrow range of experimental conditions and comparison to sequences currently available in the databases. We propose that additional sORFs will be identified as the databases expand. In continuation with our ongoing research objective we found that three of the newly identified sORFs have been identified as kinetochore components in global mass spectrometric studies. This result indicates that these sORFs likely have a role in kinetochore function and suggests the potential for the role of additional sORFs in kinetochore function and genome stability. We will analyze the sORF deletion strains for chromosome loss and defects in checkpoint function, and perform secondary genetic screens to further define the molecular role of the sORFs. In efforts to define other roles for sORFs, we have several ongoing collaborations to examine if the sORF deletion strains have phenotypes related to defects in cell cycle progression, nuclear transport, gene silencing and transcriptional regulation. We have also generated epitope tagged sORF strains and plasmids overexpressing the sORFs for public use. Our studies with sORFs will identify and establish the role of sORFs in diverse biological pathways. We propose that additional sORFs will be identified as the databases expand.. We will utilize computational, biochemical, and genomic approaches to validate the presence of sORFs and understand their biological function.