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

利用独立实验方法和计算分析的数据，我们提出了酿酒葡萄球菌基因组中存在299个sorf，约占注释orf的5%。我们确定在包括人类在内的其他真核生物中也有类似比例的sorf被注释，并且184个酿酒葡萄球菌的sorf与其他生物中的orf表现出相似性。我们与Jef Boeke（约翰霍普金斯大学）、Ron Davis（斯坦福大学）和Michael Snyder（耶鲁大学）的实验室合作，构建了140个新鉴定的sorf的基因缺失突变集合，并将它们整合到现有的缺失集合中。我们的实验室对sORF缺失菌株进行了全面分析，并确定了单倍体生长、高温生长、不可发酵碳源生长或DNA损伤和复制阻止剂存在下生长所需的22种sORF。我们对sorf的有限分析是基于狭窄的实验条件和与数据库中现有序列的比较。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中，我们发现三种新鉴定的sorf已在全球质谱研究中被鉴定为着丝点成分。这一结果表明，这些sorf可能在着丝粒功能中起作用，并提示其他sorf在着丝粒功能和基因组稳定性中可能起作用。我们将分析sORF缺失菌株的染色体丢失和检查点功能缺陷，并进行二次遗传筛选以进一步确定sORF的分子作用。为了确定sORF的其他作用，我们进行了几项正在进行的合作，以检查sORF缺失菌株是否具有与细胞周期进程、核转运、基因沉默和转录调控缺陷相关的表型。我们还生成了表位标记的sORF菌株和过表达sORF的质粒供公众使用。我们对sorf的研究将确定和确立sorf在多种生物学途径中的作用。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、生化和基因组方法来验证sorf的存在并了解它们的生物学功能。

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.