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

编码蛋白质的小型开放阅读框 (sORF) 的表征

基本信息

批准号：
7965734
负责人：
Munira Basrai
金额：
$ 12.69万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7965734
关键词：
Antithymoglobulin Biochemical Biological Biological Process Biology Carbon Cell Cycle Progression Chelating Agents Classification Codon Nucleotides Collaborations Collection DNA Replication Damage Data Databases Defect Energy Metabolism Epitopes Eukaryota Exhibits Functional RNA Gene Deletion Gene Silencing Genes Genetic Screening Genome Genome Stability Genomics Growth Haploidy Heat shock proteins High temperature of physical object Human Investigation Ions Kinetochores Laboratories Mediating Metals Modeling Molecular Mutagenesis Open Reading Frames Organism Partner in relationship Pathway interactions Phenotype Pheromone Plague Plasmids Proteins Proteolipids Publications RNA Reporting Research Ribosomal Proteins Role Saccharomyces cerevisiae Source System Thioredoxin Transcript Transcriptional Regulation Translations Universities base chaperonin chromosome loss comparative functional genomics fungus genome sequencing genome wide association study member mutant nuclease nucleocytoplasmic transport overexpression serial analysis of gene expression stress protein

项目摘要

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.

利用独立实验方法和计算分析的数据，我们提出 S 中存在 299 个 sORF。 cerevisiae基因组，约占注释ORF的5%。我们确定在其他真核生物（包括人类）和 184 个 S 中注释了类似百分比的 sORF。酿酒酵母的 sORF 与其他生物体中的 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 的基因缺失突变体集合，并将它们整合到现有的删除集合。我们的实验室对 sORF 缺失菌株进行了全面分析，并鉴定了单倍体生长、高温生长、在不可发酵碳源存在下生长或在 DNA 损伤和复制抑制剂存在下生长所需的 22 个 sORF。我们对 sORF 的有限分析是基于小范围的实验条件以及与数据库中当前可用序列的比较。我们建议随着数据库的扩展，将识别更多的 sORF。继续我们正在进行的研究目标，我们发现三个新发现的 sORF 已在全球质谱研究中被确定为动粒成分。这一结果表明这些 sORF 可能在着丝粒功能中发挥作用，并表明其他 sORF 在着丝粒功能和基因组稳定性中发挥作用的潜力。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明确 sORF 的分子作用。为了确定 sORF 的其他作用，我们正在进行多项合作，以检查 sORF 缺失菌株是否具有与细胞周期进程、核运输、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 的研究将确定并确定 sORF 在不同生物途径中的作用。我们建议随着数据库的扩展，将鉴定更多的 sORF。我们将利用计算、生物化学和基因组方法来验证 sORF 的存在并了解其生物学功能。