MOLECULAR AND BIOINFORMATIC IDENTIFICATION AND MAPPING

分子和生物信息学识别和绘图

• 批准号: 2630784
• 负责人: Manuel Ares
• 金额: $ 14.18万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA CRUZ
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-15 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2630784
• 关键词: RNA splicing; fungal genetics; genetic library; genetic mapping; genome; introns; molecular cloning; nucleic acid sequence; precursor mRNA; protein biosynthesis; yeasts

Completion of the DNA sequence of the yeast genome has made accessible a large number of questions about the organization and expression of eukaryotic genomes. Important among these questions is defining a complete minimum protein set necessary for eukaryotic cell growth and regulation, key to understanding human cancer. A hallmark of the eukaryotes is the abundant presence of introns, internal gene sequences not found in the mature messenger RNAs (mRNAs) that specify the protein coding capacity of the genome. The presence of introns clouds our ability to see open reading frames in the genomic sequence. To understand the complete coding capacity of the yeast genome, and of other eukaryotic genomes, we must first be able to recognize introns in the genomic sequence. With the complete sequence of the yeast genome in hand, we have the opportunity to map the positions of all the nuclear pre-mRNA introns in the yeast genome, and thus reveal its protein coding capacity. At this writing 220 yeast introns are known or predicted, but these have been identified in a biased, ad hoc fashion. We have developed a powerful molecular approach to the direct detection of introns in a manner not biased by the contents of the gene in which it is embedded. Oligonucleotides complementary to the unique lariat sequence formed during splicing ("branchmers") specifically prime reverse transcription of lariat intron RNA. Mutations that inactivate the lariat debranching enzyme cause dramatic accumulation of intron RNA in yeast. Thus branchmer oligonucleotides will be used to generate expressed intron probes. Our aims are (1) to create and screen libraries of "expressed intron tag" clones derived from strains of yeast that accumulate large-amounts of intron RNA. These clones will be sequenced to generate a database of expressed intron sequences, (2) to identify genomic sequences similar to known introns using informatic approaches and test these for splicing potential in vivo, and (3) to refine repeated applications of each approach until a complete set of confirmed introns is mapped to the sequence of the genome. Finding all the introns will be essential to the complete understanding of the coding capacity of the genome.

酵母基因组 DNA 序列的完成使人们能够 大量关于组织和表达的问题 真核基因组。这些问题中重要的是定义一个 完整的真核细胞生长所需的最低蛋白质组和 调节，了解人类癌症的关键。的一个标志 真核生物具有丰富的内含子、内部基因序列 在指定蛋白质的成熟信使 RNA (mRNA) 中未发现 基因组的编码能力。内含子的存在使我们的 能够看到基因组序列中的开放阅读框。到 了解酵母基因组的完整编码能力，以及 其他真核基因组，我们首先必须能够识别内含子 在基因组序列中。拥有酵母基因组的完整序列 在手中，我们有机会绘制所有位置的地图 酵母基因组中的核前mRNA内含子，从而揭示其 蛋白质编码能力。在撰写本文时，已知 220 个酵母内含子 或预测，但这些都是在有偏见的、临时的情况下确定的 时尚。我们开发了一种强大的分子方法来直接 以不受基因内容影响的方式检测内含子 它嵌入其中。与独特的寡核苷酸互补 剪接过程中形成的套索序列（“分支体”） 套索内含子RNA的主要逆转录。突变 使套索脱支酶失活，导致大量积累 酵母内含子RNA。因此将使用分支寡核苷酸 产生表达的内含子探针。我们的目标是 (1) 创造和 筛选源自菌株的“表达内含子标签”克隆文库 积累大量内含子RNA的酵母菌。这些克隆将 进行测序以生成表达内含子序列的数据库，(2) 使用信息学识别与已知内含子相似的基因组序列 方法并测试它们的体内剪接潜力，以及（3） 完善每种方法的重复应用，直到形成一套完整的方法 确认的内含子被映射到基因组序列。查找全部 内含子对于完整理解至关重要 基因组的编码能力。