MOLECULAR AND BIOINFORMATIC IDENTIFICATION AND MAPPING

分子和生物信息学识别和绘图

• 批准号: 2749001
• 负责人: Manuel Ares
• 金额: $ 14.35万
• 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 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2749001
• 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序列的完成使得 关于组织和表达的大量问题 真核生物基因组这些问题中重要的是定义一个 真核细胞生长所必需的完整最小蛋白质组， 调节，理解人类癌症的关键。的标志 真核生物中存在大量的内含子， 在指定蛋白质的成熟信使RNA（mRNA）中没有发现 基因组的编码能力。内含子的存在使我们的 在基因组序列中看到开放阅读框的能力。到 了解酵母基因组的完整编码能力， 其他真核基因组，我们必须首先能够识别内含子 在基因组序列中。有了酵母基因组的完整序列 在手中，我们有机会绘制所有的位置， 酵母基因组中的核前mRNA内含子，从而揭示其 蛋白质编码能力在这篇文章中，已知有220个酵母内含子 或预测，但这些已被确定在一个偏见，特设 时尚.我们已经开发出一种强大的分子方法， 以不受基因内容影响的方式检测内含子 它被嵌入其中。互补于独特的寡核苷酸 剪接过程中形成的分支序列（“分支聚体”）， 启动子反转录lactintron RNA。的突变 Leptin脱支酶引起显著的积累 的内含子RNA。因此，将使用分支聚体寡核苷酸 以产生表达的内含子探针。我们的目标是（1）创造和 筛选来源于菌株的“表达的内含子标签”克隆文库 积累了大量的内含子RNA。这些克隆人将 被测序以产生表达的内含子序列的数据库，（2） 使用信息技术识别与已知内含子相似的基因组序列， 方法并测试这些方法在体内的剪接潜力，以及（3） 完善每种方法的重复应用，直到形成一套完整的 确认的内含子被映射到基因组的序列。找到所有 内含子将是必不可少的完整理解的 基因组的编码能力。