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.

酵母基因组的DNA序列的完成已使得可访问 有关组织和表达的许多问题 真核基因组。这些问题中重要的是定义 完整的最小蛋白质集，用于真核细胞的生长和 调节，了解人类癌症的关键。一个标志 真核生物是内含子，内部基因序列的丰富存在 在指定蛋白质的成熟信使RNA（mRNA）中找不到 基因组的编码能力。内含子的存在使我们 能够在基因组序列中查看开放式阅读帧。到 了解酵母基因组的完整编码能力和 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。酵母基因组的完整序列 在手头，我们有机会映射所有的位置 酵母基因组中的核前mRNA内含子，因此揭示了其 蛋白质编码能力。在撰写本文中，有220个酵母内含子是已知的 或预测，但这些已在有偏见的临时性 时尚。我们已经开发了一种强大的分子方法来直接 以不受基因内容偏见的方式检测内含子 嵌入其中。寡核苷酸互补 特别是在剪接期间形成的套索序列（“分支机构”） 幼虫内含子RNA的素逆转录。突变 灭活淋亚分解酶会引起巨大的积累 酵母中内含子RNA的含量。因此，将使用Branchmer寡核苷酸 生成表达的内含子探针。我们的目标是（1）创建和 来自应变的“表达内含子标签”克隆的屏幕库 积聚大量内含子RNA的酵母。这些克隆会 测序以生成一个表达内含子序列的数据库，（2） 使用信息性识别类似于已知内含子的基因组序列 接近并测试它们是否在体内剪接潜力，（3） 优化每种方法的重复应用，直到完整 确认的内含子映射到基因组的序列。找到全部 内含子对完全理解 基因组的编码能力。