MOLECULAR AND BIOINFORMATIC IDENTIFICATION AND MAPPING

酵母基因组DNA序列的完成已成为可能 关于组织和表达的大量问题 真核基因组。在这些问题中，重要的是定义一个 真核细胞生长所需的完整最低蛋白质组和 监管，理解人类癌症的关键。这是一个标志 真核生物是丰富存在的内含子、内部基因序列 在指定蛋白质的成熟信使RNA(MRNAs)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。至 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，我们有机会绘制出所有 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、特别的 时尚。我们已经开发出一种强大的分子方法来直接 以不受基因内容偏向的方式检测内含子 它被嵌入其中。寡核苷酸互补的独一无二 在剪接过程中形成的套索序列(“分枝”) 套索内含子rna的原生反转录。突变 使套索脱枝酶失活会导致大量堆积 酵母中的内含子RNA。因此，将使用支链寡核苷酸 以产生表达的内含子探针。我们的目标是(1)创造和 菌株“表达内含子标签”克隆文库的筛选 指积累大量内含子RNA的酵母。这些克隆人将 进行测序以生成表达内含子序列的数据库，(2) 用信息学方法鉴定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) 细化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。查找全部 内含子对于完全理解 基因组的编码能力。

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.