HUMAN TRANSPOSABLE ELEMENT

人类转座元件

• 批准号: 3304859
• 负责人: HAIG H. KAZAZIAN
• 金额: $ 16.97万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-04-01 至 1994-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3304859
• 关键词: Marfan syndrome; Primates; RNA biosynthesis; RNA directed DNA polymerase; Tourette's syndrome; alleles; autosomal dominant trait; biochemical evolution; computer assisted sequence analysis; developmental genetics; gel electrophoresis; gene expression; genetic disorder diagnosis; genetic mapping; genetic models; genetic regulatory element; genetic transcription; genetic translation; hemophilia As; human population genetics; human subject; linkage mapping; molecular cloning; molecular dynamics; nucleic acid hybridization; nucleic acid sequence; oligonucleotides; open reading frames; plasmids; polymerase chain reaction; restriction fragment length polymorphism; tissue /cell culture; transfection; transposon /insertion element; yeasts

Retrotransposition events have been well documented in yeast and Drosophila for many years. In these organisms much is known about the details of this mysterious biological phenomenon. In man, until recently we could only surmise that such events occurred, although the presence of highly repeated human elements with sequence homology to retrotransposons of other species strongly suggested their existence. In 1987 our lab found two instances of de novo retrotransposition of L1 sequences causing de novo hemophilia A in man. In this application we first propose to finish tracking the functional precursor of one of these elements and to describe thoroughly its subfamily and the subfamilies' evolution. We will then isolate the functional precursor of the second L1 element which retrotransposed and compare the structure of these two elements. These studies are critical to our knowledge of the biology and propagation of L1 elements. Second, we will use short oligonucleotide probes specific for each subfamily to search for new insertions in de novo cases of autosomal dominant and X-linked dominant disorders. We should be able to clone the gene for one of these disorders through use of the sequence flanking the inserted L1 element. Third, we will study the expression of the functional element found in the first aim in vivo and in vitro. We will discover whether element found in the first aim in vivo and in vitro. We will discover whether this sequence is transcribed after transfection in a "natural state" without an overexpressing promoter, whether the open-reading frames are translated in cells, and whether the second open-reading frame can code for a natural reverse transcriptase. All of these studies should provide fundamental answers to questions as to how certain L1 elements act as retrotransposons. The second aim should lead us to a novel scientific use of these rare events, the cloning of a hitherto unknown disease gene.

逆转录事件已在酵母和果蝇中得到充分记录 多年。 在这些生物中，对此的细节知之甚少 神秘的生物学现象。 在人类中，直到最近我们才能 推测发生了此类事件，尽管存在高度重复 具有序列同源性的人类元素，可逆转其他物种 强烈建议他们的存在。 1987年，我们的实验室发现了两个实例 L1序列的从头逆转录起作用，导致NOVO HELLOBILIA A IN 男人。 在此应用程序中，我们首先建议完成跟踪 这些元素之一的功能前体，并彻底描述 它的亚科和亚家族的进化。 然后，我们将隔离 第二L1元素的功能前体，该元素逆转和 比较这两个元素的结构。这些研究对 我们对L1元素的生物学和传播的了解。 第二，我们 将使用针对每个亚科特定的寡核苷酸探针进行搜索 对于新从常染色体显性和X连锁的新插入 主要疾病。 我们应该能够克隆其中一个基因 通过使用插入的L1元素侧面的序列来疾病。 第三，我们将研究在 首先在体内和体外。 我们会发现元素是否在 体内和体外的第一个目标。 我们将发现这个序列是否 转染后以“自然状态”转录，没有 过表达的启动子，是否翻译开放式阅读帧 单元格以及第二个开读框架是否可以为天然编码 逆转录酶。 所有这些研究都应提供基本 关于某些L1元素如何充当逆转座子的问题的答案。 第二个目标应该导致我们对这些罕见的科学用途进行新颖的科学用途 事件，迄今未知疾病基因的克隆。