GENETIC AND BIOCHEMICAL ANALYSIS OF A MURINE RETROPOSON

鼠逆转录子的遗传和生化分析

• 批准号: 2180283
• 负责人: SANDRA L MARTIN
• 金额: $ 19.55万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2180283
• 关键词: biochemical evolution; developmental genetics; gel electrophoresis; gene expression; immunocytochemistry; laboratory mouse; nucleic acid hybridization; nucleic acid probes; nucleic acid sequence; polymerase chain reaction; protein biosynthesis; protein structure function; ribonucleoproteins; southern blotting; transposon /insertion element

The proposed research is directed towards gaining a more complete understanding of the biochemical and genetic behavior of the L1 (or LINE1) element in mice. The L1 family is ubiquitous in mammals and shares some characteristics with known transposable elements. Genetic considerations and DNA sequence analysis suggest that L1 is an active transposable element in mice and other mammals; its movement provides a major source of genomic flux in evolutionary time, and expression and movement of L1 is likely to occur in the germline. Characterization of the control of L1 expression and movement in the animal may lead to important insights into the general mechanisms of genome rearrangement and developmental control of gene expression. One result of these processes going awry in adult tissue may be cancer. Hypotheses based on DNA sequence analysis suggest that L1 is a retroposon-like transposable element. There is, however, no direct knowledge about the intermediates involved in the process of L1 insertion, or about the expression and control of L1 in the organsim. The proposed experiments address these unknowns. A collection of rabbit anti-L1 antisera will be made against unidentified open reading frames found in the DNA sequence of mouse L1. These antisera will be used to characterize the pattern of expression of L1 proteins. Preliminary experiments suggest that F9 teratocarcinoma cells will be a fruitful source for further study of the intermediates involved in L1 expression and movement, because a "genomic" L1 RNA is made in these cells and appears to be associated with a non-polyribosomal particle. Other cell lines will be examined as well with a goal of identifying a cell source of L1 expression products to aid further study. Antibodies will be used to identify the L1 proteins expressed in F9 and other cells, and to characterize the particle by immunoelectron microscopy. The antibodies will also be used to screen mouse tissues by immunocytochemistry, focusing particularly on expression in embryos and germ cells. One of the open reading frames found in L1 is related to reverse transcriptases; enzyme assays will be done on extracts from both mouse cells and E. coli that are known to be expressing this region of L1. Finally, the ability of L1 to move into new locations in DNA will be tested using a selection for insertional inactivation of the thymidine kinase gene of herpes simplex virus.

拟议的研究旨在获得更多 完全了解生化和遗传行为 小鼠中的L1（或line1）元素的元素。 L1家族无处不在 在哺乳动物中，并具有一些已知的特征 转座元素。 遗传因素和DNA序列 分析表明L1是小鼠的活性转座元件 和其他哺乳动物；它的运动提供了主要来源 进化时间的基因组通量以及表达和运动 L1的生殖线可能发生。 表征 控制动物的L1表达和运动可能导致 对基因组一般机制的重要见解 基因表达的重排和发育控制。 这些过程在成人组织中出现问题的结果可能是 癌症。 基于DNA序列分析的假设表明 L1是一种类似倒角的转座元件。 但是，有 没有关于涉及的中间体的直接知识 L1插入的过程，或关于L1的表达和控制 在Organsim中。 提出的实验解决了这些 未知。 将制作一组兔子抗L1抗血清 反对在DNA中发现的未识别的开放式阅读框 小鼠L1的序列。 这些抗血清将习惯 表征L1蛋白的表达模式。 初步实验表明F9 Teratocinama细胞 将成为进一步研究中间体的富有成果的来源 参与L1表达和运动，因为“基因组” L1 RNA在这些细胞中制成，似乎与 非胆小性粒子粒子。 其他细胞系将被检查为 以确定L1表达的细胞来源的目标 有助于进一步研究的产品。 抗体将用于识别 在F9和其他细胞中表达的L1蛋白，以及 通过免疫电子显微镜表征粒子。 这 抗体也将用于筛选小鼠组织 免疫细胞化学，特别关注 胚胎和生殖细胞。 在 L1与逆转录酶有关；酶测定将是 在已知的小鼠细胞和大肠杆菌的提取物上完成 表达该区域的L1。 最后，L1的能力 将使用选择测试DNA中的新位置 为了插入疱疹胸苷激酶基因 单纯性病毒。