GENETIC AND BIOCHEMICAL ANALYSIS OF A MURINE RETROPOSON

拟议的研究旨在获得更多的 完全理解生物化学和遗传行为 L1（或LINE 1）元件的基因。 L1家族无处不在 在哺乳动物中， 转座因子 遗传因素和DNA序列 分析表明，L1是小鼠中一个活跃转座因子 和其他哺乳动物;它的运动提供了一个主要来源， 基因组在进化过程中的流动， L1的突变很可能发生在生殖细胞系中。 表征 控制L1的表达和动物的运动可能导致 对基因组一般机制的重要见解 基因重排和基因表达的发育控制。 这些过程在成体组织中出错的一个结果可能是 癌 基于DNA序列分析的假设表明， L1是一个类似逆转录酶的转座因子。 然而， 没有直接的知识，涉及的中间体， L1插入的过程，或关于L1的表达和控制 在风琴上。 拟议的实验解决这些问题 未知数 收集兔抗L1抗血清 与DNA中发现的未知开放阅读框进行比对 小鼠L1的序列。 这些抗血清将用于 表征L1蛋白的表达模式。 初步实验表明，F9畸胎瘤细胞 将是进一步研究中间体的丰富来源 参与L1的表达和运动，因为“基因组”L1 RNA是在这些细胞中产生的，似乎与一种 非多聚核糖体颗粒。 其他细胞系将检查为 目的是鉴定L1表达的细胞来源 产品，以帮助进一步研究。 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颗粒。 的 抗体也将用于筛选小鼠组织， 免疫细胞化学，特别关注表达， 胚胎和生殖细胞。 其中一个开放的阅读框架， L1与逆转录酶有关;酶测定将在 对小鼠细胞和E.已知的大肠杆菌 来表达L1的这个区域。 最后，L1的能力 将使用一种选择来测试DNA中的新位置 用于疱疹胸苷激酶基因的插入失活 单纯病毒

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.