GENETIC AND BIOCHEMICAL ANALYSIS OF A MURINE RETROPOSON

拟议的研究旨在获得更多 对生化和遗传行为的全面理解 L1(或LINE1)元件在小鼠中的表达。L1家族无处不在 在哺乳动物中，并与已知的 转座因子。遗传因素和DNA序列 分析表明，L1在小鼠体内是一种活性转座元件 和其他哺乳动物；它的运动提供了 进化时间中的基因组流量以及表达和运动 L1的突变可能发生在生殖系中。刻画人物形象 控制L1在动物体内的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调控。 这些过程在成人组织中出错的一个结果可能是 癌症。基于DNA序列分析的假说表明 L1是一个逆转座子样转座子元件。然而，存在这样的问题， 对参与其中的中间体没有直接了解 L1插入的过程，或L1的表达和调控 在器乐表演中。拟议的实验解决了这些问题 未知数。将收集兔抗L1抗血清 与DNA中发现的未知开放阅读框进行比较 鼠标L1的序列。这些抗血清将被用来 描述L1蛋白的表达模式。 初步实验表明，F9畸胎癌细胞 将为进一步研究中间体提供丰富的资源。 参与L1的表达和运动，因为一个“基因组”的L1 RNA是在这些细胞中制造的，似乎与一种 非多聚核糖体颗粒。其他细胞系将被检查为 好的，目标是确定L1表达的细胞来源 帮助进一步学习的产品。抗体将被用来识别 在F9和其他细胞中表达的L1蛋白，以及 用免疫电子显微镜对颗粒进行表征。这个 抗体也将用于筛选老鼠组织，方法是 免疫细胞化学，特别关注表达在 胚胎和生殖细胞。中发现的开放阅读框之一 L1与逆转录酶有关；酶分析将是 对已知的小鼠细胞和大肠杆菌的提取物进行了研究 来表达L1的这个区域。最后，L1的能力 移动到DNA中的新位置将使用选择进行测试 对疱疹病毒胸苷激酶基因的插入失活 单纯疱疹病毒。

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.