REGULATION AND FUNCTION OF RETROELEMENTS

后勤的监管和职能

• 批准号: 6108044
• 负责人: Henry L. LEVIN
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6108044
• 关键词: RNA directed DNA polymerase; gene expression; gene mutation; genetic regulation; genetic regulatory element; genetic transcription; molecular genetics; ribonuclease H; transfer RNA; transposon /insertion element

To understand the complex functions required for the propagation of retroelements we study retrotransposons, a family of elements that are closely related to retroviruses. A significant advantage of studying retrotransposons is that they exist in hosts such as yeast that can readily be studied using sophisticated molecular genetic techniques. In the process of characterizing transposition in Schizosaccharomyces pombe, we have obtained strong evidence that the reverse transcriptase of the retrotransposon Tf1 uses a novel mechanism of self-priming to initiate cDNA synthesis. This is in contrast to the tRNA mechanisms thought to be used by all other LTR-containing elements. The first 11 bases of the Tf1 transcript anneal to the primer binding site and a cleavage reaction between the 11th and 12th nucleotides results in the initiation of reverse transcription. This region of the mRNA forms a complex RNA structure composed of 27 basepairs that includes the primer binding site and the first 11 nucleotides. We have shown previously that the formation of the 27 basepair structure is required for reverse transcription. We have identified an additional structure composed of a 7 basepair stem and a 25 nucleotide loop. Extensive mutagenesis of this stem-loop indicated that it too is required for reverse transcription in vivo. The position, size and function of this stem-loop in the mRNA of Tf1 is analogous to the U5-IR stem-loop of Rous sarcoma virus. The U5-IR stem-loop has also been identified in several other retroviruses suggesting that this structure has been highly conserved. In an effort to identify host factors that contribute to the transposition of Tf1, we have isolated mutated strains of S. pombe that are defective for transposition. Mutations in three genes, hop1, hop2, and hop3, greatly reduce the frequency of Tf1 transposition. As the result of large scale complementation screens, two of these genes have now been cloned. The sequence of hop3 indicated that it is a homologue of the sin3 genes found in S. cerevisiae and mammals. Because sin3 proteins are known to contribute to the deacetylation of histones and the condensation of chromatin, we are studying the possibility that Tf1 integration requires specific types of chromatin structure. The sequence of hop5 indicated that this gene is closely related to a class of nucleoporins that contain multiple copies of FXFG repeats. We are pursuing the model that hop5 is a nuclear pore factor that is required for the nuclear import of Tf1 integrase and reverse transcripts.

了解传播所需的复杂功能 我们研究返回座子的追溯元素，一个元素家族 与逆转录病毒密切相关。 学习的重要优势 逆转座子是它们存在于宿主中，例如酵母菌 很容易使用复杂的分子遗传技术研究。 在 表征schizosacchomyces pombe中的换位的过程， 我们已经获得了有力的证据，表明 Retrotransposon TF1使用一种新型的自我宣传机制来启动 cDNA合成。 这与被认为是的tRNA机制相反 所有其他含LTR的元素都使用。 TF1的前11个基础 对底漆结合位点的转录本退火和裂解反应 在第11和第12个核苷酸之间导致启动 逆转录。 mRNA的该区域形成复杂的RNA 由27个基础底座组成的结构，包括底漆结合位点 和前11个核苷酸。 我们之前已经证明了 反向需要27个基本结构的形成 转录。 我们已经确定了一个由A组成的额外结构 7基台茎和25个​​核苷酸环。 大量的诱变 茎环表明，在 体内。 该茎环在mRNA中的位置，大小和功能 TF1类似于Rous肉瘤病毒的U5-IR茎环。 U5-ir 在其他几个逆转录病毒中也发现了茎环 表明这种结构是高度保守的。 努力 为了确定有助于TF1转位的宿主因素，我们 具有孤立的突变菌菌菌菌菌株有缺陷 换位。 三个基因的突变，hop1，hop2和hop3 降低TF1换位的频率。 大规模的结果 互补筛，其中两个基因已被克隆。 这 hop3的序列表明它是发现的SIN3基因的同源物 在酿酒酵母和哺乳动物中。 因为SIN3蛋白已知 有助于组蛋白的脱乙酰化和凝结 染色质，我们正在研究TF1整合需要的可能性 特定类型的染色质结构。 霍普5的顺序指示 该基因与一类包含的核孔蛋白密切相关 FXFG重复的多个副本。 我们正在追求Hop5是 TF1核进口所需的核孔因子 集成酶和逆成绩单。