INTEGRATION SPECIFICITY OF THE TY3 RETROTRANSPOSON

Ty3是酵母中的一种类似吉普赛人的反转录转座子，它整合了 靠近聚合酶III转录起始点。Ty3是 全长5.4kBP，由340bp的长末端重复序列组成 位于内部域两侧。它被转录成一个5.2kb的片段， 多腺苷化的RNA，类似于猪的基因组RNA 逆转录病毒。GAG3和POL3阅读框编码结构 蛋白质--衣壳和核衣壳--一个50 nm的核蛋白颗粒和 催化蛋白质--蛋白酶、逆转录酶和整合酶(IN)-- -元素的处理、复制和集成所必需的。 Ty3的组织和蛋白质与动物逆转录病毒相似。 逆转录病毒在体内并不是随机整合的，但宿主和 除IN以外的逆转录病毒蛋白已被鉴定为存在于 整合站点。这个项目的长期目标是 利用酵母中可用的分子遗传学方法 相对明确的Ty3的位置特异性来研究 将逆转录病毒元件传递到整合部位的机制：1)和 早期的近期目标是记录超过一年的IN物理变化 时间进程，并确定它们与一体化的关系。这些 变化包括磷酸化和蛋白质降解过程。2)主持人 参与整合的蛋白质将在GAL4杂交种中被鉴定 筛选与IN相互作用的蛋白质，并通过克隆和 补充寄主突变体Ty3基因的特性研究 整合。3)对逆转录病毒和Ty3插入的研究表明 基因组背景影响整合。5S RNA基因携带于 质粒，但不在其天然的染色体环境中，是Ty3的靶标 换位。酵母核仁结构、超卷曲和 将对染色质进行检查，以确定基因组排斥是否可以 被分配到5S基因的核环境。4)体外培养； 逆转录病毒IN足以实现分子的随机整合 类似于复制的基因组进入裸露的DNA，但在体内，它不是 知道IN是否在核心粒子中，或者它是否独立作用 在整合过程中。Ty3in将在一个异源基因下表达 启动子，单独和融合到Ty3粒子靶向和核- 靶向序列，其活性将通过基因和 身体上的手段。活动不仅表明IN的程度 细胞的自主性，但将为未来提供不可或缺的工具 不同于复制的整合研究。5)体外试验 模仿逆转录病毒系统的集成系统，但合并了 宿主蛋白，将被开发以识别相互作用 负责将Ty3集成综合体对接在目标上。6) 将构建和选择Ty3 IN突变体，以便识别 负责与集成终端交互的域 前体(在所有逆转录病毒分子中保守)和影响 集成专用性。异源DNA结合的能力 还将研究赋予新插入特异性的结构域。 这些在简单的真核生物中的实验应该会导致 可以在逆转录病毒系统中明确测试的模型。

Ty3 is a gypsy-like retrotransposon in Saccharomyces that integrates close to the site of polymerase III transcription initiation. Ty3 is 5.4 kbp in length and is composed of long terminal repeats of 340 bp flanking an internal domain. It is transcribed into a 5.2kb, polyadenylated RNA which is analogous to the genomic RNA of retroviruses. GAG3 and POL3 reading frames encode the structural proteins--capsid and nucleocapsid--of a 50 nm nucleoprotein particle and catalytic proteins--protease, reverse transcriptase, and integrase (IN)- -required for processing, replication and integration of the element. Ty3 organization and proteins resemble those of animal retroviruses. Retroviruses do not integrate randomly in vivo, yet neither host nor retroviral proteins, other than IN, have been identified as present at the integration site. The long-term objective of this project is to exploit the molecular genetic approaches available in yeast and the relatively defined position specificity of Ty3 to investigate the mechanism of delivery of a retroid element to an integration site: 1) An early immediate objective is to document physical changes in IN over a timecourse, and to determine how they relate to integration. These changes include phosphorylation and proteolytic processing. 2) Host proteins involved in integration will be identified in GAL4 hybrid screen for proteins which interact with IN and by cloning and characterization of genes which complement host mutants for Ty3 integration. 3) Studies of retroviral and Ty3 insertion suggest that genomic context affects integration. The 5S RNA gene carried on a plasmid, but not in its native chromosomal context, is a target for Ty3 transposition. Yeast mutants in nucleolar structure, supercoiling, and chromatin will be examined to determine whether genomic exclusion can be assigned to the nuclear environment of the 5S gene. 4)In vitro, retroviral IN is sufficient for random integration of molecules resembling the replicated genome into naked DNA, but in vivo, it is not known whether IN is in the core particle or if it acts independently during integration. Ty3 IN will be expressed under a heterologous promoter, alone and in fusions to Ty3 particle-targeting and nuclear- targeting sequences, and its activity will be tested by genetic and physical means. Activity would not only indicate a degree of IN autonomy in the cell, but would provide an indispensable tool for future studies of integration distinct from replication. 5) An in vitro integration system modeled on the retrovirus system, but incorporating host proteins, will be developed in order to identify the interactions responsible for docking the Ty3 integration complex at the target. 6) Ty3 IN mutants will be constructed and selected in order to identify domains responsible for interaction with the termini of the integration precursor (conserved in all retroid elements) and domains that affect integration specificity. The ability of a heterologous DNA binding domain to confer novel insertion specificity will also be examined. These experiments in a simple eukaryote should lead to the development of models which can be explicitly tested in retroviral systems.