INTEGRATION SPECIFICITY OF THE TY3 RETROTRANSPOSON

Ty 3是酵母属中的吉普赛样逆转录转座子，其整合 靠近聚合酶III转录起始位点。 Ty 3是 5.4长度为340 bp，由340 bp的长末端重复序列组成 侧接内部结构域。 它被转录成5.2kb， 多聚腺苷酸化的RNA，其类似于 逆转录病毒 GAG 3和POL 3阅读框编码结构化的 50 nm核蛋白颗粒的蛋白质-衣壳和核衣壳， 催化蛋白-蛋白酶，逆转录酶和整合酶（IN）- - 元素的处理、复制和集成所需。 Ty 3组织和蛋白质类似于动物逆转录病毒。 逆转录病毒不会在体内随机整合，但宿主和 除了IN以外的逆转录病毒蛋白已经被鉴定为存在于 整合网站。 该项目的长期目标是 利用酵母中可用的分子遗传学方法， 相对确定的Ty 3的位置特异性，以研究 将逆转录酶元件递送至整合位点的机制：1） 早期的直接目标是记录IN在一段时间内的物理变化， 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水解加工。 2)主机 将在GAL 4杂合体中鉴定参与整合的蛋白质 筛选与IN相互作用的蛋白质， Ty 3的互补宿主突变体的基因的表征 一体化3)逆转录病毒和Ty 3插入的研究表明， 基因组环境影响整合。 5S RNA基因携带在一个 质粒，但不是在其天然染色体环境中，是Ty 3的靶标 换位 酵母突变体的核仁结构，超螺旋， 将检查染色质以确定基因组排除是否可以 分配给5S基因的核环境。4）在体外， 逆转录病毒IN足以使分子随机整合 类似于复制的基因组进入裸露的DNA，但在体内，它不是 已知IN是否在核心粒子中或是否独立作用 在整合过程中。 Ty 3 IN将在异源条件下表达。 启动子，单独的和与Ty 3颗粒靶向和核融合的启动子， 靶向序列，其活性将通过遗传和 物理手段。 活动不仅表明IN的程度 在细胞中的自主性，但将提供一个不可或缺的工具， 研究整合不同于复制。5)体外 整合系统模仿逆转录病毒系统，但纳入 宿主蛋白质，将被开发，以确定相互作用 负责将Ty 3整合复合物对接在靶标上。六、 将构建和选择Ty 3 IN突变体以鉴定 负责与整合末端相互作用的结构域 前体（在所有逆转录酶元件中保守）和影响 整合特异性 异源DNA结合 还将检查赋予新的插入特异性的结构域。 这些在简单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.