REGULATION OF RETROTRANSPOSITION IN S. CEREVISIAE

酿酒酵母逆转录转座的调控

• 批准号: 6525771
• 负责人: M Joan CURCIO
• 金额: $ 22万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6525771
• 关键词: Retroviridae; SDS polyacrylamide gel electrophoresis; Saccharomyces cerevisiae; autoradiography; enzyme induction /repression; eukaryote; fungal genetics; gene complementation; gene expression; gene mutation; genetic regulation; genetic transcription; host organism interaction; immunoprecipitation; laboratory rabbit; northern blottings; nucleic acid repetitive sequence; polymerase chain reaction; telomerase; telomere; transposon /insertion element; western blottings

Long terminal repeat (LTR) containing retrotransposons are mobile elements capable of synthesizing and integrating a DNA copy of their RNA genome into a new location in the host DNA. Many structural and mechanistic features of LTR-retrotransposons are shared with retroviruses. The long-term goal of the proposed research is to understand how the eucaryotic cell regulates the mobility of retrotransposons and retroviruses. The Ty1 retrotransposon of Saccharomyces cerevisiae is used as a model system. Most of the Ty1 elements in the genome are functional for transposition and efficiently transcribed. However, transposition events are rare because post-transcriptional steps in transposition are tightly regulated. Recently, Est2, a component of the reverse transcriptase that synthesizes telomeres, and Te11, a kinase involved in telomere length regulation and a homolog of the human disease gene, ATM, have been identified as inhibitors of Ty1 transposition. These and other findings have led to the hypothesis that Ty1 transposition is regulated by cellular pathways whose normal role is to maintain the integrity of the genome. We propose to systematically identify RTT (regulator of Ty1 transposition) genes in order to characterize cellular pathways that maintain the transpositional dormancy of Ty1 elements. In addition, we will investigate the mechanism of inhibition of transposition by Est2 and Te11. The specific aims of this proposal are to: 1.Perform a genome-wide search for yeast mutants with elevated levels of Ty1 transposition (rttdelta mutants). Prove that the putative rttdelta mutation is the cause of increased Ty1 transposition. 2. Quantify the levels of Ty1 RNA, protein, cDNA and integration upstream of tRNA genes in rttdelta mutants to classify RTT genes. 3. Analyze different steps in Ty1 transposition in es2delta and te11delta mutants to investigate the mechanisms of inhibition of Ty1 transposition by EST2 and Te11. Determine if Est2 and Te11 directly inhibit Ty1 replication or if telomere shortening acts as a signal to derepress Ty1 transposition. 4. Determine if Ty1 transposition or the Ty1 replication machinery can promote protection of chromosome ends in the absence of telomerase. AIDS and adult T-cell leukemia/lymphoma are the consequences of retroviral infection in humans, and cancer and various inherited disorders have been attributed to insertion of retrotransposons. It is essential to understand how the host cell interacts with retroelements because of the potential of retroelements to cause disease. We will determine if a specific type of damage to the genome, loss of telomere, can result in the induction of Ty1 transposition. Loss of normal telomere function is associated with cellular senescence. Hence, the proposed experiments will determine if there is a connection between cellular aging and an inability to maintain the dormancy of retrotransposons or by analogy, the latency of retroviruses.

含有逆转录转座子的长末端重复序列 (LTR) 是能够合成 RNA 基因组的 DNA 副本并将其整合到宿主 DNA 中新位置的移动元件。 LTR-逆转录转座子的许多结构和机制特征与逆转录病毒相同。该研究的长期目标是了解真核细胞如何调节逆转录转座子和逆转录病毒的移动性。使用酿酒酵母的 Ty1 逆转录转座子作为模型系统。基因组中的大多数 Ty1 元件都具有转座功能并有效转录。然而，转座事件很少见，因为转座的转录后步骤受到严格调控。最近，Est2（合成端粒的逆转录酶的一个组成部分）和 Te11（一种参与端粒长度调节的激酶，也是人类疾病基因 ATM 的同源物）被确定为 Ty1 转座的抑制剂。这些和其他发现导致了这样的假设：Ty1 转座受到细胞途径的调节，其正常作用是维持基因组的完整性。我们建议系统地鉴定 RTT（Ty1 转座调节因子）基因，以表征维持 Ty1 元件转座休眠的细胞途径。此外，我们还将研究Est2和Te11抑制转座的机制。该提案的具体目标是： 1. 对 Ty1 转座水平升高的酵母突变体（rttdelta 突变体）进行全基因组搜索。证明假定的 rttdelta 突变是 Ty1 转座增加的原因。 2. 定量 rttdelta 突变体中 Ty1 RNA、蛋白质、cDNA 和 tRNA 基因上游整合的水平，以对 RTT 基因进行分类。 3. 分析es2delta和te11delta突变体中Ty1转座的不同步骤，研究EST2和Te11抑制Ty1转座的机制。确定 Est2 和 Te11 是否直接抑制 Ty1 复制，或者端粒缩短是否充当去抑制 Ty1 转座的信号。 4. 确定 Ty1 转座或 Ty1 复制机制是否可以在端粒酶缺失的情况下促进染色体末端的保护。艾滋病和成人T细胞白血病/淋巴瘤是人类逆转录病毒感染的后果，而癌症和各种遗传性疾病则归因于逆转录转座子的插入。由于逆转录因子有可能引起疾病，因此了解宿主细胞如何与逆转录因子相互作用至关重要。我们将确定特定类型的基因组损伤（端粒丢失）是否会导致 Ty1 转座的诱导。正常端粒功能的丧失与细胞衰老有关。因此，拟议的实验将确定细胞衰老与无法维持反转录转座子的休眠或反之亦然的反转录病毒的潜伏期之间是否存在联系。