REGULATION OF RETROTRANSPOSITION IN S. CEREVISIAE

酿酒酵母逆转录转座的调控

• 批准号: 6199703
• 负责人: M Joan CURCIO
• 金额: $ 21.15万
• 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/6199703
• 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.

含有反转录转座子的长末端重复序列(LTR)是一种可移动的元件，能够合成并整合其RNA基因组的DNA拷贝到宿主DNA中的新位置。逆转录转座子的许多结构和机制特征与逆转录病毒是相同的。这项拟议研究的长期目标是了解真核细胞如何调控反转录转座子和逆转录病毒的移动。以酿酒酵母Ty1反转录转座子为模型系统。基因组中的大多数Ty1元件都具有转座和高效转录的功能。然而，转座事件是罕见的，因为转座中的转录后步骤受到严格的调控。最近，合成端粒的逆转录酶的成分est2和参与端粒长度调节的激酶Te11被确定为Ty1转座的抑制剂。Te11是人类疾病基因ATM的同系物。这些和其他发现导致了一种假设，即Ty1转座是由细胞途径调控的，而细胞途径的正常作用是维持基因组的完整性。我们建议系统地鉴定RTT(调节Ty1转座)基因，以表征维持Ty1元件转座休眠的细胞通路。此外，我们还将探讨Est2和Te11抑制转座的机制。这项建议的具体目标是：1.在全基因组范围内寻找Ty1转座水平升高的酵母突变株(rttDelta突变体)。证明推测的rttDelta突变是导致Ty1转座增加的原因。2.定量检测rttDelta突变体中Ty1RNA、蛋白质、cDNAs和tRNA基因上游的整合水平，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，探讨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，或者端粒缩短是否起到抑制Ty1转座的信号作用。4.确定在没有端粒酶的情况下，Ty1转座或Ty1复制机制是否可以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，癌症和各种遗传性疾病被归因于逆转录转座子的插入。了解宿主细胞如何与逆转录病毒相互作用是至关重要的，因为逆转录病毒有可能致病。我们将确定一种特定类型的基因组损伤，即端粒丢失，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老化与无法维持逆转录转座子的休眠或逆转录病毒潜伏期之间是否存在联系。