Regulation of retrotransposition in S.cerevisiae

酿酒酵母逆转录转座的调控

• 批准号: 7098850
• 负责人: M Joan CURCIO
• 金额: $ 28.87万
• 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 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7098850
• 关键词: Retroviridae; SDS polyacrylamide gel electrophoresis; Saccharomyces cerevisiae; autoradiography; complementary DNA; enzyme induction /repression; eukaryote; fungal genetics; gene complementation; gene expression; gene mutation; genetic regulation; genetic transcription; host organism interaction; immunoprecipitation; northern blottings; nucleic acid repetitive sequence; nucleic acid structure; polymerase chain reaction; telomerase; telomere; transposon /insertion element; western blottings

DESCRIPTION (provided by applicant): Long terminal repeat (LTR)-retrotransposons are mobile elements that resemble retroviruses. RNA synthesized from LTR-retrotransposons is copied back into DNA, and this "copy DNA" (cDNA) is then integrated at a new location in the genome. LTR-retrotransposons are widespread throughout eukaryotes, where they activate and inactivate host genes, promote genome rearrangements, and produce and disperse copies of host genes. Hence, they play an important role in the structure and evolution of the eukaryotic genome. Ty1 LTR-retrotransposons of the yeast, Saccharomyces cerevisiae, are typically functional and expressed at high levels, yet transposition occurs rarely because cDNA synthesis is inhibited by numerous host factors. The long-term goal of our work is to understand how the dormancy of functional LTR-retrotransposons is maintained. Our recent work has demonstrated that telomere erosion in the absence of telomerase triggers the "lesion-induced Ty1 activation pathway", a DNA-damage signaling pathway that mobilizes Ty1 elements. In this proposal, we will test the hypothesis that a large group of genes necessary for genome stability and for the suppression of tumor formation in higher eukaryotes (known as caretaker genes) blocks the formation DNA lesions that trigger the "lesion-induced Ty1 activation pathway". In their absence, the Ty1 activation pathway stimulates the synthesis of Ty1 cDNA, which results in excessive transposition. Moreover, the proposed experiments will test the hypothesis that enhanced Ty1 reverse transcriptase activity in telomerase-negative mutants results in the production and dispersal of cDNA copies of a subtelomeric repeat called Y'. Amplification of Y' repeats is one characteristic of telomerase-independent, recombination-dependent alternative telomere structures. Alternative telomere structures allow both yeast cells and human cancer cells to grow continuously in the absence of telomerase. The specific aims are: 1. Screen previously identified inhibitors of Ty1 transposition for those that repress the lesion-induced Ty1 activation pathway. Identify and characterize the components of the lesion-induced Ty1 activation pathway. 2. Perform biochemical characterization of specific intermediates in retrotransposition in wild-type and mutant strains to identify the mechanism of stimulating cDNA synthesis in mutants in which the lesion-induced Ty1 activation pathway is activated. 3. Determine the structure of Y' cDNA in telomerase-negative mutants, and determine how and where Y' cDNA is inserted into the genome. Determine the role played by Ty1 gene products in the synthesis and mobility of Y' cDNA.

描述（由申请方提供）：长末端重复序列（LTR）-逆转录转座子是类似于逆转录病毒的移动的元件。从LTR反转录转座子合成的RNA被复制回DNA，然后这个“拷贝DNA”（cDNA）被整合到基因组中的新位置。LTR-反转录转座子广泛存在于真核生物中，它们激活和复制宿主基因，促进基因组重排，并产生和分散宿主基因的拷贝。因此，它们在真核基因组的结构和进化中起着重要作用。Ty 1 LTR-逆转录转座子的酵母，酿酒酵母，通常是功能性的，并在高水平表达，但转座很少发生，因为cDNA合成被许多宿主因子抑制。我们工作的长期目标是了解功能性LTR反转录转座子的休眠是如何维持的。我们最近的工作表明，端粒侵蚀在端粒酶的情况下触发“病变诱导的Ty 1激活途径”，DNA损伤信号通路，动员Ty 1元件。在这个建议中，我们将测试的假设，即基因组稳定性和抑制肿瘤形成在高等真核生物（称为看守基因）所必需的一个大的基因组阻止形成DNA损伤，触发“损伤诱导的Ty 1激活途径”。在它们不存在的情况下，Ty 1活化途径刺激Ty 1 cDNA的合成，这导致过度转座。此外，所提出的实验将检验这样的假设，即在端粒酶阴性突变体中增强的Ty 1逆转录酶活性导致亚端粒重复序列Y '的cDNA拷贝的产生和分散。Y'重复序列的扩增是端粒非依赖性、重组依赖性的替代端粒结构的特征之一。不同的端粒结构允许酵母细胞和人类癌细胞在没有端粒酶的情况下连续生长。 具体目标是： 1.筛选先前鉴定的Ty 1转座抑制剂，以抑制病变诱导的Ty 1活化途径。识别和表征损伤诱导的Ty 1激活途径的组分。 2.对野生型和突变株中逆转录转座中的特定中间体进行生化表征，以确定刺激损伤诱导的Ty 1活化途径被激活的突变体中cDNA合成的机制。 3.确定端粒酶阴性突变体中Y' cDNA的结构，并确定Y' cDNA如何插入基因组以及插入基因组的位置。确定Ty 1基因产物在Y' cDNA的合成和迁移中所起的作用。