Regulation of retrotransposition in S.cerevisiae

酿酒酵母逆转录转座的调控

• 批准号: 6825537
• 负责人: M Joan CURCIO
• 金额: $ 28.04万
• 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/6825537
• 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(申请人提供)：长末端重复(LTR)-逆转录转座子是类似逆转录病毒的可移动元件。从反转录转座子合成的RNA被复制回DNA中，然后这个“复制DNA”(CDNA)被整合到基因组中的一个新位置。逆转录转座子广泛存在于真核生物中，激活和失活宿主基因，促进基因组重排，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的作用。酿酒酵母Ty1 LTR-逆转录转座子是典型的功能性转座子，高水平表达，但转座很少发生，因为大量宿主因子抑制了cdna的合成。我们工作的长期目标是了解功能LTR-逆转座子的休眠是如何维持的。我们最近的工作证明，在没有端粒酶的情况下，端粒侵蚀会触发“损伤诱导的Ty1激活通路”，这是一条动员Ty1元件的DNA损伤信号通路。在这项提案中，我们将检验一种假设，即在高等真核生物中，一大组基因组稳定和抑制肿瘤形成所必需的基因(称为看守基因)阻止DNA损伤的形成，从而触发“损伤诱导的Ty1激活途径”。在没有它们的情况下，Ty1激活途径刺激Ty1 cDNA的合成，从而导致过度的转座。此外，拟议的实验将检验这样的假设，即在端粒酶阴性突变体中增强Ty1逆转录酶活性会导致名为Y‘的亚端粒重复序列的cDNA拷贝的产生和分散。Y‘重复序列的扩增是端粒酶非依赖、重组依赖的替代端粒结构的特征之一。另一种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持续生长。 具体目标是： 1.筛选先前已确定的Ty1转座抑制剂，寻找那些抑制病变诱导的Ty1激活途径的抑制剂。确定和表征损伤诱导的Ty1激活途径的组成部分。 2.对野生型和突变型菌株逆转录转座过程中的特定中间产物进行生化鉴定，以确定在病变诱导的Ty1激活途径被激活的突变体中刺激cDNAs合成的机制。 3.确定端粒酶阴性突变体中Y‘基因的结构，并确定Y’基因插入基因组的方式和位置。确定Ty1基因产物在Y‘cDNA合成和迁移中的作用。