GENOME DYNAMICS IN AGING

衰老过程中的基因组动力学

• 批准号: 6783973
• 负责人: JAN VIJG
• 金额: $ 16.46万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6783973
• 关键词: DNA damage; DNA repair; DNA replication; age difference; aging; antioxidants; biological models; cell senescence; cellular pathology; cytogenetics; embryo /fetus tissue /cell culture; fibroblasts; gene mutation; gene rearrangement; genetic markers; genetically modified animals; laboratory mouse; microarray technology; model design /development; mutant; oxidative stress; polymerase chain reaction; premature aging; reporter genes

During the previous grant period it was demonstrated that mutations at a lacZ-plasmid reporter locus accumulated during aging in an organ-specific manner. In liver and heart many of the accumulated mutations were large genome rearrangements with one breakpoint in the lacZ gene and the other elsewhere in the mouse genome. While mice with defects in transcription-related DNA repair (i.e., CSB and XPD mutants) did not show accelerated mutation accumulation during aging, they do display symptoms of premature aging, as demonstrated in Project 1 by the Hoeijmakers group. Mice defective in nucleotide excision repair (XPA mutant) or a combination of defective nucleotide excision repair and inter-strand crosslink repair (Erccl mutants) did show an accelerated increase in mutation frequency during aging. However, while the Erccl mutant displayed symptoms of accelerated aging, the Xpa mutant only showed increased cancer susceptibility, as demonstrated by Hoeijmakers and van Steeg. In the Erccl mutant, premature aging was accompanied by the accelerated accumulation of genome rearrangements mutations in liver, while in the Xpa mutant mainly point mutations were found to accumulate. In this renewal application we propose to first test the hypothesis that defects in the repair of double-strand lesions, such as double-strand breaks and cross-links, are associated with both accelerated aging and the accelerated accumulation of large genome rearrangements. Second, based on results obtained in collaboration with Campisi, indicating that accelerated cellular senescence in mouse embryonic fibroblasts is caused by oxidative stress, possibly through genomic instability, we will test if overexpression of genes encoding antioxidant enzymes can retard the formation of rearrangements and partly correct accelerated aging and/or cellular senescence in mice and cells derived from them. Then, to directly analyze individual cells from young and old mouse organs for the accumulation of genome rearrangements, single-cell methods will be applied, including cytogenetic methods and molecular assays based on whole genome amplification. Finally, to assess the hypothesized consequences of genomic instability, cell-to-cell variation in gene expression levels in young and old organs will be determined using real-time PCR and microarrays with amplified total mRNA as target.

在上一个赠款期间，已证明在衰老期间以器官特异性方式累积的LACZ-质量报告基因座的突变。在肝脏和心脏中，许多积累的突变是大基因组重排，在LACZ基因中有一个断点，而小鼠基因组中的另一个则是一个断点。尽管与转录相关的DNA修复缺陷（即CSB和XPD突变体）缺陷的小鼠在衰老过程中没有表现出加速突变积累，但它们确实显示出过早衰老的症状，如Hoeijmakers Group在项目1中所证明的那样。核苷酸切除修复（XPA突变体）中有缺陷的小鼠或缺陷的核苷酸切除修复和链间交联修复（ERCCL突变体）的组合确实显示出在衰老期间突变频率的加速增加。但是，尽管ERCCL突变体显示出加速衰老的症状，但XPA突变体仅显示出癌症易感性的增加，如Hoeijmakers和van Steeg所证明的那样。在ERCCL突变体中过早 衰老伴随着肝脏基因组重排突变的加速积累，而在XPA突变体中，发现点突变主要是积累的。在此续签应用中，我们建议首先检验以下假设：双链病变的修复缺陷，例如双链断裂和交联，与加速衰老和大基因组重排的加速积累有关。其次，基于与Campisi合作获得的结果，表明小鼠胚胎成纤维细胞中加速的细胞衰老是由氧化应激引起的，这可能是由于基因组不稳定性而引起的，我们将测试是否过表达编码抗氧化剂酶的基因 重排的形成和部分纠正了小鼠和细胞中的加速衰老和/或细胞衰老。然后，为了直接分析年轻小鼠器官和基因组重排积累的单个细胞，将应用单细胞方法，包括基于整个基因组扩增的细胞遗传学方法和分子测定。最后，为了评估基因组不稳定性的假设后果，将使用实时PCR和微阵列确定基因表达水平的细胞对细胞表达水平的变化，并以扩增的总mRNA为靶标。