CLONING OF THE WERNER'S SYNDROME DEFECT

沃纳综合征，一种常染色体隐性遗传病 受多种特征的加速老化，也具有明显的 缩短了体外寿命。沃纳综合征成纤维细胞展示 几种细胞复制异常--包括生长缓慢 比率，对有丝分裂原反应降低，染色体不稳定 以及DNA复制起始率的异常。然而， 导致增殖性细胞减少的分子缺陷 这些细胞中的容量未知。 这项提议旨在识别能够互补的基因 Werner综合征的复制能力降低 成纤维细胞。第二个目标是识别能够延长寿命的基因 体外培养正常人二倍体成纤维细胞的寿命。我 建议利用一种具有可选择的 用于转移正常人DNA的遗传耐药标记 二倍体细胞或转化细胞转化为Werner综合征和 正常的二倍体成纤维细胞。 基本步骤包括构建一个cdna文库。 从年轻的二倍体或转化的细胞，连接文库 进入逆转录病毒载体，包装逆转录病毒，感染 Werner综合征或正常成纤维细胞，克隆的选择 增强的生长潜力以及分离和鉴定 互补的基因。 将使用两种类型的cDNA文库作为来源 互补基因：一种来自分离自中国仓鼠的 细胞呈指数级增长，其中一种来自S早期的细胞 用消减杂交技术检测细胞周期时相 来源于GO停滞的人类二倍体细胞。 在分离出活跃增殖的克隆后，它们将 其特征是确定之前的累计倍增 衰老。逆转录病毒插入的基因能够延长 寿命将通过Southern杂交来鉴定 病毒探针或通过重叠感染辅助病毒和拯救 整合的重组逆转录病毒。我希望能辨认出荒野 类型基因，将被用作分离Werner‘s的探针 综合症缺陷序列，并识别可能的基因 调节正常二倍体细胞的寿命。

Werner's syndrome, an autosomal recessive disorder characterized by multiple features of accelerated aging, also has a markedly reduced in vitro lifespan. Werner's syndrome fibroblasts exhibit several cell replication abnormalities--including slow growth rates, decreased response to mitogens, chromosomal instability and abnormalities in DNA replication initiation rates. However, the molecular defect responsible for diminished proliferative capacity in these cells in unknown. This proposal aims to identify genes capable of complementing the diminished replicative capacity in Werner's syndrome fibroblasts. A secondary goal is to identify genes able to prolong the lifespan of normal human diploid fibroblasts in vitro. I propose utilizing an amphotropic retrovirus with a selectable genetic drug resistance marker to transfer DNA from normal diploid cells or transformed cells to Werner's syndrome and normal diploid fibroblasts. The basic procedure will involve construction of a cDNA library from young diploid or transformed cells, ligation of the library into a retrovirus vector, packaging the retrovirus, infection of Werner's syndrome or normal fibroblasts, selection of clones with enhanced growth potential and isolation and characterization of complementing genes. Two types of cDNA libraries will be used as sources for complementing genes: one from mRNA isolated from exponentially growing cells, and one from cells in the early S phase of the cell cycle by subtraction hybridization to mRNA derived from Go arrested human diploid cells. After actively proliferating clones are isolated, they will be characterized by determining cumulative doublings before senescence. Retroviarlly inserted genes capable of prolonging lifespan will be identified either by southern hybridization with viral probes or by superinfection with helper virus and rescue of the integrated recombinant retrovirus. I hope to identify the wild type gene which will be used as a probe to isolate the Werner's syndrome defective sequence, and to identify genes that may regulate the lifespan of normal diploid cells.