CLONING OF THE WERNER'S SYNDROME DEFECT

沃纳综合征，一种常染色体隐性遗传疾病， 加速老化的多重特征，也具有明显的 体外寿命缩短。 沃纳综合征成纤维细胞表现出 几种细胞复制异常--包括生长缓慢 率，对有丝分裂原的反应降低，染色体不稳定性 和DNA复制起始率的异常。 然而，在这方面， 导致细胞增殖减少的分子缺陷 这些细胞的能力未知。 这项提议旨在确定能够补充 维尔纳综合征的复制能力减弱 成纤维细胞 第二个目标是确定能够延长 正常人二倍体成纤维细胞的体外寿命。 我 建议利用嗜热逆转录病毒， 遗传耐药标记，将DNA从正常 二倍体细胞或转化细胞对沃纳综合征的影响， 正常二倍体成纤维细胞。 基本步骤包括构建cDNA文库 从年轻的二倍体或转化细胞，文库的连接 转化成逆转录病毒载体，包装逆转录病毒，感染 维尔纳综合征或正常成纤维细胞，选择具有 增强的生长潜力以及分离和表征 互补基因 两种类型的cDNA文库将用作 互补基因：一个来自mRNA， 指数生长的细胞，和一个来自早期S 通过与mRNA的消减杂交测定细胞周期的时相 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，它们将被 其特征在于， 衰老 逆转录插入的基因能够延长 寿命将通过南方杂交来确定， 病毒探针或用辅助病毒重复感染， 整合的重组逆转录病毒。 我希望能鉴别出 型基因，这将被用作探针，以分离沃纳的 综合征缺陷序列，并鉴定可能 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.