CLONING OF THE WERNER'S SYNDROME DEFECT

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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.