The purpose of this research is to determine whether structural changes in the repetitive components of the human genome are involved in the aging process. Studies will use fibroblast strains and clones derived from normal donors of varying age in vivo as these cell traverse their limited replicative lifespan in vitro. Lymphocytes from young and old normal donors will also be examined. Five specific areas of investigation will be pursued. 1. Copy numbers per genome will be assayed by saturation hybridization for hRI highly repetitious sequences in human fibroblast clones during aging in vitro, and in lymphocytes of varying donor age. Studies to determine the mechanism for loss of tandemly repeated DNA will include array size determinations by a new procedure and in situ hybridization to sister-cell nuclei. 2. A recently characterized """"""""transposable element"""""""" in human DNA will be examined for alterations in copy number or loci during senescence. 3. Circular DNA molecules will be monitored in human fibroblasts during the in vivo and in vitro lifespan. Circular molecules will be cloned and probed for content of specific repetitive DNA families. 4. Human DNA clones will be screened for repetitive sequences which alter in number during aging, using a novel double-label hybridization assay. Age-dependent changes in copy number will be correlated with presence of the same repetitive DNA in circular molecules, and the most interesting inserts will be sequenced. 5. Cloned repetitive and/or circular sequences will be micro-injected into fibroblast nuclei and examined for extrachromosomal replication, integration, and subsequent deletion and duplication. These studies may reveal alterations in genome organization which correlate with aging, and which can be implicated as primary causes of cellular senescence. Insight into the potential of human cells for genomic rearrangement, in particular via transposable elements and circular forms of DNA, will add to our understanding of aging and of the role such rearrangements may play in age-correlated diseases.
