The proposed work has three goals: (i) To develop and apply a new human cell system, GPT 3, that will permit the use of selection to detect gene mutations but that will be uniquely well suited for the detection of partial or complete loss of the human Chr. X and Chr. 16. The GPT 3 cell system will have unusual capabilities for detecting agents and conditions that promote non-disjunction and chromosome breakage, both of which contribute to congenital abnormalities and cancer causation. The special capability of the GPT 3 system lies in its ability to sensitively detect by convenient selection partial or complete chromosome losses that can't be detected by available mutation detection methods with mammalian cells. (ii) To use the numerous deletions that can be induced in GPT 3 cells by Gamma-rays to construct a high resolution linkage map of recombinant DNA clones from the human X chromosome. This map will expedite the discovery of DNA linkage markers (RFLPs) for X-chromosomal genes that cause hereditary disease and may contribute toward isolating those genes. (iii) To use tiny human centric chromosomes X and 16 that have been created by Gamma-irradiation as starting materials for cloning functional human centromeres. The ability of DNA segments from these chromosomes to mitotically stabilize extrachromosomally replicating plasmids in human cells will be used to isolate recombinant clones that have functional centromeres. The study of these clones will contribute to knowledge of processes that govern normal chromosome distribution and will be an important step toward the construction of artificial, stable human chromosomes that do not integrate into chromosomes of the cell.
