A molecular genetic approach will be used to examine whether the yeast Saccharomyces cerevisiae FLP recombinase (or coliphage P1 Cre recombinase) can mediate site-specific recombination events in mammalian cells. We will construct four different fusions of mammalian promoters to the FLP structural gene (with the lac operator sequence located at the predicted startpoint of transcription of each of the promoters) and test whether FLP mRNA is transcribed and active in the mammalian nucleus when these fusions are introduced into mouse and human fibroblasts on polyomaviral and retroviral vectors. Defective retroviral vectors carrying FLP sites (FSV's) will be constructed and transfected into mouse and human fibroblasts. We will determine whether several classes of recombination and rearrangement events catalyzed by wild-type or mutant FLP recombinases occur in mammalian cells carrying one or more FSV's. These include whether FLP can mediate (1) reciprocal translocations, (2) the integration of a circular DNa carrying a FLP site into an FSV, (3) chromosome loss, and (4) efficient ring (or deficiency) chromosome formation. We will attempt to mediate the formation of small, human centromere-bearing ring or deficiency chromosomes, to isolate (or purify) a small ring (or deficiency) chromosome with its centromere, perhaps to clone this (ring) chromosome in yeast as a linear chromosome, and to clone and begin to dissect the structure of the human centromere.
| Peddada, L B; McPherson, J D; Law, R et al. (1992) Somatic cell mapping of the human cyclophilin B gene (PPIB) to chromosome 15. Cytogenet Cell Genet 60:219-21 |
