| 摘要 |
Eucaryotic DNA replicaton origin sequences are used as cloning vectors in placing DNA sequences into eucaryotic cells. By a "shotgun" procedure, random DNA segments, connected to eucaryotic replication origin sequences, are transfected into transformed eucaryotic cells which are then selected for cells that have reverted to normal cell phenotype. From the reverted cells, plasmid DNA molecules are isolated and when attached to procaryotic plasmids are amplified in bacteria. The eucaryotic replication origin sequences are obtained by isolating scrambled and palindromic (i.e. inverted repeat) eucaryotic DNA segments, joining the palindromic segments to procaryotic plasmids, transfecttu ing the set of recombinant DNA molecules into eucaryotic cells and selecting for surviving, replicating, supercoiled recombinant molecules. These molecules are replicated in bacteria and then isolated as supercoiled plasmids. Random DNA segments are preferably obtained from the genome of eucaryotic cells of the same phenotype as the phenotypically transformed cells and are scrambled by dividing and randomly joining the segments. Sets of these scrambled segments are attached to eucaryotic replicating origin sequences and used as reverter plasmids in transformed cells. Efficiency in changing cellular phenotypes is enhanced by incorporating additional palindromic eucaryotic DNA sequences, each at least 25 base pairs long. By appropriate survival selection techniques, reverter plasmids are obtained from tumorigenic cells and from virally infected cells. Reverter plasmids are potentially obtainable from other phenotypically altered eucaryotic cells, for example, cells containing DNA sequences which code for auto-immune disease, and cells manifesting the effects of aging. |
