A shuttle system for transfer of YACs between yeast and mammalian cells.
The development of a system for shuttling DNA cloned as yeast artificial chromosomes (YACs) between yeast and mammalian cells requires that the DNA is maintained as extrachromosomal elements in both cell types. We have recently shown that circular YACs carrying the Epstein-Barr virus origin of plasmid replication (oriP) are maintained as stable, episomal elements in a human kidney cell line constitutively expressing the viral transactivator protein EBNA-1. Here, we demonstrate that a 90-kb episomal YAC can be isolated intact from human cells by a simple alkaline lysis procedure and shuttled back into Saccharomyces cerevisiae by spheroplast transformation. In addition, we demonstrate that the 90-kb YAC can be isolated intact from yeast cells. The ability to shuttle large, intact fragments of DNA between yeast and human cells should provide a powerful tool in the manipulation and analysis of functional regions of mammalian DNA.
Simpson
K
K
Department of Biochemistry and Molecular Genetics, Imperial College School of Medicine at St. Mary's, London, UK.
Huxley
C
C
D002459:Cell Fusion; D002460:Cell Line; D018244:Chromosomes, Artificial, Yeast; D004247:DNA / Q000302:isolation & purification; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D004854:Herpesvirus 4, Human / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007668:Kidney; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013104:Spheroplasts / Q000235:genetics
D019309:Epstein-Barr Virus Nuclear Antigens; D004247:DNA