INTRODUCTION OF YACS CONTAINING A PUTATIVE MAMMALIAN REPLICATION ORIGIN INTO MAMMALIAN-CELLS CAN GENERATE STRUCTURES THAT REPLICATE AUTONOMOUSLY

Yeast artificial chromosomes (YACs) containing or lacking a biochemica lly defined DNA replication origin were transferred from yeast to mamm alian cells in order to determine whether origin-dependent autonomous replication would occur. A specialized YAC vector was designed to enab le selection for YACs in mammalian cells and for monitoring YAC abunda nce in individual mammalian cells. All of eight clones made with linea r and circularized YACs lacking the origin and seven of nine clones ma de with linear and circularized YACs containing the origin region cont ained single copies of the transfected YAC, along with various amounts of yeast DNA, integrated into single but different chromosomal sites. By contrast, two transformants derived from circularized YACs contain ing the putative replication origin showed very heterogeneous YAC copy number and numerous integration sites when analyzed after many genera tions of in vitro propagation. Analysis of both clones at an early tim e after fusion revealed variously sized extrachromosomal YAC/yeast str uctures reminiscent of the extrachromosomal elements found in some cel ls harboring amplified genes. The data are consistent with the interpr etation that YACs containing a biochemically defined origin of replica tion can initially replicate autonomously, followed by integration int o multiple chromosomal locations, as has been reported to occur in man y examples of gene amplification in mammalian cells.