Introduction of YACs containing a putative mammalian replication origin into mammalian cells can generate structures that replicate autonomously
- Salk Inst. for Biological Studies, La Jolla, CA (United States)
Yeast artificial chromosomes (YACs) containing or lacking a biochemically defined DNA replication origin were transferred from yeast to mammalian cells in order to determine whether origin-dependent autonomous replication would occur. A specialized YAC vector was designed to enable selection for YACs in mammalian cells and for monitoring YAC abundance in individual mammalian cells. All of eight clones made with linear and circularized YACs lacking the origin and seven of nine clones made with linear and circularized YACs containing the origin region contained 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 containing the putative replication origin showed very heterogeneous YAC copy number and numerous integration sites when analyzed after many generations of in vitro propagation. Analysis of both clones at an early time after fusion revealed variously sized extrachromosomal YAC/yeast structures reminiscent of the extrachromosomal elements found in some cells harboring amplified genes. The data are consistent with the interpretation that YACs containing a biochemically defined origin of replication can initially replicate autonomously, followed by integration into multiple chromosomal locations, as has been reported to occur in many example of gene amplification in mammalian cells. 75 refs., 8 figs.
- OSTI ID:
- 6442150
- Journal Information:
- Somatic Cell and Molecular Genetics; (United States), Vol. 19:2; ISSN 0740-7750
- Country of Publication:
- United States
- Language:
- English
Similar Records
Autonomously replicating episomes contain mdr1 genes in a multidrugresistant human cell line
Analysis of the structural integrity of YACs comprising human immunoglobulin genes in yeast and in embryonic stem cells