Analysis of a YAC with human telomeres and oriP from epstein-barr virus in yeast and 293 cells.
One approach to the construction and propagation of a mammalian artificial chromosome is to build it up in Saccharomyces cerevisiae, using a yeast artificial chromosome (YAC) base. We have demonstrated that circular YACs carrying the Epstein-Barr virus origin of plasmid replication ( oriP ) are maintained as stable, episomal elements in human cells. We wished to determine whether this technology could be extended, to generate linear extrachromosomal elements. Here, we describe the generation of retrofitting constructs, which permit the addition of human telomeres and the oriP domain to YACs. The constructs contain 0.8 kb of human telomere sequence separated by a unique Not I site from 0.7 kb of Tetrahymena telomere sequence. These constructs seed telomere formation with approximately 40-60% efficiency in human 293-EBNA and HT1080 cells whether or not the Tetrahymena sequence is removed by Not I digestion. A detailed analysis demonstrates that YACs carrying the human telomere cassettes on both arms show instability of the telomere sequences in S.cerevisiae at a frequency of approximately 50%. Introduction of correctly retrofitted, linear oriP YACs into human 293-EBNA cells by lipofection resulted in the generation of circular extrachromosomal elements varying in size from 8 to 300 kb. However, no apparently linear YACs could be detected, suggesting that extrachromosomal maintenance of DNA with the oriP /EBNA-1 system is not compatible with linear molecules capped by telomeres.
Tolmachova
T
T
Section of Molecular Genetics, Division of Biomedical Sciences, Imperial College School of Medicine, Imperial College Road, London SW7 2AZ, UK. t.tolmachova@ic.ac.uk
Simpson
K
K
Huxley
C
C
D000818:Animals; D015139:Blotting, Southern; D002411:Cation Exchange Resins; D002460:Cell Line; D018244:Chromosomes, Artificial, Yeast / Q000235:genetics*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D004270:DNA, Circular / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D016521:Electrophoresis, Gel, Pulsed-Field; D005819:Genetic Markers / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D008055:Lipids; D016254:Mutagenesis, Insertional / Q000235:genetics* / Q000379:methods; D011995:Recombination, Genetic / Q000235:genetics; D018741:Replication Origin / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D016615:Telomere / Q000235:genetics*; D013768:Tetrahymena / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured
C505032:CFTR protein, human; D002411:Cation Exchange Resins; D004270:DNA, Circular; D004274:DNA, Recombinant; D004279:DNA, Viral; D005819:Genetic Markers; D008055:Lipids; C086724:Lipofectamine; D019005:Cystic Fibrosis Transmembrane Conductance Regulator