Complementation of replication origin function in mouse embryonic stem cells by human DNA sequences.
A functional origin of replication was mapped to the transcriptional promoter and exon 1 of the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in the mouse and human genomes. This origin was lost in mouse embryonic stem (ES) cells with a spontaneous deletion (approximately 36 kb) at the 5' end of the HPRT locus. Restoration of HPRT activity by homologous recombination with human/mouse chimeric sequences reconstituted replication origin activity in two independent ES cell lines. Quantitative PCR analyses of abundance of genetic markers in size-fractionated nascent DNA indicated that initiation of DNA replication coincided with the site of insertion in the mouse genome of the 335 bp of human DNA containing the HPRT exon 1 and a truncated promoter. The genetic information contained in the human sequence and surrounding mouse DNA was analyzed for cis-acting elements that might contribute to selection and functional activation of a mammalian origin of DNA replication.
Cohen
Stephanie M
SM
Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7525, USA. stepmi@med.unc.edu
Hatada
Seigo
S
Brylawski
Bruna P
BP
Smithies
Oliver
O
Kaufman
David G
DG
Cordeiro-Stone
Marila
M
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D040461:Gene Components; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D051379:Mice / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic / Q000235:genetics; D018741:Replication Origin / Q000235:genetics*; D016415:Sequence Alignment; D013234:Stem Cells
D017931:DNA Primers; D007041:Hypoxanthine Phosphoribosyltransferase