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Mapping of an origin of DNA replication near the transcriptional promoter of the human HPRT gene
Author(s) -
Cohen Stephanie M.,
Brylawski Bruna P.,
CordeiroStone Marila,
Kaufman David G.
Publication year - 2002
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.10136
Subject(s) - hypoxanthine guanine phosphoribosyltransferase , primer (cosmetics) , biology , gene , microbiology and biotechnology , exon , dna , dna replication , genetics , intron , chemistry , organic chemistry , mutant
Abstract A quantitative PCR method was used to map a functional origin of DNA replication in the hypoxanthine‐guanine phosphoribosyltransferase (HPRT) gene in normal human fibroblasts. This PCR method measures the abundance of specific sequences in short fragments of newly replicated DNA from logarithmically growing cells. Quantitative measurements rely on synthetic molecules (competitors) that amplify with the same primer sets as the target molecules, but generate products of different sizes. This method was first utilized to determine the position of the replication origin near the lamin B2 gene (Giacca et al. [1994] Proc. Natl. Acad. Sci. U S A. 91:7119–7123). In the present study, primer sets were tested along a 16‐kb region near exon 1 of the HPRT gene. The most abundant fragment was found to be located in the first intron of HPRT, just downstream of the promoter and exon 1 of the gene, and approximately 3.5 kb upstream of a previously reported autonomously replicating sequence (Sykes et al. [1988] Mol. Gen. Genet. 212:301–309). J. Cell. Biochem. 85: 346–356, 2002. © 2002 Wiley‐Liss, Inc.