Fine mapping of a replication origin of human DNA.

Abstract

A highly sensitive procedure was developed for the identification of the origin of bidirectional DNA synthesis in single-copy replicons of ammalian cells. The method, which does not require cell synchronization or permeabilization, entails the absolute quantification, by a competitive PCR procedure in newly synthesized DNA samples, of the abundance of neighboring DNA framents distributed along a given genomic region. Terminal differentiation of HL-60 was achieved with retinoic acid and dimethylformamide, as described (17).Transfection. Plasmid pAWTSV (=9 kb), a kind gift of Cesare Vesco (Institute of Cell Biology, Rome), carries the whole simian virus 40 (SV40) genome inserted in the BamHI site of pAT153 (18). Six 10-cm tissue culture plates, containing about 106 COS-1 cells each, were transfected with 10 pg of pAWTSV by the calcium phosphate precipitation technique. After 10 hr of incubation in calcium phosphate solution, cells were extensively washed and fresh medium was added, containing 10 nCi of [14C]thymidine per ml. After 18 hr of incubation, BrdUrd (100 uM final concentration) and[3H]deoxycytidine (1 jAM final concentration) were added.After 1 min of incubation, cells were killed by addition of sodium azide and DNA was extracted as described below.Extrction and Purification of Newly Syntez DNA.Total DNA was extracted, denatured, and size-fractionated by sedimentation through neutral sucrose gradients as described (15).In the experiment involving transfection of plasmid pAW-TSV, DNA (700 /4 final volume) was fractionated on four 5-20%6 (wt/vol) linear sucrose gradients (5 ml each) for 210 min at 200C in a Beckman SW55Ti rotor at 55 krpm; 24 fractions of 200 j4 were collected.In the experiment with synchronized HL-60 cells, DNA (2 ml final volume) was fractionated on eight 5-30% sucrose Abbreviations: DHFR, dihydrofolate reductase; SV40, simian virus 40. tTo whom reprint requests should be addressed. 7119The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Overreplication of short DNA regions during S phase in human cells

“…In agreement with previous observations, we detected a three-to fourfold enrichment of TIMM 5-6 and TOP 5-8 relative to the TIMM 10-11 and TOP 11-12 regions that were used as a baseline (Fig. 1B;Giacca et al 1994;Keller et al 2002). In contrast, when the same nascent strand-enriched fraction was used as a template and cloned DNA was used for normalization, two unexpected results were observed.…”

“…1B,C, left panels, green and red bars). The TIMM13 ORI had originally been mapped to the TIMM 5-6 region using nascent strands ranging from 800 to 1200 nt long as input for Q-PCR (Giacca et al 1994), and a site of replication initiation has also been mapped at nucleotide resolution in this region (Fig. 1A, black arrow; Abdurashidova et al 2000).…”

“…Several of the mammalian ORIs identified to date are close to CpG island promoters (Giacca et al 1994;Delgado et al 1998;Keller et al 2002;Gómez and Brockdorff 2004;Gray et al 2007). However, the well-characterized human ␤-globin ORI (Kitsberg et al 1993) does not colocalize with a CpG island, and examples of ORIs distant from known promoters have also been reported (Mesner and Hamlin 2005;Todorovic et al 2005).…”

Mapping of an origin of DNA replication in the promoter of fragile X gene FMR1

“…A standard curve using sonicated genomic DNA (∼ 1500 bp) was amplified together with the nascent DNA sample for each primer set, so that differences in amplification efficiency would not affect the determination of relative abundance. Each preparation of short nascent DNA was also tested by measuring the relative abundance of sequences at the lamin B2 origin using previously described L5 (Cohen et al, 2002) and B13 (Giacca et al, 1994) primers.…”

Dynamics of HIV-1 mRNA expression in patients with long-term nonprogressive HIV-1 infection.

“…In recent years, we have developed and extensively applied a competitive PCR technology for the exact quantitation of low abundance nucleic acids in clinical samples (30)(31)(32)(33)(34)(35), including HIV DNA and RNA in different clinical settings (36)(37)(38)(39)(40)(41). Competitive PCR and RT-PCR (using internal DNA and RNA competitors, respectively) couple the sensitivity of conventional PCR to the accuracy that is necessary for the exact quantification of the target molecules, irrespective of sample nucleic acid purity, primer pair efficiency and specificity, or amplification kinetics profile.…”