1. Volume 19, Issue 4, Pages 567-575 (August 2005)
Human Origins of DNA Replication Selected from a Library of Nascent DNA 
Vesna Todorovic, Sara Giadrossi, Cristina Pelizon, Ramiro Mendoza-Maldonado, Hisao Masai, Mauro Giacca 
Molecular Cell 
Volume 19, Issue 4, Pages (August 2005)
DOI: /j.molcel
Copyright © 2005 Elsevier Inc. Terms and Conditions

2. Figure 1 Construction of a Library of Nascent DNA
(A) Flow chart of the method for the construction of a library of nascent DNA.
(B) Schematic representation of the human Mcm4, c-Myc, and lamin B2 DNA replication origin regions, with the position of the primer pairs used for PCR quantification. The location of the origin is shown by a shadowed dot (Mcm4 and lamin B2) or block (c-Myc). In the lamin B2 scheme, the positions of the three probes (BgB8, BN1, and SB16) used for the dot-blot experiments of (C) and (D) is highlighted in gray. The histograms on the right side of the panels indicate the relative abundance of the investigated DNA segments in the nascent DNA library (the mean and standard deviation, indicated by error bars, of at least three independent quantifications).
(C) Hybridization of Lamin B2 subclones to the nascent DNA library by dot-blot experiments. Two-fold dilutions of the nascent DNA library were spotted on nylon filters along with 2-fold dilutions of total genomic DNA and hybridized to three subclones of the Lamin B2 region.
(D) Quantification of the hybridization signals of Lamin B2 subclones to newly synthesized DNA.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
The Nascent DNA Library as a Hybridization Probe: The SHOC Assay
(A) Hybridization experiments to evaluate the origin-enriched library as a probe against Lamin B2 plasmids immobilized on filter. Only the origin-containing DNA fragment (pBN1) scored positive for hybridization.
(B) Flow chart of the SHOC procedure.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 Identification of Origins in the IL-3/GM-CSF Locus
(A) Human genomic region containing the IL-3 and GM-CSF genes; numbering is according to the GenBank sequence AC The different letters indicate the restriction fragments obtained after enzymatic digestion. On the bottom, the set of overlapping cosmids used for the SHOC selection is indicated.
(B–D) Restriction pattern (ethidium bromide-stained gels on the left sides of each panel) and Southern hybridization of the indicated cosmids using total genomic DNA or ori-library probes (middle and right pictures in each panel, respectively). While the genomic DNA probe mostly hybridized to highly repetitive sequences, the library probe recognized specific fragments belonging to the region located downstream of the GM-CSF gene. The letters on the right side of the gels indicate the positions of the fragments identified in the restriction map shown in panel (A). v represents the 8.2 kb cosmid vector; * and # mark additional hybridization signals of the library probe, belonging to fragments of cosmids 1, 2, and 3, located upstream of the analyzed 50 kb region. These signals probably indicate the presence of an additional origin of DNA replication in the upstream region. The symbol “<” before a given fragment indicates that only a portion of that fragment is contained in the cosmid.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Mapping DNA Replication Origins in the IL-3/GM-CSF Region by Competitive PCR Quantification of Nascent DNA
(A) Schematic representation of the restriction pattern of the IL-3/GM-CSF region on chromosome 5q31.1. The shadowed fragments indicate those scoring positive in the hybridization experiments of Figure 3 using the nascent DNA library.
(B) Quantification of the relative abundance of 24 different genomic markers from a ∼30 kb region encompassing the IL-3 and GM-CSF genes in a sample of nascent DNA from HL-60 cells. The results are expressed as the enrichment of nascent DNA over an unrelated nonorigin region (Lamin B2, B13 region). The amplifications of regions 13–24 have been performed at least three times on independent nascent DNA extractions; the mean and standard deviations (indicated by error bars) are shown.
(C) Representative gels showing the competitive PCR amplification results using the indicated primer pairs and four 5-fold dilutions of competitor DNA, as indicated. C: PCR band corresponding to competitor DNA; N: PCR band corresponding to nascent DNA. Below the gels, the ratios between the amount of competitor and nascent DNA amplification products (C/N) are reported.
(D) Quantification of the abundance of the indicated genomic segments amplified in panel (C). For each amplification, the C/N ratios were plotted against the input amount of competitor DNA, and the line fitting the experimental data was calculated. According to its equation, the number of nascent DNA molecules was evaluated at C/N ratio = 1 (second column). This value was then corrected for the different efficiency of primer amplification on total genomic DNA (shown in column 3, relative to the efficiency of primer set B13). Column 4 reports the corrected number of molecules at C/N = 1 and column 5 the final evaluated enrichment over the B13, nonorigin region. This is one of the values used for the graph in (B).
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Protein-DNA Interactions at GM-CSF Ori1 and Ori2 and at the Lamin B2 Origin
(A) Genomic region containing the two GM-CSF origins. Primers are indicated by converging arrows.
(B) Quantification of crosslinked DNA immunoprecipitated by ChIP. Experiments were performed on asynchronous HeLa cells. Below each gel, the ratio between the amplification products of competitor (C) and immunoprecipitated DNA (Cp) are shown. The histograms report the results (mean and standard error of the mean, indicated by error bars) of at least three different experiments. The results are presented as the enrichment of each given genomic marker with respect to markers #21 and B10 for the GM-CSF and Lamin B2 experiments, respectively.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions