Description
The subtracks within this annotation show replication origins identified using
the nascent strand method (Ori-NS), the bubble trapping method (Ori-Bubble)
and the TR50 local minima method (Ori-TR50).
Tracks are available for HeLa cells (cervix carcinoma) for all methods and
GM06990 cells (lymphoblastoid) for Ori-NS.
Display Conventions and Configuration
This annotation follows the display conventions for composite
tracks. To show only selected subtracks within this annotation,
uncheck the boxes next to the tracks you wish to hide.
Nascent Strand Method (Ori-NS)
Description
ENCODE region-wide mapping of replication origins was performed.
Origin-centered nascent-strands purified from HeLa and
GM06990 cell lines were hybridized to Affymetrix
ENCODE tiling arrays.
Methods
Cells in their exponential stage of growth were labeled, in culture, with
bromodeoxyuridine (BrdU) for 30 mins. DNA was then isolated from the cells.
Nascent strands of 0.5-2.5 kb synthesized with incorporation of BrdU,
representing the replication origins, were purified using a sucrose
gradient followed by immunoprecipitation with BrdU antibody (Giacca
et al., 1997). The purified nascent strands were amplified and
then hybridized to Affymetrix ENCODE tiling arrays, which have 25-mer
probes tiled every 22 bp, on average, in the non-repetitive sequence of the
ENCODE regions. As an experimental control, genomic DNA was hybridized to
arrays independently.
Replication origins were identified by estimating the
significance of the enrichment of nascent strands DNA (treatment) signal over
genomic DNA (control) signal in a sliding window of 1000 bp. An estimate of
significance in the window was calculated by computing the p-value using the
Wilcoxon Rank-Sum test over all three biological replicates and control signal
estimates in that window. The origins (Ori-NS) represented in the subtrack are
the genomic regions that showed a signal enrichment pValue <= 0.001.
Verification
The origin mapping experiments were completed for three biological sets.
Credits
Data generation and analysis for the subtracks using the Ori-NS method were
performed by the DNA replication group in the Dutta Lab at the University of
Virginia: Neerja Karnani, Christopher Taylor, Ankit Malhotra, Gabe Robins
and Anindya Dutta.
Christopher Taylor and Neerja Karnani prepared the data for presentation in
the UCSC Genome Browser.
References
Giacca M, Pelizon C, Falaschi A. Mapping replication origins by quantifying relative abundance
of nascent DNA strands using competitive polymerase chain reaction.
Methods. 1997;13(3):301-12.
Bubble Trapping Method (Ori-Bubble)
Description
ENCODE region-wide mapping of replication origins in HeLa
cells was performed by the bubble trapping method. Replication origins were
identified by hybridization to Affymetrix ENCODE tiling arrays.
Methods
The bubble trapping method works on the principle that circular plasmids can be
trapped in gelling agarose followed by the application of electrical current
for a prolonged period of time (see Mesner et al. 2006 for more
details). Entrapment occurs by an apparent physical linkage of the circular
DNA with the agarose matrix. The circular bubble component of the DNA
replication intermediates was therefore enriched by agarose trapping. After
recovery from the agarose gel, a library of the entrapped DNA was formed by DNA cloning. Subsequently, DNA from the library was labeled and hybridized to
Affymetrix ENCODE tiling arrays, which have 25-mer probes tiled every 22 bp
on average in the non-repetitive ENCODE regions. As an experimental control,
genomic DNA was hybridized to arrays independently.
Replication origins were identified by estimating the significance of the
enrichment of the bubble-trapped DNA (treatment) signal over genomic DNA
(control) signal in a sliding window of 10,000 bp. An estimate of significance
in the window was calculated by computing the p-value using the Wilcoxon
Rank-Sum test over all three biological replicates and the control signal
estimates in that window. The origins (Ori-Bubble) hence represented in the
UCSC browser track are the genomic regions that showed a signal enrichment
pValue <= 0.001.
Verification
The origin mapping experiments were completed for two biological sets.
Credits
Data generation and analysis for the subtrack using the Ori-bubble method
were performed by the DNA replication group in the Dutta Lab and Hamlin Lab
at the University of Virginia: Neerja Karnani, Larry Mesner, Christopher
Taylor, Ankit Malhotra, Gabe Robins, Anindya Dutta and Joyce Hamlin.
Neerja Karnani and Christopher Taylor prepared the data for presentation in the UCSC Genome Browser.
References
Mesner LD, Crawford EL, Hamlin JL. Isolating apparently pure libraries of replication origins
from complex genomes. Mol Cell. 2006 Mar 3;21(5):719-26.
TR50 local minima method (Ori-TR50)
Description
ENCODE region-wide mapping of replication origins in HeLa
cells was performed by the TR50 local minima method. Replication
origins were identified by hybridization to Affymetrix ENCODE tiling arrays.
Methods
The experimental strategy adopted to map this profile involved isolation of
replication products from HeLa cells synchronized at the G1-S boundary by
thymidine-aphidicolin double block. Cells released from the block were labeled
with BrdU at every two-hour interval of the 10 hours of S-phase. Subsequently,
DNA was isolated from the cells. The heavy-light (H/L) DNA representing the
pool of DNA replicated during each two-hour labeling period was separated from
the unlabeled DNA by double cesium chloride density gradient centrifugation.
The purified H/L DNA was then hybridized to a high-density genome-tiling
Affymetrix array comprised of all unique probes within the ENCODE regions.
The time of replication of 50% (TR50) of each microarray probe was
calculated by accumulating the sum over the five time points and linearly
interpolating the time when 50% was reached. Each probe was also classified
as showing temporally specific replication (all alleles replicating
together within a two-hour window) or temporally non-specific replication
(at least one allele replicating apart from the others by at least a two
hour difference).
The TR50 data for the temporally specific probes was then
smoothed within a 60 kb window using lowess smoothing. Local minima (within
a 30 kb window) on the smoothed TR50 curve were identified which
had at least 30 probes in the window on both sides of the minimum to locate
possible origins of replication. A confidence value was calculated for each
site as the average difference from the value of the local minimum of all
TR50 values falling into the 30 kb window.
Verification
The replication experiments were completed for two biological sets and a
technical replicate in the HeLa adherent cell line.
Credits
Data generation and analysis for the subtrack using the Ori-TR50 method
were performed by the DNA replication group in the Dutta Lab at the
University of Virginia: Neerja Karnani, Christopher Taylor, Hakkyun Kim,
Louis Lim, Ankit Malhotra, Gabe Robins and Anindya Dutta.
Neerja Karnani and Christopher Taylor prepared the data for presentation in the UCSC Genome Browser.
References
Jeon Y, Bekiranov S, Karnani N, Kapranov P, Ghosh S, MacAlpine D, Lee C,
Hwang DS, Gingeras TR, Dutta A.
Temporal profile of replication of human chromosomes.
Proc Natl Acad Sci U S A. 2005 May 3;102(18):6419-24.
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