Whole
genome high density oligonucleotide arrays were used to simultaneously
map Saccharomyces cerevisiae origins of replication in a single
set of hybridizations. Yeast DNA samples enriched for origin activity
were obtained using a variation of the Meselson-Stahl experiment. Yeast
( MATa, cdc7, bar1 ) grown for seven generations in minimal medium
containing 13 C glucose and
( 15 NH 4 ) 2 SO 4 were allowed to
enter a synchronous S phase after transfer to medium containing light
isotope. Samples were collected through S phase and newly replicated DNA
(hybrid of heavy and light DNA was separated from unreplicated DNA
(heavy-labeled) by density gradient centrifugation. The heavy-labeled
and hybrid-density DNA from each time point was fragmented, labeled with
biotin, and hybridized to whole genome yeast arrays. These arrays
contain 20 or more oligonucleotide probes for every identified open
reading frame in the yeast genome. Hybridization was detected by
fluorescence using a confocal laser scanning device. The normalized
signal intensity for each of the 157,112 oligonucleotide features was
computed and plotted against the oligonucleotide's chromosomal
coordinate for each time point for both fractions. Analysis of the data
for chromosome V identified 14 regions containing origin activity, 13 of
which has been previously mapped, using a plasmid-based ARS assay
(Tanaka et al., Yeast 12:101, 1996). The timing of each origin's firing
within the cell cycle was also determined. In addition to mapping
replication origins for the entire genome, the direction and rates of
replication fork movement was determined.
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