Isolating Apparently Pure Libraries of Replication Origins from Complex Genomes

Because of the complexity of higher eukaryotic genomes and the lack of a reliable autonomously replicating sequence (ARS) assay for isolating potential replicators, the identification of origins has proven to be extremely challenging and time consuming. We have developed a new origin-trapping method...

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Bibliographic Details
Published in:Molecular cell 2006-03, Vol.21 (5), p.719-726
Main Authors: Mesner, Larry D., Crawford, Emily L., Hamlin, Joyce L.
Format: Article
Language:English
Subjects:
Animals
CHO Cells
Cricetinae
Cricetulus
Electrophoresis, Gel, Two-Dimensional
Gene Library
Genomics - methods
Replication Origin - genetics
Rhodopsin - genetics
Tetrahydrofolate Dehydrogenase - genetics
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Summary:Because of the complexity of higher eukaryotic genomes and the lack of a reliable autonomously replicating sequence (ARS) assay for isolating potential replicators, the identification of origins has proven to be extremely challenging and time consuming. We have developed a new origin-trapping method based on the partially circular nature of restriction fragments containing replication bubbles and have prepared a library of ∼1000 clones from early S phase CHO cells. When 15 randomly selected clones were analyzed by a stringent two-dimensional (2D) gel replicon mapping method, all were shown to correspond to active, early firing origins. Furthermore, most of these appear to derive from broad zones of potential sites, and the five that were analyzed in a time-course study are all inefficient. This bubble-trapping scheme will allow the construction of comprehensive origin libraries from any complex genome so that their natures and distributions vis-a-vis other chromosomal markers can be established.
ISSN:1097-2765
1097-4164
DOI:10.1016/j.molcel.2006.01.015