Figure 1. SMARD. (A) Exponentially growing cells were labeled with the halogenated nucleosides IdU for 4 h followed by CldU for 4 h. Genomic DNA was then isolated. To avoid the breakage of large molecules, the cells were embedded in agarose. We used rare cutting restriction endonucleases to obtain unique large molecules of genomic DNA, which were separated by pulse field gel electrophoresis to enrich by size for the particular subtelomere segments of interest. Southern blotting was performed to identify the target segment within the gel, which was excised. The gel slice containing the segment of interest was melted and the enriched DNA in the melted gel solution was stretched on silanized glass slides. Fluorescent antibodies were used to identify regions where IdU (red) or CldU (green) were incorporated into the DNA. Immunodetected biotinylated FISH probes (blue) were used to identify the target molecules and to align the images of individual molecules to produce a composite profile of replication. (B) Specific replication events are identified by characteristic patterns in the aligned molecule images. DNA molecules with increasing red stain from one end indicate replication forks progressing in a single direction through the aligned region (ii and iii). Initiation events are indicated by a red tract flanked on both sides by green (i), whereas a green tract flanked on both sides by red (iv) indicates a termination event. Molecule alignments can be graphically depicted as histograms of the percentage of IdU incorporation along each 5 kb of a segment. Specific replication events produce characteristic features in these replication profiles. Positions of the centers of initiation zones are indicated by peaks (i), whereas termination events are indicated by valleys (iv). Replication progressing through the segment primarily in one direction (5′ to 3′ or 3′ to 5′) from an external origin is observed as a progressive decrease in the percentage of IdU incorporation from one end to the other across the segment (x axis; ii and iii).
Figure 1.

SMARD. (A) Exponentially growing cells were labeled with the halogenated nucleosides IdU for 4 h followed by CldU for 4 h. Genomic DNA was then isolated. To avoid the breakage of large molecules, the cells were embedded in agarose. We used rare cutting restriction endonucleases to obtain unique large molecules of genomic DNA, which were separated by pulse field gel electrophoresis to enrich by size for the particular subtelomere segments of interest. Southern blotting was performed to identify the target segment within the gel, which was excised. The gel slice containing the segment of interest was melted and the enriched DNA in the melted gel solution was stretched on silanized glass slides. Fluorescent antibodies were used to identify regions where IdU (red) or CldU (green) were incorporated into the DNA. Immunodetected biotinylated FISH probes (blue) were used to identify the target molecules and to align the images of individual molecules to produce a composite profile of replication. (B) Specific replication events are identified by characteristic patterns in the aligned molecule images. DNA molecules with increasing red stain from one end indicate replication forks progressing in a single direction through the aligned region (ii and iii). Initiation events are indicated by a red tract flanked on both sides by green (i), whereas a green tract flanked on both sides by red (iv) indicates a termination event. Molecule alignments can be graphically depicted as histograms of the percentage of IdU incorporation along each 5 kb of a segment. Specific replication events produce characteristic features in these replication profiles. Positions of the centers of initiation zones are indicated by peaks (i), whereas termination events are indicated by valleys (iv). Replication progressing through the segment primarily in one direction (5′ to 3′ or 3′ to 5′) from an external origin is observed as a progressive decrease in the percentage of IdU incorporation from one end to the other across the segment (x axis; ii and iii).

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