Analysis of the Replication Timing of a Contig from Human Chromosomal Band 22q13 (2004)

Undergraduates: Michael Kelso, Bruna P. Brylawski, Stephanie M. Cohen, Marila Cordeiro-Stone

Faculty Advisor: David Kaufman
Department: Biology

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DNA replication is initiated in cells at specific sites along chromosomes at the beginning of S phase. The process of initiating DNA synthesis in higher eukaryotic cells is not well understood, but it is presumed to start at one or a few origins of DNA replication near genes. There is evidence that sites replicating very early in the S phase are more susceptible to damage when treated with carcinogens. Several chromosomal bands, including 22q13, have been found to contain regions that replicate very early during the S phase of normal human fibroblasts (NHF1 cells). However, chromosomal band 22q13 is several megabases in length, and it is unknown exactly which part of the band replicates very early in S phase. We chose an 88kb contig (GenBank number z82244) that reportedly maps to chromosomal band 22q13 to begin our analysis. NHF1 cells were synchronized at the beginning of the S phase by confluence arrest followed by incubation in aphidicolin. DNA from the NHF 1 cells was collected at 1-hour intervals for the first four hours of the S phase, and newly replicated DNA was isolated by CsCl gradient centrifugation. Using the synchronized DNA from each 1-hour interval, quantitative PCR was used to determine a more precise timing of replication for this contig. The results indicate that the 88kb contig from 22q13 is very early replicating.