NDLI: Chromosome replicating timing combined with fluorescent in situ hybridization.

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Year: 2012 Issue: 70

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Chromosome replicating timing combined with fluorescent in situ hybridization.

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Chromosome replicating timing combined with fluorescent in situ hybridization.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Smith, Leslie Thayer, Mathew
Description	Author Affiliation: Smith L ( Department of Biochemistry and Molecular Biology, Knight Cancer Institute, Oregon Health & Science University.)
Abstract	Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation(1). Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of >3 hours that affects the entire chromosome(2,3). Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome(4). Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes(5). Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from(6). In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.
File Format	HTM / HTML
e-ISSN	1940087X
DOI	10.3791/4400
Journal	Journal of Visualized Experiments
Issue Number	70
Language	English
Publisher	MyJove Corp.
Publisher Date	2012-12-10
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Discipline Physical Sciences Discipline Life Sciences Discipline Medicine Dna Replication Timing In Situ Hybridization, Fluorescence Dna Replication Research Support, N.i.h., Extramural Video-audio Media
Content Type	Text
Resource Type	Article

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