Abstract
The alteration in the location of the chromosomes within the nucleus upon action of internal or external stimuli has been implicated in altering genome function. The effect of stimuli at a whole genome level is studied by using two-dimensional fluorescence in situ hybridization (FISH) to delineate whole chromosome territories within a cell nucleus, followed by a quantitative analysis of the spatial distribution of the chromosome. However, to the best of our knowledge, open access software capable of quantifying spatial distribution of whole chromosomes within cell nucleus is not available. In the current work, we present a software package that computes localization of whole chromosomes - Image Analysis of Chromosomes for computing localization (IMACULAT). We partition the nucleus into concentric elliptical compartments of equal area and the variance in the quantity of any chromosome in these shells is used to determine its localization in the nucleus. The images are pre-processed to remove the smudges outside the cell boundary. Automation allows high throughput analysis for deriving statistics. Proliferating normal human dermal fibroblasts were subjected to standard a two-dimensional FISH to delineate territories for all human chromosomes. Approximately 100 images from each chromosome were analyzed using IMACULAT. The analysis corroborated that these chromosome territories have non-random gene density based organization within the interphase nuclei of human fibroblasts. The ImageMagick Perl API has been used for pre-processing the images. The source code is made available at www.sanchak.com/imaculat.html.
Highlights
DNA is folded and compacted in order to occupy the limited space within the cell nucleus wherein the genome replicates, transcribes and translates to form proteins [1]
The organized genome and the machineries required for its maintenance and function within the nucleus, along with other nuclear components make up the nuclear architecture [6,7,8]
In order to validate the functionality of IMACULAT, we have mapped the positions of all human chromosomes in normal human dermal fibroblasts
Summary
DNA is folded and compacted in order to occupy the limited space within the cell nucleus wherein the genome replicates, transcribes and translates to form proteins [1]. This non-random chromosomal positioning within the architectural framework of the nucleus is thought to be a critical dimension of genome function [5,8,9,10,11]. Chromosome territories occupy a non-random and a radial distribution within interphase nuclei [2,3,4,5,10,12,13]. One of the major hurdles in performing 2D-FISH is the lack of open access software that could be used to quantify the localization of chromosomal territories.
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