If we hope to one day understand this connection it will be necessary
to obtain high resolution information about the native path of chromosomes
and the spatial relationships among multiple genetic loci to other nuclear
structures. Current methods of fluorescence in situ hybridization (FISH)
in combination with 3D optical microscopy represent a huge advance over
previous technology with respect to structural resolution, precision and
specificity . Still, the limited number of spectrally distinguishable fluorophores
limits our ability to sample the position of the chromosome fiber at many
loci simultaneously (hence limiting our evaluation of long range structure).
To overcome this limitation we have modified existing FISH techniques such
that each fluorophore can be used to localize many discrete loci. The color
barcode method involves labeling a set of ordered probes with one of 3
or more fluorophores such that a color barcode is embedded along the linear
chromosome. After co-hybridizing the entire probe set and then imaging
samples, a dataset is generated with multiple spots in each wave per nucleus.
Distinguishing among identically labeled probes (and tracing the chromosome
path) is possible because the barcode, when well designed, augments already
powerful distance-geometrical constraints intrinsic to the linear chromosome.
As a result, all but a few potential solutions are excluded. |
Using this technique two types of useful information can be obtained.
First, given the distance between many adjacent loci we can evaluate local
variations in compaction, which are hypothesized to correlate with changes
in expression. Second, by measuring all inter-probe distances (including
those between homologs), and the distance of all probes to the nuclear
envelope, we can identify structural motifs which define higher order nuclear
organization. As data from multiple studies are integrated, we hope to
understand how the global configuration of chromosomes influence biological
processes such as replication, transcription, the cell cycle and development.
We have recently completed the path tracing analysis for a data set
containing a patch of about 80 nuclei localized to the surface of mitotic
cycle 14 D. melanogaster embryos. The chromosomes were labeled using a
3 color, 13 probe barcode which maps to Drosophila chromosome 2L (26 distinct
loci for diploid nuclei). This represents the first time we have been able
to trace the majority of paths within a given data set with reasonable
confidence. These experiments serve as proof of principle for the barcoded
FISH method and also hint at some very interesting structural features.
To evaluate these possibilities will require cluster analysis based grouping
of similar structures and then correlation of these clusters with functional
and metabolic processes. Despite the promise of our preliminary results,
we decided not to continue with the structural analysis at this time. Instead
we are working on a second generation barcode method which addresses many
of the unexpected obstacles encountered in our initial experiments. We
are quite optimistic that these changes will aid in increasing the resolution
and quality of the structural data while also simplifying the experimental
and analytical aspects of the method. Our hope is that these developments
will prove useful in our continuing efforts to understand the interplay
between structure and function in nuclear biology.
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