|
Visualizing the central dogma in
living cells:
DNA → RNA → Protein
We have developed a cell line in which we can observe a stably integrated genetic locus as well
as its mRNA and protein products using live cell microscopy and various GFP fusion proteins. This locus can be induced to initiate
transcription. In the movie you will be able to see the fluorescent signal from the chromatin at the genetic locus as well as the
protein product in the left panel and you will be able to see the mRNA synthesized when the genetic locus is turned on in the right
panel. Both movies are showing the same cell.
When the genetic locus is not transcriptionally active the chromatin is condensed (tight blue
dot in left panel) and there is no protein product seen in the cytoplasm of the cell. At this time the mRNA binding protein is
diffusely distributed throughout the nucleus (right panel), as it has no substrate to bind to. When you start the movie you will see
the chromatin in the left panel decondense over time and eventually you will see the protein product encoded for by the genetic locus,
distributed in peroxisomes in the cytoplasm (blue dots in the cytoplasm). The protein has encoded within it a peroxisome-targeting
signal. In the right panel you will notice that the mRNA binding protein concentrates at the genetic locus (transcription site) as the
chromatin decondenses, since mRNA is being synthesized that has a binding site for the fluorescently tagged mRNA binding protein, and
the nucleoplasmic pool now looks granular. The granular structures represent mRNPs that are moving away from the genetic locus in all
directions by diffusion. They will end up in the cytoplasm where the mRNA will be translated into the protein that is targeted to
peroxisomes.
From: Janicki, S.M., Tsukamoto, T., Salghetti, S.E., Tansey, W.P., Sachidanandam, R., Prasanth, K.V., Ried, T.,
Shav-Tal, Y., Bertrand, E., Singer, R.H. and Spector, D.L. 2004. From silencing to gene expression: Real-time analysis in single
cells. Cell 116, 683-698.
|
