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 Salivary glands of Drosophila larvae contain giant polytene chromosomes whose characteristic banded structure is readily visible by light microscopy. Usually these chromosomes are visualized by breaking the nuclei and "spreading" the chromosomes in two dimensions. Using two-photon laser-scanning microscopy, these chromosomes can be examined in living salivary gland tissue in 3D and in real time with similar high resolution. This image shows a series of optical sections where the DNA is stained with Hoechst (pseudo-colored red). Pic: Jie Yao, Copyright © Cornell University.
The discovery was the result of cross-disciplinary collaboration
between Webb and John Lis, Cornell's Barbara McClintock Professor of
Molecular Biology and Genetics. Jie Yao, who recently earned his Ph.D.
at Cornell, initiated and facilitated the work.
"This technology will revolutionize the way we see gene expression
in organisms," said Lis. "We're watching transcription in real time in
living cells."
The research was described in the Aug. 31 issue of the journal Nature.
The team's experiments focused on gene regulatory mechanisms:
specifically, what happens in a cell's nucleus when an external
stimulus (heat) prompts specific genes to activate, and how those
activated genes direct the production of proteins that protect the fly
against the stress of heating.
"Whenever a cell is stressed -- bingo, it will produce proteins that
will help the cell resist stress," said Webb, Cornell professor of
applied physics and the S.B. Eckert Professor in Engineering. The
process is triggered by a molecule called heat shock factor (HSF),
which interacts with genes to cue the synthesis of new proteins. But
this well-known process had never been seen in living cells.
Yao used multiphoton microscopy (MPM) to image living salivary gland tissue of Drosophila
(fruit flies). Unlike other methods, which lack penetrating power and
can damage the specimen, MPM delivers crisp, clear images, even in
thicker tissue samples like Drosophila salivary glands.
The research was ultimately possible thanks to the unique
composition of the fruit flies' polytene cells -- giant, multistranded
chromosomes with hundreds of sets of the genome instead of the usual
two sets in conventional cells. This enlarges the usual nuclear
dimensions by about 10 times, making them large enough to image the
detail.
The results were stunning. "Within two weeks we had spectacular
pictures," said Lis. The images included pictures of the genes (hsp70
genes) that protect flies from the effects of extreme heat. By cranking
up the heat, the researchers could activate these genes, and by using
fruit flies specifically bred to carry fluorescent proteins on HSF,
they could watch the transcription factors in action.
"This is the first time ever that anyone has been able to see in detail, at native genes in vivo, how a transcription factor is turned on, and how it then is activated," said Webb.
Using another method that Webb engineered at Cornell, called
fluorescence recovery after photobleaching, the researchers also
discovered that HSF activators bind to hsp70 genes much longer than
previously thought before being replaced with new HSFs, which raises
new questions about the mechanisms of gene transcription.
The technique also may offer a new tool for researchers across the
biological sciences. Webb says it marks the success of an
interdisciplinary trend that offers new potential for researchers in a
variety of fields.
"Interaction between the physical sciences and the life sciences is
very powerful," said Webb. "And it's becoming more powerful as a tool
for advancing our understanding of the life sciences."
Better understanding transcription in lower organisms will help
understand the processes in higher organisms, Yao added. "We hope to
push the limits to human cells. That's the goal in the next 20 years."
Source: Cornell University
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