Ithaca, New York
January 10, 2008
By Lauren Gold
 |
|
Watching genes
turn on: Multiphoton microscopy images of living
cells show the transcriptional activation of
heat shock loci in real time. (Provided) |
|
The molecular machinery behind
gene transcription -- the intricate transfer of information from
a segment of DNA to a corresponding strand of messenger RNA --
isn't stationed in special "transcription factories" within a
cell nucleus, according to Cornell researchers. Instead, the
enzyme RNA polymerase II (Pol II) and other key molecules can
assemble at the site of an activated gene, regardless of the
gene's position.
The findings, published in the Dec. 28, 2007, issue of the
journal Molecular Cell,
are the result of an ongoing collaboration between the
laboratories of John T. Lis, the Barbara McClintock Professor of
Molecular Biology and Genetics, and Watt W. Webb, professor of
applied physics and the S.B. Eckert Professor in Engineering.
Jie Yao, the paper's lead author, recently finished his Ph.D. at
Cornell under Webb.
Using multiphoton microscopy, a technique developed by Webb that
allows high-precision 3D imaging in living cells, the
researchers observed polytene chromosomes -- giant,
multistranded chromosomes in the salivary gland tissue of fruit
flies that have hundreds of sets of the genome instead of the
usual two sets in conventional cells.
They activated heat shock genes, which protect cells from sudden
rises in temperature, and watched them in real time as they
began to be transcribed. The researchers also tagged Pol II with
a fluorescent marker to track its movements within the nucleus.
While some reports have suggested that activated genes move to a
specific nuclear location for transcription, the Cornell
research supports the traditional view that gene activation is
not dependent on movement to special locations, or so-called
"transcription factories," said Lis.
"You see the genes decondense and fill up with polymerase, but
they're not moving anywhere -- they don't collect in a single
place," he said. Instead, the transcription machinery assembles
at the called-upon locus, regardless of its position in the
nucleus.
To test the generality of the findings beyond polytene nuclei to
common (but much smaller and more difficult to test) diploid
cells, the researchers used a technique called fluorescence in
situ hybridization, which allowed them to detect the location of
specific DNA sequences along a chromosome in fixed cells.
Looking at the location of co-regulated heat shock genes (genes
that are transcribed simultaneously), they found that
co-regulated pairs that occupied distinct sites before heat
shock were no closer together after heat shock. As in the
polytene chromosomes, the genes did not move to a single site
for transcription.
And using fluorescence recovery after photobleaching -- another
method engineered by Webb -- the researchers found that over
time Pol II began to recycle itself within newly formed
"compartments" around the activated gene.
"At some point you accumulate enough polymerase that it feeds
back, so in a sense you've created a factory de novo" said Lis.
"This is, to our knowledge, the first demonstration of Pol II
recycling at a specific gene in vivo."
Lis and colleagues are now looking at other molecules involved
in transcription to see if they behave similarly. "We're hoping
to develop new ways to really see, in vivo, how gene regulation
works mechanistically," he said.
|
|
