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By
collecting and analyzing thousands of points of light within a single
cell, researchers at the Albert Einstein College of Medicine and their
colleagues have for the first time visualized individual transcripts
of messenger RNA.Their technique also permits the accurate quantitation
of multiple RNA molecules, such as those that are found at transcription
sites, the places on chromosomes where the genetic information is read
out. The innovation, which relies on advanced imaging techniques, sophisticated
algorithms, and the power of parallel computing, is described in the
April 24, l998 cover story of Science. The lead author of the
paper is Andrea Femino. The corresponding author is Robert H. Singer,
head of the laboratory in which the research was conducted.
The
new methodology holds promise for disclosing the operations of the cell
at a heretofore unthought-of level of detail. For instance, using the
technique scientists can study how many molecules of mRNA are transcribed
from a particular gene during a particular unit of time. (Messenger
RNA is the molecular intermediary from which information encoded in
genes is translated into proteins.) The Einstein researchers have already
quantitated the kinetics of mRNA transcription from the beta-actin gene,
and have found that the transcription is cyclical despite the constant
presence of a stimulating signal."To our amazement, we found that transcription
hits its peak in 30 minutes. By 60 minutes transcription falls off,
and two hours later, it's gone." This indicates the existence of an
as-yet-unknown regulatory feedback mechanism, says Singer.
The
new technique has also enabled the researchers to show that the rate
at which genes are transcribed is limited not by the speed at which
the copying enzymes (polymerases) move down the gene with their growing
mRNA transcript attached, but rather by the number of polymerase/mRNA
units located at the gene's stopping point. At that genetic location,
says Singer, the molecules "really pile up as if they're waiting their
turn to go through a toll booth."
In
yet another discovery, the scientists have shown that completed mRNA
transcripts leave the gene one at a time, rather than in multiple units.
In addition, the researchers infer that the transcript exits the nucleus
and reaches the cell cytoplasm within minutes, because, as Singer reports,
"We don't see anything accumulated inside the nucleus."
The
technique that allows such detailed views of molecular functioning employs
multiple oligonucleotide probes that are complementary to mRNA and that
have been conjugated with fluorochromes. Hit with light, the probes
that have linked up (hybridized) with their RNA partners fluoresce and
are detected using light microscopy. Digital images of the fluorescent
points are acquired through "optical sectioning" of the hybridized cell.
The thousands of points of light (photons) acquired in this way go in
and out of focus. The out-of-focus light is reassigned to its position
of origin by means of a novel algorithm developed in collaboration with
Frederic Fay, now deceased, and his colleagues at the University of
Massachusetts Medical School. This algorithm, called Exhaustive Photon
Reassignment, requires the power of parallel computer processing to
handle the prodigious computational tasks involved in the analysis.
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