"A Thousand (And More) Points Of Light" Yield New View Into Cells

April 24, 1998

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 a parallel computer processing to handle the prodigious computational tasks involved in the analysis.
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Albert Einstein College of Medicine

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