Kathy Kieva

ENL 641 - Technical & Scientific Journalism

Chris Eisenhart
2/26/09

Audience: Educated, interested (not attentive) in science in general but not scientists
Publication: Boston Globe
Word count: 1181

50,000 Jellyfish Lead to Nobel Prize in Chemistry

Osamu Shimomura spent the summer of 1961 in Friday Harbor, north of Seattle, WA,
cutting off the edges of about 50,000 jellyfish and squeezing them through a filter to get what he
called "squeezate." He probably couldn't have imagined that forty-seven years later he'd receive a
Nobel Prize in chemistry for his efforts, which eventually led to his discovery of green
fluorescent protein, or GFP, an exciting new tool for scientists seeking to understand the inner
workings of active cells and the mechanism of gene expression.
Shimomura, who is Professor emeritus at Marine Biological Laboratory (MBL), Woods
Hole, and Boston University Medical School, shares this year's prize with Martin Chalfie, the
Chairman of the Biology Department at Columbia University, who paved the way for expressing
GFP's fluorescent properties in bacteria and worms, and Roger Tsien, a professor of the
Department of Chemistry and Biochemistry at University of California, San Diego, who
expanded the palette of colors available to researchers.
Chalfie’s research focused on the millimeter-long transparent roundworm known as
Caenorhabditis elegans, or C. elegans. He first heard about green fluorescent protein, or GFP, at
a seminar in 1988 and immediately grasped the implications of using a protein that fluoresces
bright green when exposed to blue or UV light. "I admit I completely ignored the rest of the
seminar," he said in a Nobel interview. "All I could think of was that this would be a wonderful
tool for visualizing life." Tsien took the research further by modifying the gene that makes GFP
to create an artist's palette of colors that glow longer and with more intensity than the original
green.
What made GFP different, and what excited researchers like Chalfie so much, was the
fact that GFP was a "stand-alone" protein. In other words, no other chemical or protein or
enzyme was required to create the bioluminescent effect, nor is there a need to continually re-
energize the protein as is the case in most other bioluminescent organisms like the firefly. This
characteristic meant that GFP could be used to study how genes are expressed and where
proteins operate in the cell without introducing additional biological chemicals that may
adversely affect cell function. Further, says Chalfie, "GFP is heritable, relatively non-invasive,
small and monomeric, and visible in living tissue," making the ideal tracer protein for studying
the dynamic processes within a cell.

From Jellyfish to GFP

Having completed his studies in post-World War II Japan, Shimomura was recruited to
work at Princeton by Dr. Frank Johnson. Their research originally focused on the jellyfish
Aequorea victoria, which glowed green around its outer rim when agitated, and they were
eventually able to isolate the bioluminescent protein aequorin. Contrary to expectation, however,
Shimomura was surprised to find that aequorin glows blue, not green. They were missing some
crucial step.
What was missing, Shimomura found, was his ability to transcend existing scientific
knowledge regarding bioluminescence in nature. Common belief among scientists at that time
was that bioluminescence is a complex biochemical process requiring at least two different
chemicals, one that produces the light, generically called "luciferin," and the one that drives the
chemical reaction, an enzyme called "luciferase." If either chemical is missing, no
bioluminescence.
“I was convinced that the cause of our failure was the luciferin hypothesis that dominated
our minds,” Shimomura said in his Nobel lecture. “I did a lot of soul searching, trying to find
what was missing in my experiment.” That soul searching led to a rift with Johnson, who
continued to work on the luciferin angle while Shimomura shifted his attention to the "green
protein" that he and Johnson had described in their aequorin paper as being “slightly greenish in
sunlight, yellowish in the light from a light bulb and fluorescent green in UV light.”
What he found was that the within the protein there is a chemical group of amino acids
that absorb and emit light called a chromophore. When the aequorin in the jellyfish emits blue
light, it excites the GFP chromophore, which in turn emits green light. What Shimomura found
was that he could eliminate the need for aequorin altogether by simply shining blue or UV light
directly on the GFP, causing it to glow green.

Glowing Bacteria and Worms

Almost thirty years after Shimomura and Johnson first described GFP, the gene for the
protein was discovered, sequenced and cloned by Dr. Douglas Prasher, working then at Woods
Hole Oceanographic Institution in Massachusetts. Unfortunately his grant funding ran out before
he could successfully express the gene in bacteria and demonstrate that GFP could serve as a
tracer molecule in living cells. He sent the results of his work to Chalfie and Tsien, both of
whom had contacted Prasher independently, unbeknownst to the other. It was Chalfie and his
team who discovered that the gene Prasher sequenced was off by 20 base pairs, resulting in
incorrect folding of the protein and thus inhibiting its fluorescent abilities. Once they had the
correct sequence, it didn't take long to express the gene in E. coli bacteria and obtain the
signature green glow from the bacteria.
Using DNA technology, Chalfie and his team then inserted the gene for GFP behind a
promoter that is active in six touch receptor neurons in a mature C. elegans worm and allowed it
to reproduce. Sure enough, they were able to visualize those touch receptor neurons in the
offspring of that mature worm - they were glowing bright green.

Creating Rainbows and “Brainbows”

Tsien wasn't content with green. By identifying the crystal structure of GFP, he and his
team were able to re-engineer the protein to fluoresce in a wide range of colors by exchanging
various amino acids in different parts of GFP. By experimenting with the amino acid
composition, Tsien was able to develop new variants of GFP in colors ranging across the
spectrum with such mouth-watering names as mHoneydew (m for "mutant"), mStrawberry,
mGrape1 and MGrape2, and mPlum. Using different colors, researchers can now selectively tag
proteins in cells and follow different proteins as they fluoresce in shades of blue, green, yellow,
orange and red. For example, researchers at the Harvard Brain Center have created transgenic
mice with fluorescent multicolored neurons in their brains, creating what they call a "brainbow"
that allows them to distinguish between individual neurons. With this color-coded tool,
"researchers will now be able to map the neural circuits of the brain. The individually colored
neurons will help define the complex tangle of neurons that comprise the brain and nervous
system. By creating a wiring diagram of the brain, researchers hope to help identify the defective
wiring found in neurodegenerative diseases such as Altzheimer's and Parkinson's disease."
(http://www.conncoll.edu/ccacad/zimmer/GFP-ww/cooluses0.html)

And the Prize Goes To…

It was basic curiosity about what made a particular species of jellyfish glow that started
Shimomura on the path to the discovery of GFP. From tracking the growth of cancer cells and
the path of the HIV virus to the ability to identify individual neurons in the brain and trace gene
expression, GFP is now providing researchers with an unprecedented tool for visualizing life in
all its complexity.