n
this interview, in-cites correspondent Gary Taubes talks with
Dr. Martin Chalfie of Columbia University about his highly
cited work. Dr. Chalfie is the lead author of the paper
"Green fluorescent protein as a marker for gene
expression" (Science 263[5148]: 802-5, 11 February
1994), which is among the 20 most-cited papers in the field of
Molecular Biology & Genetics over the past decade, with
1,793 total citations to date. In the ISI
Essential Science Indicators
Web product, Dr. Chalfie has 13 papers listed in Molecular
Biology & Genetics, with a total of 2,154 citations to
date, as well as 3 papers in the Multidisciplinary category,
with 157 total citations to date. Dr. Chalfie is the William
R. Kenan Jr. Professor of Biological Sciences at Columbia
University in New York City.
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Why did you start working on green fluorescent protein,
considering your primary research interest is the worm C. elegans?
Well, the murky beginnings were in about 1988, when Paul Brehm,
who is now at Stony Brook, gave a seminar in our department. He was
talking about bioluminescent organisms, in particular a coelenterate
called Obelia geniculata. Coelenterates are the source of the
protein aequorin, and the name comes from the jellyfish, Aequoria
victoria. Aequorin is a bioluminescent protein that gives off
blue light when calcium is added in vitro. But many
coelenterates are not blue-light emitters. They emit a green light.
Brehm said the reason for the color change was a protein called
green fluorescent protein, GFP, and that shining blue light on the
organism was sufficient to have the cells fluoresce green. At that
point I thought to myself, "What an absolutely wonderful,
wonderful compound this would be. I would love to put it into C.
elegans and look at gene expression." In fact I imagined a
really large number of things I would like to do if I had such a way
of marking gene expression and protein localization in living cells.
I got very excited and didn’t listen to another word of the
seminar.
I spent the rest of the day and maybe the next day trying to find
out who was working on the molecular biology or biochemistry of this
protein and eventually found Douglas Prasher at the Woods Hole
Oceanographic Institute. He was in the process of trying to clone
the cDNA for GFP. I called Prasher and it turned out he was as
excited about this as I was. "This is terrific," I told
him. "We have the right organism to put it in. It’s a
transparent organism. We have cell-specific promoters to drive it.
This is going to be lot of fun." But he hadn’t finished the
sequencing or isolation. I think maybe he only had a partial cDNA.
He said he’d get back to me.
There seems to be a four-year gap between that discussion and
the publication of your seminal paper in Science. What
happened?
I didn’t hear from Doug Prasher for a while. And then I went on
sabbatical and it turned out that Doug called while I was away,
because he had the DNA ready, but nobody told him how to get in
touch with me. Whoever answered the phone just said I wasn’t there
anymore. Finally in about September 1992, a graduate student, Ghia
Euschirken, one of the co-authors on the Science paper,
rotated through the lab and I told her the GFP story. I said I
really would have liked to have used GFP but that Doug never got in
touch with me. So we went to Medline to see if there was anything on
GFP, and Doug had just published a paper in the journal Gene saying
he had the cDNA for GFP. l went and got the paper from the library
and it had his phone number and I called him up and said, "What
happened to our collaboration? Do you still want to do this?"
Of course, he did. But for Doug, the protein wasn’t fluorescent.
He said he could send the clone to me, and I talked to Ghia about
how to make the construct. Maybe a week or two later, she came back
and said she wanted to show me something. I went and looked in the
microscope and saw green fluorescent E. coli. It had worked.
That was pretty exciting. And I immediately got on the phone and
told Doug and we started doing other experiments.
Why did GFP fluoresce for you and not for Prasher or other
researchers? What was the trick? Was it serendipity?
I have to set the stage for you; what the problems were at the
time. GFP was first identified by Osamu Shimomura, who was at
Princeton. He was the one who purified aequorin and had a paper in
the early 1960s identifying another protein that fluoresced green.
By the time I first spoke to Doug Prasher, it was understood that
the fluorophore that made the protein fluorescent was actually
formed from the primary amino acid sequence, but nobody knew how
many enzymes, if any, were needed to make the final product. In Doug’s
paper on the cloning of the cDNA, the protein product is referred to
as apoGFP to signify this concern. I was curious to see if the
protein could work on its own. Although others saw Doug’s paper
and were trying to do the same experiments, we were successful
because of the way we did the experiment. I was very concerned that
we only use the coding sequence and no flanking DNA in our
construct. We used PCR to obtain just the coding sequence and that
worked terrifically. But the others who tried to use GFP simply cut
the original lambda cDNA clone with restriction enzymes, and got
additional bases associated with each end. These extra sequences
turned out to be quite detrimental; expression wouldn’t occur. So
we were fortunate in the way we designed the experiment.
Why choose Science for the announcement?
Actually, since I am in the worm field, the first written report
about GFP was in the C. elegans newsletter in October 1993,
and, of course, we had also told several people. So even before the Science
paper came out, we were sending out samples to people to let them
try their hands with it. The reason we published in Science
was to reach a broad range of scientists, because we were pretty
sure that GFP was going to useful in many contexts.
What is it that makes this protein and your Science paper
so significant?
There are a couple of things. First of all, because GFP needs no
substrate, it works just by shining a light on it; you don’t have
to permeabilize the tissue to allow it to work. You can simply look
at the cells and see whether they are expressing it with the
virtually non-invasive technique of shining light on it. The second
and maybe even more important factor is that this can be done in
living cells, and so you can watch the processes occur in real time
or even sped up using time-lapse photography. You can look at the
localization of proteins in living cells as was first done in Tulle
Hazelrigg’s lab, and whether proteins relocalize, e.g., from the
nucleus to the cytoplasm, over time. That’s very useful. Then
there are some other technical aspects that are quite nice. One is
that GFP has a very high quantum efficiency. You get a lot of light
out for what you put in. And there has been a lot of work since then
to make the molecule fold more efficiently and so utilize light even
more efficiently. Of all these, the real advantage is being able to
look in living cells. Another advantage is that GFP is a relatively
small molecule. GFP has 238 amino acids and exists as a monomer,
except at very high concentrations. These properties mean, for
example, that GFP can diffuse throughout cells. In contrast, beta-galactosidase
forms a tetramer. It’s a very large molecule and it normally stays
sequestered in the cell body. It won’t diffuse throughout nerve
cell processes, for example, unless you do some tricks. GFP is able
to diffuse throughout cells.
How has GFP technology changed since your first paper?
People have tinkered with the molecule and changed things and
improved it in a variety of ways. Several people, among them Roger
Tsien and Brendon McCormack in Stanley Falkow’s lab, and Jim
Haseloff, have mutated GFP and obtained variants that excite at
different wavelengths, emit different colors, or fold more
efficiently at high temperature. Sergey Lukyanov’s group used the
sequence of GFP to find a wonderful set of fluorescent proteins from
corals that give us even more colors to work with. In addition, some
really wonderful work was done by Roger Tsien in developing other
aspects of the molecule. For example, his lab has pioneered the use
of cameleons, which are molecules with two different color
fluorescent proteins on them and a calcium-binding site, that fold
when calcium is bound in such a way that the fluorescent energy from
one GFP is transferred to the other. The result is a color shift
and, thus, and indicator of calcium concentration. Although GFP is
inherently pH-sensitive, Gero Miesenböck has modified this to
develop pH indicators. These are only some of the modifications;
people are continuing to modify and develop the protein .
How do you use it in your lab?
We use it in quite a number of different ways. We identify cells
by using promoter fusions to drive GFP expression to determine
patterns of gene expression. We use it to look at where proteins
localize within cells. We use it as the basis of mutant hunts. We
have used it to characterize mutants. We use it to label nerve cells
so we can then electrically record from those cells. That is one of
the first things I said it would be used for. C. elegans is
an animal in which every cell is identifiable, but once you cut open
the animal everything gets a little messy. With GFP, you can label a
particular cell or subset of cells and then go in and locate those
cells and actually record from them.
What’s the most imaginative use of GFP that you’ve heard
of, in your opinion?
One of the most intriguing applications that is currently in
progress is work by Bob Burlage, who is expressing GFP in bacteria
off of a promoter that responds to TNT. The potential here is that
these bacteria could be used to detect land mines because they often
leak the explosive. Eliminating these remnants of war would be
terrific. A lot of different things have been tried with GFP. It’s
been into virtually every organism that you can think of. It’s
been used in plants. It’s been used in prokaryotes and in
eukaryotes, including mammals. It has been a lot of fun.
Considering how significant GFP has been, why do you think no
one else came up with it, while you were waiting for Doug Prasher to
clone it?
That’s a very important point. In hindsight, you wonder why 50
billion people weren’t working on this. But I think the field of
bioluminescence or, in general, the research done on organisms and
biological problems that have no immediate medical implications, was
not viewed as being important science. People were working on this,
but it was slow and tedious work, and getting enough protein from
jellyfish required rather long hours at the lab. They had to devise
ways of isolating the cells that were bioluminescent and then
grinding them up and doing the extraction on them. It’s not like
ordering a bunch of mice and getting livers out and doing an
experiment. It was all rather arduous. It’s quite remarkable that
it was done at all. It was mostly biochemists doing it, and they
were not getting a lot of support. In fact, as I remember it, Doug
Prasher had some funding initially from the American Cancer Society,
and when that dried up he could not get grants to pursue the work. I
never applied for a grant to do the original GFP research. Granting
agencies would have wanted to see preliminary data and the work was
outside my main research program. GFP is really an example of
something very useful coming from a far-outside-the-mainstream
source. And because this was coming from a non-model-organism
system, these jellyfish found off the west coast of the U.S., people
were not jumping at the chance to go out and isolate RNAs and make
cDNAs from them. So we’re not talking about a field that was
highly populated. It was not something that was widely talked about.
At the time, there was a lot of excitement about molecular biology,
but this was biochemistry. The discovery really was somewhat
orthogonal to the mainstream of biological research.
Did you expect it to have the kind of impact it has had?
Yes, I thought it would be pretty important. I certainly hoped it
would be important and that people would see it as an extremely
useful tool. What’s really been quite wonderful is how many people
have put their input into modifying, changing, and improving GFP so
that it has become more and more useful every year.
Would you consider GFP your greatest professional achievement
or is there some aspect of your C. elegans work, your primary
research, that you would rate above it?
I’m certainly very proud of this. There’s nothing quite as
enjoyable as having an idea and finding out that it works. We have a
lot of ideas that are best forgotten and usually are. But when you
actually think of something and it works, that’s extremely
thrilling. And then you always hope to have a breakthrough someday
that will have implications or usefulness to a large audience, and
this really did.
Martin Chalfie, Ph.D.
Columbia University
New York, NY, USA
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