Диссертация (1173136), страница 61
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All of these could answer the question that cells activated what particular gene? Butthere were problems with these methods.First, they actually required a fair amount of work. We had to prepare the tissues, and that meantkilling them, fixing them, permeabilizing them so that we could get the antibody or the substrate forbeta-galactosidase, or the probe for in situ hybridization into the tissue. But the main problem I thinkwas the fact that although we could tell at the moment we prepared the sample that genes were beingexpressed there, we couldn't look at more than just this static picture. If we wanted to look over time,we'd have to do preparations for each time point. Then on April 25 th,1989 , I heard a seminar thatreally changed my life. It was a talk being given by Paul Brehm, who at the time, was aneurobiologist at Tufts University.
And in the introduction of his talk, he started talking about thework of this man, Osamu Shimomura, and the work he had done in isolating proteins from thejellyfish, Aequorea victoria. In the early 1960s, Shimomura had discovered the protein aequorin,which was a bioluminescent protein. It produced light. What he found was that when aequorin andcalcium were together, they would produce light, and this was the problem he was trying to address.But he had one slight difficulty, and that was that the light that was produced by this reaction wasblue.
The jellyfish, however, gave off a light that was green. And he knew that there had to besomething else. So he went back to his protein fractions to look at what was there, and he found thatthere was another protein, that when added to aequorin and calcium, now gave green light. And thisprotein, he called the green protein. We now call it GFP or green fluorescent protein. And because Iwas working on a transparent animal, Caenorhabditis elegans, I was looking at gene expression, Isuddenly realized that this protein, GFP, would make a terrific marker for our experiments. And Ispent the whole rest of the seminar fantasizing about this work. I actually don't remember what therest of the seminar was about.
The next day, I got in touch with this man, Douglas Prasher, and foundout that he was in the process of cloning the cDNA for GFP from the jellyfish. We had a wonderfulconversation, culminating in a decision to collaborate to see whether GFP could be used in otherorganisms. A few years later, I was lucky enough to get a very talented graduate student into my lab,Ghia Euskirchen, who came to the lab having just finished a Master's degree in chemical engineeringworking on fluorescence. She came into the lab, to do a rotation, and this was the project that shewas given. We got back in touch with Douglas who had cloned the gene by this time; he sent it to us,and Ghia proceeded to see whether this would work in, at first, E.
coli. When she did theseexperiments, there was hanging over us a problem. And that problem was that we weren't really surethat it was going to work because of what was known about the GFP molecule. By this timeinvestigators had found that GFP had a rather unusual post-translational modification. The peptidebackbone in GFP cyclized, and the making of this five-membered ring was real mystery. Peoplespeculated that it might take one, two, or maybe even more converting enzymes to make the matureprotein from the translated product. So it wasn't a sure thing that this was going to work.Nonetheless, Ghia did try the experiment, and to our great joy and excitement, it worked very nicely.This is a page from her lab notebook that day, approximately one month after she entered graduateschool where she found strongly fluorescent E.
coli. and took this picture. She was also lucky inanother sense, and that is how we did the particular experiment. Douglas's clone had a cDNA fromthe jellyfish that was really the coding sequence which I have diagrammed here in green, and noncoding sequence, which is in red. He had gotten this as an EcoRI restriction fragment. Now we couldhave simply taken that restriction fragment and used that in our experiments, but I decided that Ididn't want to get extra stuff, and that it would be better to use PCR to amplify just the coding region.I wasn't sure what the rest was going to be, but this has turned out to be a very lucky choice, becauseit turns out that at least three other labs that I know of tried the experiment, but used the EcoRIfragment.
And when they did that, they never got any fluorescence, so there's something in theseextra sequences that seemed to interfere with the production of a fluorescent protein. We didn't dothat. We didn't have that problem. Within one month we knew that this was going to work. We werevery excited, and it now meant that we had to do… We were going to go off and do many otherthings. We put this into worms, and we decided that this was time now to publish the material, andwe had a little bit of difficulty with publishing the paper. So we sent the paper in to be reviewed in296Science, and the first thing we found was that the editors were not going to send it out to reviewersfor consideration because they did not like the title.
The original title was "Green Fluorescent ProteinNew Marker for Gene Expression" and the editors told me that all the papers in the journal were newand novel and so we couldn't use new in the title and would I change the title. I was a little bit miffedat this, so in retaliation, the title I gave them was considerably longer, "The Aequorea Victoria GreenFluorescent Protein Needs No Exogenously-Added Component to Produce A Fluorescent Product inProkaryotic and Eukaryotic Cells". This is essentially the entire paper. The paper got reviewed; thereviewers liked it.
And then the copy editor got in touch with me and said, "You know your title is alittle long, could you possibly shorten it?" And I said, "I think I can do that," and I changed it so thefinal title is "Green Fluorescent Protein as a Marker for Gene Expression". This was not the end ofour troubles though, we had sent in this picture that you see here that eventually made it to the coverof Science and I was very proud of this picture because what it shows is a growing nerve cell in aliving larva of the animal. And I wanted to point out the fact that GFP could be used in living tissue.The art editor, the cover editor called me up and said, that they really liked the picture, that theywanted to use it on the cover but there was one problem.
And that problem was that they never liketo use the color green on the cover, and would I consider changing the color of the picture. I said,"no, I really wanted it to be green," and fortunately they kept it that way. The final problem that wehad in publication was by this time we had already given out samples of the plasmid to people to tryfor themselves.
and we were getting wonderful reports back from people saying that it had worked.So I wanted to put that as personal communication into our paper, and the people that we asked wereall uniformly very generous, and one person, however, asked for some additional considerations,specifically, and you probably can't read it in this letter, but the letter asked that I prepare coffeeevery Saturday morning for two months, prepare a special French dinner, and take out the garbagenightly for the next month, these were requirements set out by my wife, Tulle Hazelrigg. But I reallywanted to use her work, and although we still debate about whether I have actually paid up on this.The work she did was really wonderful, and it was published a few months later.
But she's the personthat made the first protein fusion with GFP, so she was able to show that GFP could be linked toother proteins, and that one could follow those other proteins, check their localization, but also seetheir movements within cells and tissues. A beautiful example of the use of GFP protein fusions canbe seen in this movie by Rosalind Silverman-Gavrila of the nuclear divisions in an embryo ofDrosophila. I have taken this from the cell image library of the American Society for Cell Biology.It's on their website.
It's a really beautiful picture showing, in this time-lapse film, cell division inwhich you can see the spindle labeled and you can see the spindle forming and then dissolving andforming once again. GFP, other fluorescent proteins, and their derivatives, have been used forthousands upon thousands of experiments throughout biology. Their small size and inheritabilityprovide a dynamic and essentially a non-invasive means of following biological processes in livingcells.We've already learned much, and we will continue to learn more by using these molecules in thefuture.
I want to close with some more general lessons that I take from the story of the developmentof GFP. First, many discoveries are accidental. Certainly the discovery of GFP is one of those cases.Shimomura was not looking for a fluorescent protein; he was looking for a bioluminescent protein.But his experiments led him to this rather wonderful molecule.Second, scientific progress is cumulative. It is not the product of just Shimomura or myself orDouglas Prasher, or Tulle Hazelrigg, or many other people- Roger Tsien, who developed the firstmolecules with different emission colors, who also developed the first FRET based molecularmonitors using the fluorescent proteins, or the Lukyanovs or Mikhail Matz who discovered the firstred fluorescent protein by looking at corals.
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