Green Mice and GFP

Green Mice and GFP

Charlie, my late friend and colleague, left me a large pile of

magazines to peruse and, as usual, I have found fodder for this

column in one of them, the November/December 1999 issue of

Biophotonics International. I was impressed that many of the

articles in this issue mentioned a substance known as “green

fluorescent protein” (GFP). One article stated that GFP is

responsible for a revolution in molecular biology. After

searching the term “green fluorescent protein” on the Internet and

coming up with over a hundred Web sites on GFP, I have no

reason to doubt this statement.

GFP is not new. It is the compound that”s responsible for the

green-glowing jellyfish known at least as far back as the heyday

of the Roman Empire. This jellyfish, formally known as

Aequorea victoria, will be referred to here more informally as A.

Vicki, in honor of my wife, whose name is also Victoria/Vicki.

Well, A. Vicki actually only emits this green light when its GFP

is hit by blue light emitted by another protein in the jellyfish

called aequorin. The emission of blue light in aequorin is

stimulated by calcium ions. Recently, other proteins, many

derived from various species of coral, have been found that

fluoresce yellow-green and orange-red, or even bright blue.

All these colorful proteins sound nice, but why should they cause

a revolution in molecular biology? Let”s look briefly at the

structure of the GFP, a string of some 238 amino acids. It has

been found that these amino acids wrap themselves around in

such a way that the structure is sort of like a barrel. Inside the

barrel is the so-called “chromophore”, the part of the protein that

emits the light. This chromophore, nestled safely within a helix

inside the barrel, is nicely protected against changes in its

environment. Because of this, the GFP will still fluoresce when

attached to other proteins and it can be injected into genes or

cells as glowing “markers”. Now all you have to do is look

through your microscope and watch what happens to the green

glow to follow the “host” protein to which the GFP is attached.

Surprisingly, the attachment of the fluorescent marker proteins

generally doesn”t interfere significantly with the normal behavior

of the host protein. This tagging allows the host protein”s

behavior to be studied under whatever conditions are of interest.

Some strange things can be done with GFP and its colorful

cousins. For example, in 1997 some Japanese scientists created

“green” mice, that is, mice that glowed green under artificial

light. They created these creatures by injecting GFP from A.

Vicki into the genes of lab mice. Even more bizarre, some

Russian workers attached GFP to one RNA protein and a red

fluorescent protein to another RNA protein. (RNA and DNA are

two of the most important proteins in us living creatures.) They

then stuck the “green” RNA into one cell of an 8-cell frog

embryo and the “red” RNA into another cell of the same embryo.

When the 8-cell embryo grew up to become a tadpole, the

tadpole, when illuminated with the proper type of light, glowed

red on one side, green on the other side and yellow in the middle

where the red and green overlapped!

You might rightly say that the call for multicolored tadpoles and

green mice is pretty limited. Let”s step back a little and suppose

we tag a protein in a cell with red fluorescent protein and another

protein in that cell with GFP. Now let”s follow what happens

when the cell divides. We can follow the two different proteins

within the same cell just by observing the red and green spots as

development of an embryo takes place. We might also follow

what happens to a tagged protein during an attack on a cell by a

virus or bacterium. Or we might observe the change in behavior

when an experimental drug is added to the mix. The GFP and

cousins can provide a window into the various reactions within

cells or among cells to all kinds of biological, chemical or

environmental factors.

Let”s look at another specific application of GFP. With the past

weeks” troubles at Los Alamos, it”s fitting we consider something

positive, the Los Alamos “Rapid Protein Folding Assay”. If you

look through any magazine or journal that talks about proteins,

you”re bound to come across very complicated models of various

proteins. I confess to my eyes glazing over when I look at these

models, filled with ribbons folding every which way in complex

fashion. Nevertheless, how proteins fold is key to their proper

functioning in the body and the study of “protein-folding” is a

big deal in biology these days. As a result, there is a demand for

techniques to determine whether or not a newly synthesized

protein folds properly. Possible areas of usefulness include drug

development and the study of diseases such as Alzheimer”s,

which some think is linked to misfolded proteins.

The Los Alamos technique employs the sensitivity of GFP to

proper folding of proteins. What the workers at Los Alamos did

was to attach the genetic code for GFP production to the code for

various other proteins. Typically, the more properly the protein

folds the more soluble it is. It turns out the more soluble it is, the

brighter the fluorescence of GFP. So, just by comparing the

brightnesses of the various proteins, the brightest, best proteins

can be separated from their duller, less suitable counterparts.

In 1997, there was an International Symposium on GFP

sponsored by Rutgers University, where I spend some time most

weeks. Just to show the wide range of applications for GFP and

other fluorescent proteins, the topics included marking viral

infections, assaying mutation rates, probing the intracellular

environment relating to apoptosis (cell suicide discussed in an

earlier column), calcium concentrations, monitoring of HIV

infections, development of drug screening methods, tracing of

evolutionary paths of organisms, olfactory receptor studies, yeast

mitochondria, follow microbial populations in their native

environment, etc. Quite a list!

We noted above that new fluorescent proteins are being

discovered in corals. You”ve probably read over the years of the

concern about the bleaching and resulting destruction of coral

reefs in various areas over the globe. Fluorescence can be used

to measure the state of a given coral species through an

understanding of the photosynthesis. During photosynthesis

light is absorbed and chemical processes such as the formation of

sugars occurs. Not all the energy of the absorbed light goes into

chemical processes, however. Instead some of the energy is lost

in the emission of light, i.e., fluorescence. By measuring the

fluorescence, the health of the photosynthesis process can be

determined. Higher than normal water temperatures have been

implicated in coral bleaching and, of course, global warming is

lurking as a possible important factor.

This just gives a taste of the colorful potentials of these

compounds. If you”re interested in actually seeing movies of

some of them in action, just search the term “GFP” and some of

the sites have short little movies of various cellular processes

involving tagged proteins. ……..Whoops. I just tried “GFP” and

instead of green fluorescence proteins, I came up with a couple

sites such as Gays for Patsy, a site for gay country western

dancing enthusiasts. I have nothing against gays or country

western dancers but, depending on your own interests, you might

want to search the full “green fluorescent protein”!

Allen F. Bortrum