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05/23/2000

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










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Dr. Bortrum

05/23/2000

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