(no subject)

C. S. Prakash Prakash at ACD.TUSK.EDU
Sun Mar 31 17:34:30 EST 1996


Here I have reproduced the draft of an article I wrote on "GFP" for the N=
BIAP/ISB News Report. =20
To receive this newsletter in print or electronic versions, send an email=
 to "P. L. Traynor"=20
<traynor at gophisb.biochem.vt.edu>

-C. S. Prakash
Tuskegee University

=20
        Green Fluorescent Protein- A New Reporter Gene Making its Mark on=
 the Transgenic=20
Research

       =20
        Reporter genes or screenable markers have proven very useful in b=
iotechnology research=20
as they provide a visual means to identify genetically engineered cells. =
 Transgenic plants,=20
animals and microorganisms are now routinely identified using such genes.=
  Currently the most=20
popular reporter genes are GUS (=DF-glucuronidase; uidA) and lacZ (=DF-ga=
lactosidase) from E. coli=20
which confer blue color to the transformed cells, and the luciferase gene=
 from firefly or bacteria=20
which produces luminescence.  Detection of these gene products, however, =
 often necessitates an=20
assay that results in the destruction of the sample and requires the addi=
tion of substrates.  A new=20
reporter gene obtained from the jelly fish (Aequorea victoria) called Gre=
en Fluorescent Protein=20
(GFP) is thus attracting considerable attention because it can be detecte=
d without destroying the=20
tissue.  Cells transformed with the GFP gene exhibit bright fluorescence =
under ultraviolet or=20
blue light, and this luminescence requires only oxygen but no other subst=
rates .  The GFP is a=20
highly stable protein with a small molecular weight and shows very little=
 photobleaching. =20

        Douglas Prasher of USDA/APHIS (Prasherd at Delphi.com) first cloned =
the GFP gene in=20
1992.  Along with Martin Chalfie and associates at Columbia University in=
 1994, he showed that=20
E. coli and C. elegans  exhibit bright green fluorescence when transforme=
d with the GFP gene. =20
Since then, GFP has been expressed  in many  organisms including yeast, D=
rosophila,=20
vertebrate  (including human) cell lines and plant cells (Kain, et al, 19=
95 BioTechniques 19:=20
650-655).  Because it can be detected non-invasively and with little disr=
uption of the cell=20
ultrastructure, GFP gene has many potential applications in molecular bio=
logy research and=20
commercial biotechnology:  measuring gene expression in vitro, selecting =
transgenic cells,=20
studying fusion proteins, studying intracellular protein traffic (and thu=
s identifying signal=20
sequences), determining cell lineage, assessing promoter activity, develo=
ping cell- and=20
tissue-specific markers, investigating the pathogen movement and disease =
development,=20
biomonitoring of organisms released into the environment, developing bioi=
ndicators for=20
detecting environmental pollutants, ensuring the containment of genetical=
ly modified organisms=20
and in evolutionary and ecological studies of transgenic organisms (Prash=
er 1995 Trends in=20
Genetics 11: 320-323).

        Several variations and improvements have now been made in the GFP=
 gene sequence.  By=20
substituting amino acids in the chromophore, proteins which yield blue an=
d red  fluorescence=20
have been developed (Heim, et al 1994 PNAS   91:12501-12504 ; Delagrave, =
et al. 1995=20
Bio/Technology 13:151-154).  As the GFP mRNA is spliced into smaller frag=
ments after=20
transcription in certain plant species, Jim Haseloff (jph at MRC-LMB.cam.AC.=
UK) at MRC=20
Laboratories in U. K. has created an improved GFP by removing the cryptic=
 intron splice sites=20
from the coding region.   Jen Sheen and Brian Seed (Sheen at opal.mgh.Harvar=
d.edu) at=20
Massachusetts General Hospital have developed a completely synthetic GFP =
based on the=20
optimum codon usage for plants, and this new gene results in an impressiv=
e 120-fold brighter=20
fluorescence than the original GFP (1995 The Plant Journal 8:777-784).  A=
 similar 'humanized'=20
GFP gene has also been developed at Florida State University.  David Galb=
raith and associates=20
at University of Arizona along with Jen Sheen have developed protocols fo=
r the identification=20
and sorting of GFP-expressing cells using the flow cytometry equipment. =20

        Roger Beachy and colleagues at the Scripps Research Institute hav=
e recently employed the=20
GFP gene fused with the tobamovirus movement protein gene to conduct some=
 very innovative=20
studies on the cell-to-cell movement of this virus in plants and to show =
that the movement=20
protein interacts with the microtubules of the plant cytoskeleton (1995 S=
cience 270:1983-1985). =20
Baulcombe, et al. (1995) also employed the GFP gene fusion to confirm the=
 role of viral coat=20
protein in the intercellular movement of potato virus X (1995 The Plant J=
ournal 7:1045-1053). =20

        A few potential problems in the use of GFP marker include its wea=
k expression as it is not=20
an enzyme and thus its signal is not amplified; green autofluorescence of=
 certain samples (e.g.=20
lignin in plants); potential toxicity to cells from excited GFP (GFP may =
inhibit plant cell=20
regeneration and growth); and the variability in the intensity of fluores=
cence.

        A biotechnology company at U. K. is now developing transgenic pla=
nts with the GFP gene=20
fused to various stress-promoters to eventually market such indicator pla=
nts for farmers to help=20
them detect heat, pathogen, drought and other stress situations on the fa=
rm.  In the not so distant=20
future, Santa Clause may find himself placing a gift under the Christmas =
trees=20
'glowing-in-the-dark'!=20

        Several GFP gene clones, antibodies and bibliography are availabl=
e from Clonetech=20
Laboratories (Tech at Clonetech.com).  The optical filters for GFP detection=
 are available from=20
Chroma Technology Corp. (Sales at Chroma.com).  An electronic newsgroup ('Fl=
uorescent=20
Proteins') devoted to discussion on research issues related to GFP  is on=
 the Internet=20
(http://www.bio.net).   Send an email message to biosci at net.bio.net to re=
ceive information on=20
accessing this and other newsgroups.


C. S. Prakash
Center for Plant Biotechnology Research
Tuskegee University
Prakash at Acd.Tusk.Edu

        =20






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