PI.ANT MOL|:CIII.AR I:11()I.

O(;Y REI>ORTER
Volume %, Number i , 1987 Pages :187-.i()5

EXPERIMENTAL PROTOCOLS

Assaying Chimeric Genes in

Plants: The GUS Gene
Fusion System
R i c h a r d A. J e f f e r s o n
Z)~partmeplt ~!/-klo/ecH/m Ge~letk:~, P/met Breec/ip~<h~.~tittlte, Camhri~/q,e, Ell~/apt~t
CB2 2LQ

I n t r o d u c t i o n to G e n e F u s i o n s

DeJi~eitio, r Ge~e lrlt.~irm

Much of tile attention and interest in modern molecular biology is fi~cussed
on the regulation of gene expression. Factors influencing or mediating such
regulation are often best studied using gene Alsions. Gene fusions can be
defined its D N A constructions (perfi3rmed ill vitro or i~e Hvo) that result in the
coding sequences from one gene (r@o,ter) being transcribed and/or translated
under the direction of the controlling sequences of another gene (cmltrr
Gene fusions can be of two general types, with many wtriatiuns within
types. Transcriptional fusions are defined as fusions in which all protein cod-
ing sequences are derived from the reporter, with none from the cmm,//e~.
Thus, although the m R N A produced may consist of sequences from both
control/o and re/;o~ter, the protein synthesized will be encoded only by the
reporter. Translational fusions, in contrast, are defined as those in which the
polypeptide produced is the result of coding information provided by both
copraoiler and reporter.

I*~qlti~'i~ t,,. Richard A. [ct~L.rson. Plant l',rc~'dmg Institute, Caml.~ridge, England (:[:12 2LQ
388 The' Pknl! /~,D#~'cn/,oBk~hJW Rq~,n't~,r

Why Use Gene Fusions?

It is now clear that control nfgene activity can be manifested at many levels,
including the initiation of transcription or translation, the processing, trans-
port or degradation of m R N A or protein. The use of precise gene fusions can
simplify analysis of this complex process. For example, it is possible to de-
lineate the contribution of transcriptional control ofgene expression by elim-
inating all the specific signals for post-transcriptional controls and replacing
them with sequences from a readily assayed reporter gene. Further careRd
gent fusion constructions can then be perfnrmed to assay the eft:cots of inclu-
sion of additional controller sequences, for instance the "untranslated leader
sequences" or the sequences surrounding the site of translational initiation.
Gene fusions need not be confined to promoter analysis, since h~ctors affecting
m R N A processing and stability, (such as polyadenylatinn signals or intronsL
or translational efficiency (such as the context of the initiator codnn or m R N A
secondary structure), will inevitably affect reporter enzyme levels. \X/ith the
appropriate controls, many of these regulatory steps can also be analyzed with
gene fusion technology. It is importan~ to be aware of tile potential contri-
butions of these "downstream" points in the reguiatory pathway of gene
expression so they can be considered in the design and interpretation ofgene
fusinn experi men is.
In addition, many genes in plants and other higher organisms exist in
multi-gene families whose products are very similar but can be regulated
differentially during development - - in fact, members of multi-gene families
are often apparently inactive. By using gene fusions to individual members
of such fitmilies and introducing these fusions into the genome, one can study
the expression of individual genes separate and distinct from the background
of the other members of the gene family.
Analysis of mutationally altered genes in plants accessible to transfi)rma-
tinn techniques is greatly facilitated by the use of sensitive and versatile re-
porter enzymes. Many of the regulatory parameters that we would like to
study are those responsible for spatial and temporal restriction ofgene activity
- - requiring analysis methods that can resolve these issues. Moreover, the
logistics of analyzing gene function in large numbers of transgenic plants can
be overwhelming, unless routine, high resolution techniques are available.
Although many of the plant genes that have been characterized to date pro-
duce abundant products that are measurable by existing means, many more
will certainly be described whose products are of moderate or low abundance;
these will doubtlessly prove important and interesting to study, requiring
increasingly sensitive methods. By using a reporter gene that encodes an en-
zyme activity not found in the organism being studied, the sensitivity with
The GUS Germ Fu.,iopl Syste'm 389

which chimeric gene activity can be measured is limited only by the proper-
ties of the reporter enzyme and the quality of the available assays fur the
enzyme.

Reporter Genes ipe P/apJts

More than six reporter genes have been used in studies ofgene expression in
higher plants. These include the E. co/i beta-galactosidase (/aicZ), chloram-
phenicol acetyl transferase (CAT), neomycin phospotransferase (APH3'II,
NPTII), nopalinc synrhase (NOS), octopine synthase (OCS), and firefly luci-
ferase.
In spite of the remarkable success of lac fusions in other systems, beta-
galactosidasegene Alsions in plants(Helmer et al., 1984) have been of little
use, and have proved difficult to assay because of high endogenous beta-
galactosidase activity in plants. Galactosidases are present in virtually all
plants, and in most, if not all tissues.
Problems associated with endogenous reporter activity were largely over-
o)me by using the k wobatTerium tumef, aie*ts Ti-plasmid-encoded genes nopa-
line synthase(Depicker et al., 1983; Beval et al., 1983a) and octopinesyn-
thase (De Greve et al., 1982) because the opines produced by these genes are
not f])und in normal plant cells. However, these reporter genes are not widely
used because the assays are cumbersome, difficult to quantify (Otten et al.,
1978), and octopine synthase cannot tolerate anaino-terminal fusions (jones
et al., 1985).
The two most widely used reporter genes have been the bacterial genes
chloramphenicol acetyl transferase (CAT) and neomycin phototransferase
(NPTll) which encode enzymes with specificities not normally found in plant
tissues (Hererra-Estrella et al., 1983a,b; Bevan et al., 1983b; Fraley et al.,
198"3). In addition, NPTII can tolerate amino-terminal fusions and remain
enzymatically active, making it useful for studying organelle transport in
plants (win den Broeck et al., 1985). However, both CAT and NPTII are
relatively difficult, tedious and expensive to assay and suffer from variable
endogenous activities in plant cells (generally caused by enzymes with
broader substrate specificity), which limits both their sensitivity and the va-
lidity o f q u a n t i t a t i o n ( G o r m a n et al., 1982; Reisset al., 198-i). Competing
reactions catalyzed by endogenous esterases, phosphatases, transferases and
other enzymes also make quantitation of CAT or NPTII by, enzyme kinetics
difficult. Recently, the firely luciferase gene has been used as a marker in
transgenic plants (Ow et al., 1986), but the enzyme is labile and difficult to
assay with accuracy (DeLuca and McEiroy, 1978), the reaction is complex and
there is little, if any, potential for meaningful histochemical analysis or fusion
genetics.
~,9() The P/a./ Alokcn/,o Bir)&<yR~porter

Future advances in the study of plant gene expression require the develo-
ment of new gene fusion systems that are easy to quantify and are highly
sensitive, thus allowing for anlysis of genes whose products are of moderate
and low abundance. Activity of the reporter enzyme should be maintained
when fused to other proteins at its amino terminus, to alh)w the study of
translation and the processing events involved in protein transport. The re-
porter enzyme should be detectable with sensitive histochemical assays to
localize gene activity in particular cell types. Finall B the reaction catalyzed
by the reporter enzyme should be sufficiently specific to minimize interference
with normal cellular metabolism and general enough to allow the use of a
variety of novel substrates to maximize the potential for fusion genetics and
ip/l'iz', analysis.
"I~ meet these criteria, the E. oJ/i beta-glucuronidase gene was developed
as a reporter gene system. Beta glucuronidase (EC.3.2.1.31) is a hydrolase
that catalyzes the cleavage of a wide variety of beta-glucuronides (Stoeber,
1961), many of which are available commercially as spectrophotometric, flou-
rometric and histochemical substrates. The beta-glucuronidase gene has been
chined and sequenced, and encodes a stable enzyme that has desirable prop-
erties fi)r the construction and analysis o f g e n e fusions(Jefferson, 1985;Jef-
ferson et al., 1986a; Jefferson et al., 1987a; Jefferson c t a l . , 1987b; Jefferson
et al., 1987c). In this paper and in the cited refernces, I describe several
useful features of the GUS gene that make it an excellent reporter gene fur
plant studies.

Beta-G lu~vo'mJidase
The E. c,/i beta-glucuronidase (GUS) has a monomer molecular weight of
abnut 68,200 daltons, although under certain conditions of SDS-PAGE it
migrates a bit slower than would be predicted (around 74,000 daltons). The
behavior of the native enzyme on gel filtration columns indicates that it is
probably a tetramer. Beta-glucuronidase is very stable, and will tolerate many
detergents, widely varying ionic conditions, and general abuse. It is mnst
active in the presence of thiol reducing agents such as beta-mercaptoethannl
or DTT. GUS has no cofactors, nor any ionic requirements, although because
it is inhibited by some heavy divalent metal ions (Cu e+ and Zne+), it is
prudent to include EDTA when assaying (Stoeber, 1961). Glucumnidase can
be assayed at an}, physiological pH, with an optimum between 5.2 and 8.(I.
The enzyme is about 50c2; as active at pH 4.3. GUS is reasonably resistant to
thermal inactivation with a half-life at 55~ of about two hours. The purifi-
cation and properties of the enzyme and the gene cloning, vector construction
and complete nucleotide sequence have been described (Jefferson, Burgess
and Hirsh, 1986;Jeflerson, R. A., 1985).
The G US Gene Fusion S),stem 391

G US Gene Fusions
The GUS gene was developed initially as a gene fusion marker in E. coli and
in Caenorhahditis e/egans, but has more recently been used extensively to mon-
itor chimeric gene expression in plants. There is little or no detectable beta-
glucuronidase activity in Saccharomyces cerez,isiae, Dros,phi/~1 melalzogaster em-
bryos, larvae and pupae (adults were not tested), certain strains of Caenorhah-
ditis elegans (Jefferson et al., 1987a; Sebastiano et al., 1986, Ge~zetks. 1 I2."
459-468), Dico,ostelium discoidllm (R. Firtel, pers. comm, quoted in Jefferson
et al., 1986) or in almost any higher plant (Jefferson et al., 1987b), includ-
ing tobacco, petunia, potato, tomato, Bras.6ca, maize, soybean, wheat, rice,
barley, or Arahidopss The enzyme can tolerate large anaino-terminal addi-
tions, so construction of translational fusions is feasible and may be a valuable
method for assaying the behavior of transit or signal peptides either in trans-
genie systems or i,l vitro. The enzyme can be translocated across chloroplast
membranes with high efficiency (Kavanagh, Jefferson and Bevan, in press).
We have used this system successfully to study the regulation of numerous
genes in transgenic tobacco and potato, including CaMV35S promoter, ri-
bulose bisphosphate, carboxylase small subunit, (Jefferson et al., 1987), chlo-
rophyll a/b binding protein (Jefferson, Harkins, Bevan, Kavanagh and Be-
van, submitted; Kavanagh, Jefferson and Bevan, in press), patatin (Jefferson
and Bevan, in preparation), soybean phenylalanine ammonia lyase (Bevan,
Davis, Edwards, Shuch and Jefferson, in preparation), wheat glutenin genes
(Colot et ai., in preparation; Robert et al., in preparation), nopaline synthase
(Scofield, Bevan and Jefferson, unpublished), potato wound-induced genes
(Stanford et al., in preparation) and others.

Substrates
p-nitrophenyl glucuronide (PNPG) (Sigma N-1627)
4-methyl umbelliferyl glucuronide (MUG) (Sigma M-9130 or Molecular
Probes)
5-bromo-4-chloro-3-indolyl glucuronide (X-GLUC) (Research Organics,
4353 East 49th St., Cleveland Ohio)
naphthol AS-BI glucuronide (NAG) (Sigma N-1875)
resorufin glucuronide (ReG) (Molecular Probes, Inc., 4849 Pitchford Avenue,
Eugene, Oregon, 97402 (503) 344-3007).

Notes abozzt sztbstrates."

Molecular Probes Inc. of Eugene, Oregon has made a major commitment to
developing and marketing new substrates with substantially enhanced prop-
erties for fluorescence and histochemical assays as well as for novel fusion
 392 The Plant Alo/e,ul,lr Biolow Repot/o"

genetic approaches. I recommend them as sources of these compounds, and

because of my collaborations with them, as sources of information. Likewise,
Research Organics, of Cleveland Ohio, were the first (and at present the only)
company to manufacture X-glucuronide, They did this at my request, and
manufacture an excellent product.

Lysis Conditions
Tissues or cultures can be homogenized for assays in many different buffers.
We have found this Extraction Buffer to be very effective at achieving good
lysis and preserving G U S activity and D N A (for denominators - - see Jeffer-
s o n e t al., 1987b)

GUS Extraction Buffer."

50 mM NaPO.,, p H 7.()
l0 mM beta-Mercaptoethanol
l0 mM N a , E D T A
0. l ~ Sodium Laury[ Sarcosine
O. t(5~ Triton X-lO0
Use either the French press, homogenizer, cell disruptors, sonication or
grinding with sand or glass beads. Kontes Glass sells little disposable pestles
that fit into Eppendorf tubes that are satisfilctory for grinding small bits of
tissue (e.g., leaf tissue). The method of lysis will depend on the nature of the
tissue. GUS is remarkably resistant to protease action, with a very long half-
life in living cells and in most extracts, but if proteases are a potential prob-
lena, PMSF (phenylmethyl sulfonyl flouride - - a potent inhibitor of serine
prnteases) at a final concentration of 23 p,g/ml can be included in the lysis
buffer with no ill effects on the enzyme.

Sto~zlge of Extrcars
Extracts and tissues can be stored at - 7 0 ~ with no loss ot activity for a
long time, and at 4~ with very little loss (at least in tobacco leaf extracts
- this will, of course, depend on intrinsic protease levels). Avoid storage at
-

-20~ which seems to kill the enzyme m this extraction buflbr. Tissues
stored at - 20~ for a few days do not seem to lose significant GUS activity.

Hints about Extraction and Dealing u'ith Endogenous Fluorescence

Tissues or cells that are high in endogenous absorbing or fluorescent com-
pounds or that produce high levels of polyphenolics can be extracted in G U S
extraction buffer with a pinch of polyc[ar (insoluable polyvinyl pyrollidone),
centrifugation to eliminate debris and adsorbed materials, followed by a brief
The GUS Gene Fusion System 393

spin-column of Sephadex G-25 to eliminate almost all polyphenolics and low-

molecular weight flourescent contaminants from the extract (Alison Huttly
and David Baulcombe, personal communication). Huttly and Baulucombe
have found this to be extremely effective for assaying tissues as rich in absorb-
ing materials as oat aleurone protoplasts.
Occasionally callus, wounded plant cells (such as protoplasts that have been
PEG treated or electroporated) and tissues such as root, sometimes accumu-
late fluorogenic compounds (presumably secondary products, intermediates
in lignin biosynthesis, and other phenylpropanoid pathway compounds).
Some of these compounds are not fluorescent until after cell lysis - - which
can release enzymes that cleave off the glycoside or other conjugate to release
the free fluorochrome. This can be easily dealt with by the spin-column
method, or by following the kinetics of fluorescence increase. These endoge-
nous compounds and enzymes should nnt give rise to MUG-dependent fluo-
rescence.

Beta-Glucuronidase A s s a y s
Why use fluorescence?
Detection of beta-glucumnidase activity depends on the availability of sub-
strates for the enzyme which, when acted on by the enzyme, liberate a prod-
uct that is distinguishable from the substrate. A substrate that is designed to
maximize sensitivity of detection of the enzyme should have several proper-
ties. There should be a method of detecting the product very specifically. The
substrate should be cleaved only by the enzyme under study, with minimal
spontaneous cleavage. The signal-to-noise ratio of the method of detection
should be as high as possible. These properties are best achieved by using
fluorogenic substrates for beta-glucuronidase, such as ,4-methyl umbelliferyl
glucuronide (MUG). Detection of fluorescent molecules offers a very high
signal-to-noise ratio because the incident excitation light does not impinge
on the detection apparatus, and has a spectrum distinct and separable from
that of the emission. The use of fluorescence measurements to detect enzyme
activity usually allow two to four orders of magnitude greater sensitivity than
methods that rely o n spectrophotometric determination of product concentra-
tion by absorption. Absorption techniques measure a small difference be-
tween two large values, whereas fluorescence techniques measure an absolute
value over an arbitrarily small background. Whether the detection apparatus
is a quantitating optical device such as a spectrophotometer, or the human
eye, the signal to noise ratio is the limiting factor in determining the levels
of product detectable.
394 The P/ant Al~J/ecu/arBiol, gy Rep,rler

Fluorogenic Assay
Tile fluorogenic assay can be performed in Eppendorf tubes or in microtiter
plates. The recipe below is for Eppendorfs, but can easily be scaled down for
microtiter plates to readily assay numerous samples.
G U S E x t r a c t i o n B u f f e r (1 1) Stock Solutions Volumes
50 mM NaPOq, p H 7.0 1 M N % H P O , , p H 7.0 50 ml
10 mM beta-Mercaptoethanol neat (14.4 M) 0.7 ml
10 mM N a , E D T A 0.5 M Na~EDTA, p H 8.0 20 ml
0 . 1 % Sodium Lauryl Sarcosine 1 0 ~ Sarcosyl 10 ml
0.1% Triton X-100 10% Triton 10 ml
909 ml H , O

Assay Buffer (MUG):

9 I mM M U G in Extraction Buffer (50 ml)
9Dissolve 22 mg 4-Methyl umbelliferyl beta-D-glucuronide in 30 m[ GUS
extraction buffer in a 50 ml disposable polypropylene tube.

Stop Buffer:
90.2 M Na:CO~ (1 liter)
9Dissolve 21.2 grams NaeCO~ in deionized, distilled water. Make up to 1
liter.

Fluorogenic Assay Protoco/

9Incubate 0.5 ml aliquots of Assay Buffer at 37~ to pre-warm.
9Add 1-50 ILl of Extract to 0.5 ml Assay buffer.
9Mix thoroughly with pipet tip or vortexer.
9After 1-2 minutes, remove 100 ILl into Eppendorf tube containing 0.9 ml
Stop Buffer at room temperature.
9At regular time intervals after this remove successive 100 >1 aliquots into
labeled Eppendorf tubes containing 0.9 ml Stop Buffer. Typically 5 minute
intervals for high-levels of GUS, or 30-60 minute intervals for lower levels.
Take 3-4 time points.
9Determine M U concentrations with Spectrofluorimeter, excitation at 365
nm, emission at 455 nm.

Ca/ibration (~ the Fluorimeter.

Readings taken with a spectrofluorimeter are relative fluorescence, and must
be calibrated at each use with known standards.
The GUS Gene Fusion S),,~tem 395

1 mM alethylumhelliferone Stock Solution:

Dissolve 19.8 mg Na + methyl-umbelliferone (e.g., Sigma 1508) in double
distilled water. Wrap the bottle in a l u m i n u m foil and store at 4~ This stock
is good for at least one month.

IV1U Standards:
1 ~ M , 100 nM in 0.2 M Na,CO~
Add 100 btl 1 mM MU Stock Solution to 900 btl Stop Buffer, mix well.
1 btM MU: Add 100 ILl of this dilution to 9.9 ml Stop Buffer.
100 nM MU: Add 1.0 ml of 1 I~M MU to 9.0 ml Stop Buffer.
Use the l btM and 100 nM MU standards in Stop Buffer to calibrate the
fluorimeter. On a machine with a digital readout up to 9999 (such as the
Perkin-Elmer LS series) I typically establish 1 btM MU equal to 1000 relative
fluorescence. Thus the relative fluorescence reading from the samples can be
read directly as nM MU. W i t h a machine such as a Kontron (Gilford) SFM-
25, I usually set 1 btM MU equal to 100% relative fluorescence, and use the
factor control on the instrument to multiply upwards, e.g., factor 1 0 x , if
the reading is low, ordownwards,e.g. ,factor 0.1 • if the reading is too high.
Read the relative fluorescence of the time course for each extract in an
ascending order. Convert the values obtained to nanomolar MU, and then to
nanomoles MU, after correcting for the assayed volume. The preferred units
to express G U S activity are nmoles MU produced/minute. The normalization
(per mg protein, per p.g D N A , or per unit flesh weight, etc., is at your
discretion).

Co111111ellt.~."
This assay is the most sensitive and versatile. Fluorescence methods are in-
trinsically lO0 to 1000 times more sensitive than colorimetric methods, for
reasons discussed above. For a good reference that will cover the basics of
fluorescence measurements, I recommend "Practical Fluorescence" by George
G u i l b a u l t , Dekker, New York, 1973. Although the book is a bit out of date,
the coverage is very thorough on most aspects of fluorescence spectroscopy
that will be of interest to molecular biologists (and a lot that will not).
The only commercially available fluorogenic substrate that has been de-
scribed in the literature for assay of beta-glucuronidase activity is 4-methyl
umbelliferyl glucuronide. This compound is not fluorescent until cleaved by
beta-glucuronidase to release 4-methyl umbelliferone (7-hydroxy-4-methyl
coumarin). The product is fluorescent only when the hydroxyl group is ion-
ized. The PK, of this hydroxyl is between 8 and 9, and maximal fluorescence
will only be obtained if the product is in solution at a p H greater than the
 7;96 The Pktnt glo/ec#hzr 13i0/r Rq~r

pK.,; fluorescence is relatively low at the neutral pH of the living cell. Better
and more sensitive substrates are being developed.
We have observed that GUS activity remains linear (even in crude extracts)
for a very long time, sometimes days. Hence tile " t i m e = 0" point does not
need to be at tile m o m e n t of addition of extract to substrate. In fact, I often
allow the reaction to proceed at 37~ fi)r several minutes to equilibrate and
to achieve V ...... bef;,/re taking tile initial time point (as abm, e). Thus, I will
actually take 5, 13, 25, and 33 minute time points, and always find true
linearity is maintained. The resulting slope can therefore be measured inde-
pendent of tile intrinsic fluorescence of the extract. Another major advantage
to using kinetics (as opposed to single stopped reactions, is the greatly en-
hanced significance of a slope generated from several points rather than a
single end-pnint.
Linearity of the assay is excellent from as low as the machine can measure
(usually 1 nanomolar MU or less) up to 5-1() micromular .i-methyl umbelli-
femne. Measurements of values greater than this with a conventional 1 cm
path length will be artifacrually low, due to inner-cell effects and other f:ac-
mrs. Values up to ten times higher are actually linear with respect to enzyme
activity, but need to be diluted in ().2 M Na,CO~ before reading on most
f l u o r i m e t e r s . . i - M U is nut particularly photo-labile, but it is probably wise
to protect it from strong light, and keep the stock soiuthm in the refrigerator.
The stock solution is stable for a month, but be carcAd of its tendency to
crystallize out of solution with time. The diluted solutions and the stopped
reactions are stable fiJr at least a few days, if kept cold and dark. Hence, if
necessary a large set of reactions can be run and stopt~ed together, but kept
fbr later analysis with the fluorimeter.
The addition of N a , C O , serves the dual purpose of stopping the reaction
and maximizing the fluorescence of the product, 4-MU, as described above.
The initial levels of fluorescence in the extract (at time = 0) are often largely
contributed by the traces of pre-hydrolyzed substrate (i.e., methyl umbelli-
ferone) in commercial substrate preparations, or coumarins in the extract. For
ultra-sensitive measurements, an initial cleanup of the substrate could be
performed, but in general, we never bother. It should be pointed out, in s
that the G U S produced by one transformed plant cell can easily be measured
using unpurified commercial M U G and a relatively "low-tech" fluorimeter
(Jefferson, Harkins, Bevan, Kavanagh and Galbraith, submitted). If you are
going to push the detection limits, purchase M U G (ultrapure) from Molecu-
lar Probes, Inc. To minimize the levels of zero-time fluorescence (the MU in
the M U G ) keep the solid stocks of M U G frozen and dessicated, and don't
open the bottle when it is still cold (condensation of water from the air will
stimulate hydrolysis in the bottle). The M U G in Extraction Buffer (i.e., As-
The GUS Gene Fusion S),stem 397

say Buffer) is fine in the refrigerator For a couple of weeks, but will eventually
begin to show increased fluorescence.
A very convenient and sensitive qualitative assay can be done by setting
the tubes on a long-wave U.V. light box and observing the blue fluorescence.
This assay can be scaled down easily to assay very small volumes (reaction
volume 30 I*1 or less, stopped by addition of 25 btl 1M NaeCO 0 in microtiter
plates or Eppendorf tubes. For those without access to a fluorimeter, the use
of the long-wave U.V. box can actually give very sensitive and accurate esti-
mates of activity. Use a series of known standards of MU in 0.2 M NaeCO ,,
and interpolate the MU concentration of your assays by eye. You may be
surprised how accurate this method is. We have found that if we can't see tile
fluorescence by eye, it is usually not significant, even with the machine.
If the intrinsic fluorescence or absorption (resulting in quenching) of the
extract is a serious problem limiting sensitivity, one can either extract the
fluorescent compounds prior to analysis (see above) or use one of tile other
fluorogenic substrates synthesized by Molecular Probes. In particular, reso-
rufin glucuronide yields resorufin, which has an extraordinarily high extinc-
tion coefficient and q u a n t u m efficiency, and its excitation (570nm) and emis-
sion (590) are conveniently in a range where plant tissue does not heavily
absorb or fluoresce. In addition, it fluoresces maximally at neutral pH, mak-
ing it unnecessary to stop the reaction. Note that resorufin undergoes ready
reduction to rezaurin (non-fluorescent) in the presence of mercaptoethanol.
Hence, if you use resorufin glucuronide, omit beta-mercaptoethanol from tile
assay buffer.
An additional and occasionally useful trick to convince yourself that a low
level of linear fluorescence increase is due to GUS, is to use the specific glu-
curonidase inhibitor saccharo-lactone (Sigma S-0375), saccharic acid, t-4 lac-
tone) to prove the GUS-dependence of the fluorescence increase. This inhibi-
tor will eliminate glucuronidase activities at concentrations less than one
millimolar, but the compound is unstable at neutral p H , so beware of lengthy
assays. Because of this instability, I use saccharo-lactone at up to 5 mM for
assays up to half an hour. Alternatively, perform the reaction and the inhib-
ited reaction at p H below 6.0. GUS is active at low p H , and saccharo-lactone
is much more stable at acid pH. Note that this will give good evidence that
a glucur0nidase is responsible for the activity, but not necessarily the E. a~li
enzyme.

Spectrophotometric Assa3,
This assay is very straightforward and moderately sensitive. Because of the
remarkable stability of GUS, one can enhance the sensitivity by using very
long assays. We find overnight assays can give very good, linear results. It is
398 The Plant MdecMar Bio/oO' Reporter

also very cheap, easy to automate and easy to quantitate without sophisticated
instrumentation. Its limitations are the intrinsic lack of sensitivity of meth-
ods based on absorption of light (which measure a small difference in two
large numbers), and the problems caused by light absorption by pigments in
extracts. For very long assays (overnight or longer), add 0.02~, N a N , to
prevent microbial growth, and 100 gtg/ml BSA to stabilize the enzyme.
9U s eI m M p-nitrophenyl beta-D-glucuronide in Extraction Buffer.
9Perform the reaction in 1 ml volume.
9Incubate at 37~
9Stop the reaction by addition of 0..4 ml 2.5 M 2-amino-2-methyl propane-
diol (Sigma A-9754)
Absorbance is measured at .~ 15 nm against a substrate blank (or if turbidity
of the extract is a problem, against a stopped blank reaction to which an
identical amount of extract has been added).
Under these conditions the molar extinction coefficient of p-nitrophenol is
assumed to be 14,000, thus in the 1.4 ml final volume, an absorbance of
0.010 represents one nanomole of product produced. One unit is defined as
the amount of enzyme that produces one nanomole of product/minute at
37~ This represents about 5 ng of pure beta-glucuronidase.
This assay can very easily be automated using commercially available
ELISA plate readers, but is much less sensitive than the fluorescence assay.

Histochend,zd Assay
An excellent detailed description of laboratory methods for histological and
microscopic manipulation of plants, including sectioning tissues by hand is
given by O'Brien and McCully (1981). The book is hard to find, but well
worttl the effort.
The best substrate currently available for histochemical localization of beta-
glucuronidase activity in tissues and cells is 5-bromo-.-~-chloro-3-indolyl glu-
curonide (X-Gluc). This substrate works very well, giving a blue precipitate
at the site of enzyme activity. There are numerolts variables that affect the
quality of the histochemical localization, including all aspects of tissue prep-
aration and fixation as well as the reaction itself.
It is worthwhile understanding the nature of the reaction to better elimi-
nate the variables. The product of glucuronidase action on X-Gluc is not
colored. Instead, the indoxyl derivative produced must undergo an oxidative
dimerization to form the insoluble and highly colored indigo dye. This di-
merization is stimulated by atmospheric oxygen, and can be enhanced by
using an oxidation catalyst such as a K + ferricyanide/ferrocyanide mixture
(Lojda, 1970. Histochemie 23." 266-288; reviewed in Pearse, 1976). Without
The GUS Gene Fusion S),.rtem 399

such a catalyst, the results are often very good, but one must be concerned
about the possibility that localized peroxidases may enhance the apparent
localization ofglucuronidase. One will not get false positives, but the relative
degree of staining may not necessarily reflect the concentrations of glucuron-
idase. We rarely use a catalyst for routine work, however.
Fixation conditions will vary with the tissue, and its permeability to the
fixative. Glutaraldehyde does not easily penetrate leaf cuticle, but is imme-
diately available to stem cross-sections. One should test fixation empirically
in any new system. Formaldehyde seems to be a more gentle fixative than
glutaraldehyde, and can be used for longer times.
We have successfully used 0.3c~ formaldehyde in 0.3 M mannitol, 10 mM
MES, p H 3.6 for 30-45 minutes at room temperature for protoplasts or stem
cross sections. This should be followed by several washes, usually in phos-
phate buffer or osmoticum.

Histochemical Staining with X-glue.

9Dissolve 5 mg X-gluc in 30 b~l dimethyl formamide
9Add 50 mM N a P O , , p H 7.0 to 5-10 nal.
9Incubate freshly cut or formaldehyde fixed sections or tissues at 37~ for
times from a few minutes to overnight. The quality of the localization does
not decay much with lengthy assays.
W h o l e plants, callus, suspension culture cells and protoplasts can also be
stained in this manner, but survival of the stained cells is not by any means
certain. Reasonable staining can be achieved at much lower substrate concen-
trations (50 ~xg/ml) with enhanced recovery.
After staining, clearing the tissue sections with 709; ethanol seems to im-
prove contrast in many cases (Maud Hinchee, personal communication).
An alternative (and cheaper) histochemical assay for GUS uses naphthnl
ASBl-glucuronide, cleaved to liberate the free naphthol ASBI, then coupled
to a diazo dye (e.g., Jefferson et al., 1987a).
Incubate the formaldehyde or glutaraldehyde fixed tissue or whole mounts
in 0. l M NaPO., p H 7.0 containing 1 mM napthol ASBI glucuronide in a
moist chamber at 37~ For very low amounts of enzyme lengthy incubation
may be necessary, but gives poorer localization of activity due to diffusion of
the primary reaction product. The specimen is then washed in phosphate
buffer and coupled using a fresh solution of diazotized dye in phosphate
buffer.
Post-coupling with a 1-5 m g / m l solution of Fast Garnet GBC in phosphate
buffer, p H 7, gives a very nice result alter as little as thirty seconds coupling,
but many different coupling agents can be used. It is extreme/), important to
4OO The P/ant iAl./ecHklr Bio/~D' Rq~orter

do controls with diazo coupling agent alone. Perform an identical mock re-
action with Napthol ASBI glucurnnide, but perform the coupling reaction.
There is almost always a definite staining from the diazo reagent by itself,
but this staining is usually easily distinguished from the napthol coupled
product.
There is a voluminous literature about these histochemical methods as ap-
plied to mammalian glucuronidases (reviewed in Pearse that I recommend to
anyone embarking on a serious histochemical analysis.

S,ur,-es ,/-"'Bach~,r, und"

There are occasionally reports of "background" activity that can often be
traced to one of several potential sources. Often the "background" is simply
intrinsic fluorescence that has nothing to do with GUS, but simply shows the
levels of endogenous fluorescent compounds. This can be dealt with as de-
scribed above, using better extraction methods and perf;,)rming simple en-
zyme kinetic measurements. Another source of"background" comes from labs
using protoplasts to study transient gene expression. Be careful! Some enzyme
mixtures (especially Rhozyme) used to prepare protoplasts have a very large
amount of beta-glucuronidase activity. The usual flotation and KC1 washes
work fine to eliminate all residual activity from tobacco protoplasts, but other
types of cells may be more or less difficult to clean up. There are also reports
that some cell types, especially upon lengthy incubathm with prutoplasting
enzymes, endocytose material from the medium, if this occurs, only the ap-
propriate controls will minimize the "background," its washing will not help.
The other source of "background" is simply the mutability of chemicals.
When one performs extremely lengthy assays (overnight or hmger) with very
concentrated extracts, there is the risk that the substrate is being converted
into another compound, and then cleaved to produce a fluorescent signal.
This possibility must arise for an), chemical reaction, but is easily dealt with
by (again) using enzyme kinetics. I generally find that long assays don't help
much. If you can't see activity in a few hours (at least in a reasonably concen-
trated extract), you probably don't have anything to be excited about.

An add#iona/ noteJin"d~oseushl~ AgrohacterJ/nnam/RhJz~J~Jum:

We have found that AgrohacterJum containing s o m e of the GUS plasmids
shows significant OUS activity (Jefferson, unpublished Y. Komeda, unpub-
lished). This seems to be due to read-through transcription from the /acZ
transcript of pBIN 19 into the GUS coding region, fbllowed by translational
initiation at the GUS i n i t i a t o r - Agrohacterium and RhizeJhium without these
plasmids shows little, if any, detectable GUS activity. When the GUS cassette
The GUS Gene Fusion System dO 1

is inverted with respect to the lac promoter, this background activity drops
more than 20 fold (R. Green and R. Jefferson, unpublished). Hence it is
worth being very careful to only use sterile tissue, and/nr do the appropriate
controls.

Sequencing Ol~q,onuc/eotide:
The 22 base olign ( 5 ' - G - A - T - T - T - C - A - C - G - G - G - T - T - G - G - G - G - T - T - T - C -
T-3') is very useAH for p r i m i n g D N A sequencing of fusion junctions. This
oligonucleotide is complementary to the GUS coding strand, and terminates
14 bp downstream of the A of the initiator ATG. W h e n constructing gene
fusions, this primer can be successfully used for double strand sequencing of
mini-prep D N A to confirm construction details. It is also useful for primer-
extension analysis of chimeric transcripts.

Some f f the P&smh/s A z'ai&hle JiJr lmplemepitm~ the G US system:

In general, it is probably wise to use a recA strain for propagation of these
plasmids whenever possible, as the G U S gene is encoded by the E. coli uidA
lOCUS,

pRAy255 8_,I,RAd26~
pRAJ255 has been described (Jefferson et al., 1986). It consists of a 1.87 kb
Pst I - - Eco RI fragment containing the GUS gene inserted into the poly-
linker site of pEMBLg, pRAJ260 is the same its pRAJ255, but with the Eco
RI site filled in and at Pst 1 octameric linker added. Cloned into the Pst I site
of pEMBL9, in-frame to lacZ. Hits the advantage of being able to remove the
entire coding sequence with a single enzyme.

pRA}275
pRAJ275 is at derivative of pRAJ255 in which the 5' sequences of GUS were
removed by progressive BAL31 deletion and replaced with a syndletic oligon-
ucleotide that has constructed a "consensus" translational initiator as defined
by M. Kozak (e.g., Kozak, 1983. ,~licrJ~H. RevJeu's. 4v." 1--i5~ (Jefferson
and Bcvan, unpublished; Sleat et al., 1988, Gene, in press). This plasmid has
a unique Ncu I site {CCATGG) positioned at the initiator ATG co&m, with
the surrounding context G T C G A C C A T G G T C . It has been shown both m
vitr, and m vh'o that the context around the initiator can have a profound
effect on the levels of translated product from a given amount of m R N A .
This construction has been adjusted to maximize this effect. It is provided as
a Sal I - - Eco RI tragment in p U C I 9 .
,402 The PLant Alo/ecnko Bio/o<~, R@orter

p B l l O l . 1:
pB[ 101 is a "promoter-less" G U S cassette in the Agrohacterinm binary plasmid
vector p B I N 1 9 (Bevan, 1984. Nud. Acidv. Res. 12:8711-8721; Jefferson, et
al., 1987b). It consists of the G U S gene from pRAJ260 blunt-end ligated
into a filled-in Asp718 (Kpn I) site of the p B I N I 9 polylinker (the same as
the pUC19) polylinker) but upstream of a 260 bp Sst I - - Eco RI fragment
containing the polyadenylation signal from the nopaline synthase gene of the
Agrohacterium tumefaciem Ti plasmid (e.g., Bevan et al., 1983). This allows
plant promoters to be easily cloned upstream of GUS and transferred to phmts
with all the advantages of the binary vector systems. This vector has a low-
copy RK2 origin of replication, and confers kanamycin resistance, both in
bacteria and in plants. It should be cautioned that while there are no "spu-
rious" ATG codons on the GUS fragment of this vector, any promoters cloned
into tile H i n d III site may have problems with tile ATG from tile polylinker's
Sph I site (GCATGC). All strains containing plasmids derived from p B I N 19
MUST be grown on kanamycin, as the replicon is very unstable in tile absence
of selection.
pBlIO].2 & p B l ] O l . 3 :
These are plasmids resulting from the PB1101 construction by T. Kavanagh
that provide the other two reading frames of GUS relative to the pnlylinker
sites.

GUS fusion junctions:

The GUS initiator Z~hold/ace, the Barn HI size Z; underlined."
pBll01.1: AAG CTT GCA TGC CTG CAG GTC GAC TCT AGA GGA
TCC CCG G G T G G T C A G TCC CTT A T G TTA . . .
p B l l 0 1 . 2 : A A G C T T G CAT GCC T G C A G G T C G ACT CTA G A G G A T
CCC C G G GTA G G T C A G TCC CTT A T G T T A . . .
p B I 1 0 1 . 3 : AA G C T T G C ATG CCT G C A G G T C G A CTC TAG A G G ATC
CCC G G G TAC G G T C A G TCC CTT A T G TTA . . .
p B I 1 2 1 . 1 : T h i s is simply p B l l 0 1 . 1 into which an 800 bp fragment cuntain-
ing the 33S promoter from Cauliflower Mosaic Virus has been cloned. That
gives high levels of G U S upon transformation of tobacco (Jefferson et al., in
press).
pBI121.2:
As pB1121.1, but with the "consensus" initiator codon context and with the
entire transcriptional cassette inverted with respect to t i l e / m Z transcriptional
unit in p B I N 1 9 . This vector has greatly reduced GUS levels in E. col~ and
Agrohacterinm, but gives excellent expression in tobacco and potato (Jefferson
The GUS Gene Fusion System 4O3

and Bevan, in preparation; A. Goldsbrough, Jefferson and Bevan, unpub-

lished).
pBIl31.1:
A tobacco rbcS promoter (Mazur and Chui, 1985; Jefferson et al., 1987b)
placed in the pB1101, l cassette to make a transcriptional fdsion.
pBI221.1
This is a vector in which the 3.0 kb Hind lII - - Eco RI fragment o f p B l l 2 1
containing the CaMV 35S promoter-GUS-NOS poly A site has been cloned
into the corresponding sites of pUC19 to facilitate high-yield plasmid prep-
arations for direct D N A expression methods such as electroporation and pro-
toplast fusion.
pBI221.2:
A CaMV-GUS fusion in pUC19 making use of the observation that the
pRAJ275 initiator context gives enhanced translational yields, e.g., a "con-
sensus" CaMV-GUS-NOS cassette.

Acknowledgements
I would like to thank David Hirsh, in whose lab I started development of the
GUS fusion system. I also thank Mike Bevan, Dick Flavell and Tony Kava-
nagh for their participation in the system development in plants. Thanks also
to the various lab members and members of the plant molecular biology
community who have explored the benefits and limitations of the system and
who have communicated them to me. I was supported by N I H GM 10789-
02, and a grant from the Gatsby Foundation.

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