Analytical Biochemistry 285, 1–15 (2000)
doi:10.1006/abio.2000.4672, available online at http://www.idealibrary.com on
REVIEW
Uses of lacZ to Study Gene
Function: Evaluation of
␤-Galactosidase Assays Employed
in the Yeast Two-Hybrid System
Ilya G. Serebriiskii and Erica A. Golemis 1
Division of Basic Science, Fox Chase Cancer Center,
Philadelphia, Pennsylvania 19111
The bacterial lacZ gene, encoding the enzyme ␤-ga-
lactosidase (␤-gal, EC 3.2.1.23), is a ubiquitous reagent
applied to the study of problems in genetics, cell and
molecular biology, development, and the recently
emerging fields of genomics and proteomics. Because
␤-gal activity is readily assessed in vitro or in vivo, in
hosts as evolutionarily diverse as bacteria, yeast, and
mammals, its induction has become a standard means
of assaying clonal insertion, transcriptional activation,
protein expression, and protein interaction. One con-
sequence of this omnipresence is that lacZ is rarely
reevaluated for performance in different current appli-
cations. The goal of this review is to first briefly sum-
marize the origins of lacZ, initially as a topic of study
in its own right, and subsequently as factotum en-
abling diverse biological inquiries. After a description
of the relevant biochemical properties of the ␤-gal en-
zyme, we will provide an overview of the methodologies
that have been developed that exploit ␤-gal to advance
studies in eukaryotes. Finally, focusing on one major
area of application for lacZ, as a transcriptional re-
porter in yeast and particularly the yeast two-hybrid
system, we will comparatively evaluate the ability of a
number of standardly used assays to accurately deter-
mine ␤-gal activity, and discuss variables impacting
such measurements.
THE ORIGINS OF lacZ
Few genes have as long and distinguished history of
study as lacZ. In an effort to gain insight into the early
20th century debate on “enzymatic adaptation,” the
1
To whom correspondence should be addressed at W406 Fox
Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA
19111. Fax: (215) 728 3616. E-mail: EA_Golemis@fccc.edu.
0003-2697/00 $35.00
Copyright © 2000 by Academic Press
All rights of reproduction in any form reserved.
Ilya G. Serebriiskii Erica A. Golemis
process by which organisms altered enzymatic activity
in response to certain inducing agents, the pioneering
biochemical work of Jacob Monod and associates fo-
cused on analyzing the induction of ␤-galactosidase in
response to addition of specific substrates (1, 2). ␤-Gal
induction was found to be robust, with expression lev-
els of ␤-gal increased up to four orders of magnitude by
discrete inducers, and rapid, with enhanced levels of
␤-gal detected within 3 min of addition of inducer. The
induction of ␤-gal was also found to be reversible, with
levels of expressed protein decreasing rapidly following
removal of inducers, and finally, was shown to require
de novo synthesis of the ␤-gal protein.
Around the same time, the development of tech-
niques for creating mutations and ordering genes by
bacterial conjugation led to the physical and functional
definition of the lac locus as a target of genetic study (3,
4). Pursuance of these and related approaches allowing
ordered gene transfer into transient heterozygotes pro-
vided initial insights to the mechanism by which the
induction of lacZ was controlled through action of the
lacI repressor gene (5, 6). These studies greatly ad-
vanced the arguments for a genetic view of organismal
response, playing a significant role in arguing against
the views of Lysenko. Combined with separate work on
bacteriophage immunity (7, 8), analysis of the genetic
control of lac induction provided important evidence
for the idea of the operon, thus conceptually opening
the fields of gene expression and transcription. Space
in this Review does not allow a thorough recounting of
this history. A much more detailed discussion is pro-
vided in the excellent reviews on lac in Ref. (9).
The lacZ gene encodes an open reading frame of 1024
amino acids (10, 11), and is one of the first large genes
to be completely sequenced. In E. coli, the biologically
1
2 SEREBRIISKII AND GOLEMIS

FIG. 1. Enzymatic function of ␤-galactosidase in cleaving indicator substrates. ␤-gal cleaves ␤-D-galactoside containing substrates with a
diverse range of aglycone groups, targeting between the glycosyl oxygen and anomeric carbon as indicated (scissors). Substrates shown
indicate commonly used indicators for assays on ␤-gal function on plates (X-Gal) or for liquid assay by measure of fluorescence (MU-Gal or
MUG) or color (ONPG). Top left, X-Gal is 5-bromo-4-chloro-3-indolyl-␤-D-galactoside, and when cleaved and oxidized produces the insoluble
dye 5-bromo-4-chloro-indigo, as described previously (22). Right panel, top, yeast colonies expressing ␤-gal and exposed to X-Gal (right half)
or the closely related compound Magenta-Gal (left half, see Biosynth, Inc., or Diagnostic Chemicals Limited). Middle left, MUG is
methylumbelliferyl-␤-D-galactoside, and when cleaved by ␤-gal produces the fluorescent product methylumbelliferone (first described in
(102)). Right panel, middle, shows yeast lysates expressing ␤-gal exposed to MUG, under long-wave UV. Bottom left, PNPG and ONPG are
closely related nitrophenol-␤-D-galactosides with similar assay properties, e.g., (103), whose cleavage releases the yellow product nitrophenol
(right panel, bottom); PNPG is shown.

active ␤-gal protein exists as a tetramer of four identi-
cal subunits ((12), reviewed in work of Zabin and
Fowler (9)), and migrates on nondenaturing protein
gels with an apparent molecular weight of approxi-
mately 480 –500 kDa, in accord with a predicted mo-
lecular mass for the tetramer of 465,406. The ␤-gal
enzyme functions optimally at pH 7.2. The primary
enzymatic function of ␤-gal relevant to its role as a
biotechnological tool is to cleave the chemical bond
between the anomeric carbon and glycosyl oxygen of
appropriate substrates (Fig. 1; see (13, 14) for review).
While cleaved substrates are restricted to ␤-D-galacto-
pyranosides, the enzyme is tolerant of a range of agly-
cone groups of differing chemical composition.
lacZ was chosen as a target of such extensive early
analysis in part because of specific experimental ad-
vantages accompanying work with ␤-gal, which con-
tinue to provide a rationale for use of the ␤-gal protein
in biotechnological applications today, and enable par-
ticularly desirable novel applications. To summarize
these advantages: First, as noted above, induction of
␤-gal synthesis occurs over a large dynamic range (up
to 10,000-fold over baseline levels with some inducers).
This large range is achievable in part because the ␤-gal
 ␤-GALACTOSIDASE ASSAYS IN STUDY OF GENE FUNCTION
protein can be tolerated at extremely high levels in
Escherichia coli. These levels approach 5% of total cell
protein in naturally occurring strains of E. coli, and
can reach levels of greater than 20% in partial diploids
(15). Significantly, ␤-gal is also well tolerated and func-
tional in many other organisms in addition to E. coli,
which number among them yeasts (16, 17), Caenorhab-
ditis elegans (18), Drosophila melanogaster (19), and
mammals (20)(to name a few). Further, the ␤-gal pro-
tein is readily purified by a number of relatively simple
techniques (reviewed by Zabin and Fowler in (9)), fa-
cilitating in vitro analysis of its activity.
Second, a considerable number of substrates, induc-
ers, or closely related “negative controls” for ␤-gal were
either naturally available or very easily chemically
synthesized using methodologies available in the first
half of the 20th century. The ability to probe ␤-gal
activity with an extensive panel of inducers or sub-
strates enhanced development of models for ␤-gal en-
zymatic activity, and also provided a practical tool to
finely modulate expression or dissect catalytic activity
of the ␤-gal protein product.
Third, ␤-gal activity is very easily assayed, both in
vivo and in vitro. A number of versions of enzymatic
assay and in vivo selection or reporter assay were
originally developed (reviewed in (9, 21)). Assays which
achieved considerable initial prominence, and continue
to be standardly employed today, involve the use of
colorimetric substrates in which the cleavage of specific
␤-D-galactopyranoside-coupled aglycone moieties re-
leases colored dyes. In vitro, cleavage of ortho-nitrophe-
nyl-␤-D-galactoside (ONPG) 2 produces a yellow prod-
uct, nitrophenol, which is readily monitored by a
spectrophotometer, allowing a quantitative assay (5).
In vivo, exposure of bacteria to 5-bromo-4-chloro-3-
indolyl-␤-D-galactoside (XGal) to produce a blue prod-
uct, 5-bromo-4-chloro-indigo, allows a convenient qual-
itative identification and activity ranking of colonies
positive for ␤-gal (some examples of early use have
been provided previously (22, 23)). More recently, the
panel of available colorimetric substrates has been
augmented with fluorescent (methylumbelliferyl-␤-D-
galactoside, MUG) or chemiluminescent alternative
substrates, which further expand sensitivity and ap-
plications.
Fourth, the ␤-gal protein is structurally malleable.
Work in the 1960s demonstrated that ␤-gal consisted of
three separable functional domains, alpha (␣, amino-
terminal), beta (␤, central), and omega (␻, carboxy-
terminal). Independent coexpression of separated do-
2
Abbreviations used: ONPG, ortho-nitrophenyl-␤-D-galactoside;
Xgal, 5-bromo-4-chloro-3-indolyl-␤-D-galactoside; MUG, methylum-
belliferyl-␤-D-galactoside; FACS, fluorescence activated cell sorting;
DBD, DNA-binding domain; AD, activation domain; CPRG, chloro-
phenol red-␤-D-galactopyranoside.
3
mains of the ␤-gal protein—for example, coexpressing
a peptide encompassing ␣ with a second peptide en-
compassing ␤␻ (24), or an ␣␤ peptide with an ␻ peptide
(25)—successfully reconstitutes the activity of the full
enzyme. This ability of ␤-gal to undergo transcomple-
mentation, as well as the additional capacity of ␤-gal to
function enzymatically when expressed as a transla-
tional fusion to a varied group of protein or peptide
moieties (initially described in (26)), enabled further
applications.
lacZ APPLICATIONS
To date, the lacZ gene and its ␤-gal protein product
have been exploited in a number of discrete classes of
experimental scheme. Some of these approaches in-
clude [i] monitoring an increase in ␤-gal activity as an
indirect measure of the enhanced transcription of a
lacZ reporter gene, [ii] assaying ␤-gal protein expres-
sion and activity as a means of monitoring spontaneous
or experimentally induced genetic change in lacZ cod-
ing sequence, and [iii] expressing chimeric proteins in
which heterologous protein domains are fused either to
full-length ␤-gal, or ␤-gal-derived peptides, as a means
of monitoring the activity of the fused heterologous
domains. Some prominent examples of each approach
follow, with particular emphasis on approaches that
have been adapted to eukaryotic systems.
Monitoring the Transcriptional Induction of lacZ
Expression of the ␤-gal protein is simple to detect
both in vitro and in vivo, in fixed or living cells, based
on scoring the appearance of colored or fluorescent
products arising from cleavage of appropriate sub-
strates. One early biotechnological use of lacZ as a
reporter was its adaptation into studies of promoter
function, and analysis of gene expression. The utility of
this approach was first demonstrated in Saccharomy-
ces cerevisiae (17), D. melanogaster (19), and mammals
(27) in the early 1980s. lacZ-based promoter studies
have continued to evolve in power and sophistication.
For example, “enhancer traps” were conceived of as a
means of identifying novel DNA sequences with the
power of inducing transcription. The original selection
for such enhancers included rescue of SV40 viral infec-
tivity in mammalian cell culture (28) and was thus
restricted in applicability. However, with the adapta-
tion of lacZ as a reporter gene in combination with
manipulable transposable mutagens such as P-ele-
ments (29) or retroviruses (30), enhancer trap strate-
gies have become primary tools for analyzing changes
in transcription that occur in regulation of develop-
ment (31).
In complementary approaches to the study of higher
eukaryotic development, others have placed the control
4 SEREBRIISKII AND GOLEMIS
of lacZ under a constitutive promoter and introduced
the promoter–lacZ construct stably into cells which
were in turn introduced into embryos. Such experi-
ments have enabled the detection of patterns of cell
movement and division, providing fine-resolution visu-
alization of cell lineage in vertebrate nervous system
development (32) and other tissues (recently reviewed
in 33). Such developmental analysis was made possible
because of the ease of detecting ␤-gal at single cell
resolution, a property that has similarly made the pro-
tein a useful reporter in studies of the metastasis of
cancerous cells (34).
On a more mechanistic level, the ability to visualize
␤-gal in individual cells has provided important in-
sights into the basic mechanisms of transcription. For
example, assays for transcriptional activation are gen-
erally performed on harvested populations of cells, ei-
ther by direct measurement of total induced transcript
or indirectly by measurement of a reporter gene. While
strong activators induce high levels of transcription,
and weak activators induce a low level of transcription,
population-based assays do not yield insights into
whether the sum of transcripts produced arise from a
graded but homogeneous level of transcriptional activ-
ity in each cell, or from the aggregate of some cells with
high levels of activity, and others with little or no
activity. The availability of fluorimetric ␤-gal sub-
strates in conjunction with fluorescence activated cell
sorting (FACS) was applied to the analysis of tran-
scriptional responses in individual cells in vivo in mon-
itoring transcriptional induction dependent on NF-␬B
and NF-AT in transcriptional activation (35, 36).
Through this analysis, it was found that transcription
was “threshold-gated” (i.e., demonstrated a bimodal
pattern of high activation versus no activation that
strongly correlated with the intracellular levels of tran-
scription factors), arguing for a strong cooperative fac-
tor in the assembly of functional transcriptional com-
plexes.
Evolving separately from its role as a tool for per-
forming structure–function analysis of naturally occur-
ring promoters (37, 38), lacZ was utilized as a conve-
nient reporter in yeast to assess the functionality of de
novo constructed transcriptional control systems.
Early work involved the identification and character-
ization of DNA-binding and transcriptional activation
domains in proteins such as GAL4 and LexA (39, 40).
Building on the information and tools developed in
these studies, the yeast two-hybrid system (41) was
developed as a wholly artificial transcriptional system
designed to identify and study protein–protein interac-
tions. In this system, one protein of interest is fused to
a DNA-binding domain (DBD), while a second protein
or proteins encoded by a cDNA library are expressed as
fusions to a transcriptional activation sequence (AD,
activation domain). Interaction of the two proteins
brings DBD and AD into proximity, allowing transcrip-
tional activation of reporter genes containing cognate
binding sites for the DBD component. The initially
described two-hybrid system incorporated lacZ as pri-
mary reporter: as multiple groups developed variants
of two-hybrid system, lacZ remained common in all
systems (42– 45). Because of the large-scale adaptation
of the two-hybrid system as a standard laboratory tech-
nique, it has provided an excellent test case to compar-
atively evaluate the performance of different forms of
analysis of lacZ, as is discussed in detail below (under
The Yeast Two-Hybrid System: Methodologies for As-
sessment of lacZ Reporter Function).
Interruption of lacZ Coding Sequence as a Monitor of
Genetic Change
For a number of reasons, including the simple tech-
nical point that it is easier to detect blue colonies
against a white background than the converse, the lacI
repressor gene, rather than its lacZ regulatory target,
has been most exploited in strategies to identify spon-
taneous mutation (reviewed in 21). However, lacZ has
also found use in this work. In one example, the fre-
quency of conversion to either a normal codon versus
nonsense codon of lacZ was used as an effective mon-
itor of the efficiency of mismatch repair in yeast (46).
Another useful application for studies of mutagenesis
in mammals has been the construction of transgenic
strains of mice containing integrated tandem copies of
a bacteriophage ␭ vector encompassing the lacZ coding
sequences (47, 48). Harvest and lysis of the organs of
such mice releases bacteriophage which can subse-
quently introduced into E. coli, allowing rapid analysis
of ␤-gal expression and function by observing the ap-
pearance of blue colonies following plating on medium
incorporating IPTG and X-Gal. Notably, treatment of
lacZ mice with mutational agents results in a strong
enhancement of white versus blue clones, reflecting the
presence of mutations which inactivate ␤-gal function.
Quantification of the appearance rate of such plaques
allows sensitive and physiologically significant gauge
of strength of prospective mutagens.
␤-Gal Chimeras, ␣-Complementation, and Protein
Interaction
In contrast to the first group of strategies discussed
above, which utilize induction of a native lacZ product
to monitor transcription, it is alternatively possible to
use a (gene-of-interest)-␤-gal chimeric protein as a
means of monitoring protein expression, stability, and
localization. In eukaryotic organisms, and for fusions
to native lacZ, this application is now mostly of histor-
ical interest, as the explosion of convenient immuno-
logical and fluorescent tags has superceded the desir-
 ␤-GALACTOSIDASE ASSAYS IN STUDY OF GENE FUNCTION 5

ability of ␤-gal as a “tagging” moiety. However,

particularly in the 1980s and early 1990s, this was a
common and powerful technique (see 49, 50). A review
of examples and considerations of use is provided in
Ref. (51).
Other techniques that remain highly useful exploit
the fact that the amino-terminal ⬃50 amino acids of
␤-gal, encoding the ␣-peptide, are essential for the
tetramerization that allows enzymatic activity (dis-
cussed in 52). On a simple level, this observation has
been exploited for two decades as basis of selection for
inserts in many standard cloning vectors. Insertion of
DNA fragments into a polylinker embedded in the
␣-peptide results in gross disruption of sequence, and
appearance of white colonies against a background of
blue colonies following plating on media containing
X-Gal/IPTG (53).
A separate application takes advantage of the fact,
noted above, that the function of the amino-terminal ␣
peptide can be supplied in trans to molecules of ␤-gal
made inactive through the deletion of corresponding
amino-terminal sequences, effectively reconstituting
the activity of the enzyme (24, 54, 55). Following the
demonstrations that ␣-complementation functioned ef-
fectively in mammalian cells (56, 57), and that the
complementing peptides, similarly to the native ␤-gal, FIG. 2. The yeast two-hybrid system. A typical set of yeast two-
tolerated the presence of fused additional sequence (56, hybrid components is shown. Required reagents are an activation-
58), ␣-complementation provides the basis of a novel domain-fused prey (AD–prey), a DNA binding domain-fused bait
(DBD– bait), and reporters with a cognate-binding site (CBS) for the
protein interaction detection strategy. In this system
DBD directing expression of the lacZ and auxotrophic (aux) selection
(58, 59; reviewed in 60), specific mutant forms of ␣ and genes. Interaction between bait and prey leads to transcriptional
␤␻ peptides that complement poorly because they re- activation of lacZ and auxotrophic reporters, reflected as blue color
tain minimal competency to interact are fused to pro- following exposure to X-Gal, and growth on appropriate selective
tein domains that supply a compensating dimerization media (discussed more fully in (104)). Detailed information about
two-hybrid system usage is provided at http://www.fccc.edu/
function. The resulting appearance of ␤-gal enzymatic research/labs/golemis/InteractionTrapInWork.html.
function can be readily detected and provides a quan-
titative and sensitive monitor of protein interaction
strength. Although this is a very recently developed olated to other eukaryotic organisms. Of immediate
technique, it has considerable potential for augment- practical use, over the 11 years since the initial de-
ing studies of protein–protein association. scription of the two-hybrid system, a large number of
different techniques designed to facilitate analysis of
THE YEAST TWO-HYBRID SYSTEM: METHODOLOGIES ␤-gal activity have been devised for yeast lacZ report-
FOR ASSESSMENT OF lacZ REPORTER FUNCTION ers, and comparison of the effectiveness of these differ-
ent techniques should provide insight into optimizing
The Yeast Two-Hybrid System as Model experimental design.
In performing a critique of the effectiveness of ␤-gal To review the key parameters of two-hybrid sys-
as a bioindicator, and establishing critical parameters tem function that are important for interpreting the
for the evaluation of ␤-gal activity, an analysis of data subsequent analysis, a typical two-hybrid schematic
arising from use of the yeast two-hybrid system has is shown in Fig. 2. A much more detailed review of
distinct advantages. The two-hybrid technique is ex- two-hybrid system function is presented in (61).
tremely commonly used, with over 3000 papers citing Two-hybrid systems contain three essential ele-
the system in Medline as of summer 2000, creating a ments, and generally a fourth element. Host yeast
large data set for evaluation. The use of yeast as or- invariably contain a first construct allowing the ex-
ganismal host for the system enables the analysis of pression of a DBD-fused “bait” protein, a second con-
large numbers of cells and colonies expressing ␤-gal, struct expressing an AD-“prey” protein component,
addressing issues of reproducibility; at the same time, and a third construct expressing a cognate DBD-
established points about ␤-gal function can be extrap- binding site motif encompassed in a minimal pro-
6 SEREBRIISKII AND GOLEMIS

TABLE 1
␤-Galactosidase Assay Variations Utilized in the Yeast Two-Hybrid System

Basic protocol Control points Variations Advantages/limitations

In liquid Cell treatment during
lysis
Normalization
Selection of substrates
a
Microplate assay
Growth on plate/filter Types of filter used for
lift lift
Freezing technique
Exposure to X-Gal
Growth and X-Gal Exposure to X-Gal
assay on plate
Freeze–thaw lysis (cycle ⫺80°C to
room temp)
Detergent lysis (e.g., Y-PER)
Solvent-assisted permeabilization
Based on OD 600 of culture mass
per mg of protein
Chromogenic: ONPG, PNPG
Chromogenic: CPRG
Chemiluminescent: Galacton,
Galacton-Star
Fluorescent: 4-MU-␤-D-gal
Fluorescent: resorufin-␤-D-gal
Yeast retained throughout assay
Cells removed before enzymatic
assay
Whatman paper, nitrocellulose,
nylon
⫺80° freezer; immersion in liquid
nitrogen
On agar pad; on soaked filter;
direct addition of liquid
X-Gal incorporated in plate
X-Gal provided after CHCl 3
treatment in Z- buffer/agarose
overlay
Faster if many samples; minimal
manipulation
If only a few samples, speed of
cell lysis compensates for
greater manipulation.
Convenient, rapid; usually
sufficient.
Preferred in comparison between
different strains of yeast, or if
cell viability an issue
Detection limit ⬃3 ⫻ 10 8
molecules; inexpensive
Detection limit ⬃3 ⫻ 10 7
molecules; greater cost
Detection limit ⬃4000 molecules;
high cost
Detection limit ⬃6 ⫻ 10 5
molecules; inexpensive
Current best detection limit,
⬃900 molecules
Streamlined technique, but loss of
dynamic range
More accurate data, but requires
more time or robotic handling
Qualitatively similar, differences
in sensitivity (see Fig. 5)
Essentially equivalent for speed/
effectiveness
Comparable; solid support
prevents colony dispersing and
loss of signal
Easy; no solvents involved/ takes
long time
Rapid; very sensitive; need to
handle toxic solvents

Note. Liquid assays are standardly performed by growing yeast in microbiological culture tubes, performance of assays in 1.5 ml Eppendorf
tubes. For assays of ␤-gal activity in microtiter plates, yeast can be grown and assayed in microtiter plates, or grown in tubes and transferred
to plates for assay, with equivalent results: most of the options described as suitable for “in liquid” handling can be adapted to microtiter
variants. Galacton and Galacton-Star are proprietary compounds made by Tropix, Inc. (Bedford, MA). Y-PER (Yeast protein extraction
reagent) is a propietary compound made by Pierce, Inc. (Rockford, IL).
a
Alternatively, microtiter plate-based assays can be used to enable high-throughput assessment of LacZ activity.

moter, directing the expression of a lacZ reporter.
Finally, most current systems use at least one addi-
tional DBD-binding site-responsive reporter, with
genes such as HIS3 or LEU2 that allow growth on
suitable selective media generally favored for this
purpose. The bait and prey constructs are almost
invariably present on plasmids containing sequences
stipulating segregation and plasmid maintenance at
a copy number of 10 – 40 per cell. The auxotrophic
(HIS3 or LEU2) reporter is invariably integrated.
Depending on the system variant, the lacZ reporter
is either plasmid-borne or integrated, with the
former option somewhat more common.
Methodologies Commonly Used to Assay lacZ Levels
in Yeast
Table 1 summarizes a number of techniques that
have been developed to assess ␤-gal enzymatic activity
in yeast. The available technical options allow variance
of elements impacting all levels of the assay process.
These include, first, growth conditions for yeast con-
taining lacZ reporters. Growth can be on solid support
(agar plates) or in liquid culture (either in tubes or
microplates). Second, exposure to ␤-gal substrates can
occur while yeast are actively growing, or subsequent
to cell lysis. Third, in techniques where yeast are lysed
prior to assay, cell lysis can be achieved by multiple
 ␤-GALACTOSIDASE ASSAYS IN STUDY OF GENE FUNCTION
FIG. 3. Patterns of usage for different ␤-galactosidase assay tech-
niques. Frequency of assay use determined from analysis of 80 ran-
domly selected papers from 1999 describing ␤-gal assays in yeast,
predominantly in the context of two-hybrid studies. Most commonly
utilized are quantitation based on ONPG cleavage, and qualitative
assessment of blue-versus-white on filter lifts.
means, including freeze–thaw cycles, detergent extrac-
tion or solvent extraction. Fourth, in the case of liquid
assays, normalization of ␤-gal activity can be done to
overall population mass (e.g., extrapolated from optical
density readings for cultures), or to directly deter-
mined standard protein load. Fifth, in the case of solid
support assays, the assay can be done directly on
growth plates, versus on lifted filters of a variety of
compositions. Sixth, multiple substrates are available
for use, spanning at least five orders of magnitude in
sensitivity range, and assayed by colorimetric versus
chemiluminescence or fluorescence detection tech-
niques. As discussed in detail in the following sections,
variance of some of these points can make a consider-
able difference in adaptability of systems to high-
throughput, dynamic range of signal detection, ease of
use, and susceptibility of the system to false-positive or
false-negative readings. It is worth critically evaluat-
ing particular experimental needs before selecting a
particular modality.
In fact, although many options are available, inspec-
tion of the recent literature suggests that the majority
are not being widely employed. From a randomly se-
lected set of 80 publications utilizing the two-hybrid
technique appearing in the year 1999, nearly half ex-
pressed ␤-gal activity in terms of cleavage of the sub-
strate ONPG, as determined by liquid assay, while a
quarter assessed relative ␤-gal activity based on qual-
itative assessment of degree of blue–white coloration
following filter lifts (Fig. 3). One reason for the prefer-
ence of these techniques is undoubtedly the low cost of
the respective ␤-gal substrates they employ, and their
antiquity (for example, the ONPG-cleavage assay has
been in use since the 1950s, e.g., (5)), which theoreti-
7
cally enables more direct comparison with a large com-
mon fund of data. However, comparative analysis of
the performance of the commonly used versus more
uncommon variants suggests that the latter may in
some cases offer superior performance, as detailed in
the following section.
Issues Related to ␤-Gal Function in Vitro and in
Vivo, and Evaluation of Standard Techniques
If ␤-gal did not function well and reliably by many
standard measures, it would not have achieved the
prominence it has as a reporter. Nevertheless, the fact
that ␤-gal is expressed within living organisms for
most standard applications can potentially lead to bias
of ␤-gal assays in response to indirect factors that
affect either the viability or general metabolism of the
host organism. While assays can be performed without
taking these factors into account, experiments which
address their existence will help to maximize assay
performance and consistency. The most significant of
these biasing factors, as gauged from comparative
analysis of different techniques for assaying lacZ re-
porters in the yeast two-hybrid system, are listed in the
sections following. Although we discuss these biasing
factors in the context of the lacZ reporter in the yeast
two-hybrid system, the underlying principles pertain
in some cases to many common uses of lacZ, or to other
commonly used reporter genes.
1. Exposure to substrate before or after lysis of yeast.
Of the standard assays used to assess ␤-gal activity,
some, including measure of ONPG cleavage, colony
overlay, or colony lift (filter) assays, are performed
following lysis of yeast. Others, including growth on
plates containing X-Gal or FACS analysis of yeast ex-
pressing ␤-gal and exposed to fluorescent substrates,
occur in the context of living cells.
We have recently found that these different classes
of assay can differ substantially in measure of ␤-gal
activity (62). Analysis of the underlying causes for the
difference revealed that expression of some combina-
tions of bait and prey resulted in toxicity to the yeast,
which could be mild or severe. In some cases, it was
also associated with greater permeability of yeast cells,
as reflected by increased degree of uptake of small
molecules (such as the commonly used dye, bromphe-
nol blue) from the surrounding medium, and increased
leakage of cleaved X-Gal from cells into the surround-
ing medium. Because of the simultaneous variance in
cell viability and permeability, assays in which yeast
were grown in the presence of substrate would yield
sometimes erratic results, with individual colonies ei-
ther extremely blue or showing little blue color. Fur-
ther investigation suggested that an additional cause
of this variability was the seed density of yeast plated
onto X-Gal plates. Yeast containing toxic bait–prey
8 SEREBRIISKII AND GOLEMIS
combinations plated on X-Gal plates at high density
(with little further growth required to generate an
assayable cell mass) would demonstrate extremely
high apparent ␤-gal activity. Conversely, yeast plated
at low density would frequently not grow sufficiently to
yield discernible signal, yielding anomalously low ap-
parent ␤-gal activity. Strikingly, yeast which mani-
fested such behavior when grown on X-Gal plates dis-
played much more reproducible behavior when ␤-gal
activity was determined by short-term pregrowth in
liquid and subsequent ONPG cleavage assay, as would
be expected if permeability and viability were impor-
tant biasing factors.
The extrapolated issue from this analysis is that in
an organism, rate of uptake of substrate or release of
cleaved product is in part a factor of the organismal
viability in the presence of factors inducing the expres-
sion of lacZ, and can clearly affect the scoring of ␤-gal
activity. It is likely that this class of phenotype may
underlie the isolation of some clones considered to be
false positives in two-hybrid screen (62).
2. Clonal and temporal variability in expression lev-
els of lacZ. ␤-Gal expression assays in yeast generally
assess the behavior of populations of cells, represent-
ing colonies expanded in parallel from independent
transformation events. Different combinations of bait,
prey, and lacZ-reporter behave nonequivalently in
these assays. For example, in a hypothetical situation
in which “A–E” designate five parallel transformations
performed with different pairs of bait and prey, combi-
nations A–D will be yield highly reproducible results in
␤-gal assays, such that 10 independently picked colo-
nies containing one of these bait–prey pairs will pro-
duce similar levels of ␤-gal activity with standard de-
viation of no greater than 20% from average. However,
␤-gal activity in yeast containing combination E will be
highly variable, with some colonies white, some dark
blue, and others intermediate in color.
In explaining this variance, which is frequently
greater in yeast than that reported in assays of ␤-gal in
bacterial systems (e.g., compare (63) versus (64)), it is
important to consider some underlying causes. First,
although a population is assayed, individual cells com-
posing that population are heterogenous with respect
to activation of lacZ transcription. This has been well-
documented in single cell assays performed with fluo-
rescent lacZ substrates in mammalian cells, where a
“threshold” effect has been observed (36, 65) and ad-
dressed in theory (66), as introduced above (see lacZ
Applications). Comparable findings have been made in
yeast with fluorogenic reporters (e.g., (67)). Corre-
spondingly, the degree of activation of auxotrophic re-
porters in yeast reflects increasing activation strength
as an increase in the percentage of cells that pass a
threshold for growth into colonies, rather than changes
in the uniform rate of growth for all colonies (68).
Second, two-hybrid systems utilize at least two and
frequently three plasmids that are partitioned into
daughter cells such that copy numbers of the plasmids
vary between 10 and 40 copies per cell in sum. Follow-
ing transformation, the ratio of bait, prey, and reporter
plasmids may vary between separately transformed
colonies, particularly if one component induces nega-
tive selection. Therefore, hypothetically, one colony
may contain 15 copies of bait, 15 of prey, and 10 of
reporter, while a second might contain 15 of bait, 5 of
prey, and 5 of reporter. In a system nonsaturated for
protein–protein interaction and transcriptional activa-
tion, the second colony will produce lower levels of
productive bait–prey activation complexes, and poten-
tially be more prone to transcriptional threshold effects
for synthesis of lacZ. It is reasonable to think that
two-hybrid systems with integrated lacZ-reporter plas-
mids may be less vulnerable to this class of effect,
although no direct comparative study has been done to
test the point; however, the generally decreased sensi-
tivity of such strains offsets this potential advantage.
Third, in the case of toxic or otherwise disfavored
transcriptional activators, yeast are efficient at genet-
ically or epigenetically adapting to limit the strength of
the activator, either by modifying the core transcrip-
tional apparatus (e.g., using ada2 mutations to limit
the toxicity of GAL4-VP16 (69)) or by eliminating ex-
pression of individual disfavored proteins (which could
be either the bait or prey, or both (62)). We note that
the set of “disfavored” proteins does not completely
overlap with the putative false positives defined in the
preceding section 1. Some baits which are difficult to
express in yeast (such that only ⬃10 –20% of yeast
colonies containing the expression plasmid for the bait
produce detectable levels of protein) have little if any
toxic effect on growth in yeast which do produce the
protein. It is likely that part of this variability of ex-
pression may derive from destabilizing sequences (e.g.,
degradation targeting motifs) on the bait in question.
These considerations are indirectly related to a
fourth group of points, describing yeast growth status.
Since yeast are maintained for days to weeks on
growth plates, expression of lacZ and ␤-gal varies over
time from the starting levels obtained following the
initial transformation of yeast with expression plas-
mids. Cells pass from active growth phases through a
diauxic shift to stationary phase growth (70), resulting
in global declines in transcription and translation. Sig-
nificantly, in yeast grown on plates, growth status var-
ies between centers and edges of colonies (71), suggest-
ing transcription rate of lacZ will differ in different
areas of yeast streaked on plates. ␤-Gal itself possesses
a very long half-life in yeast, estimated to be on the
order of 20 h (72). Summing these variables, it is not
 ␤-GALACTOSIDASE ASSAYS IN STUDY OF GENE FUNCTION
unreasonable to imagine that the relative ratios of
␤-gal activity observed in assays run on yeast grown
for short periods of time can differ from those obtained
in yeast grown for longer periods, and this is in fact
observed. Hence, it is important to control for growth
status, time of assay, and growth in liquid (homoge-
nous) versus solid (spatially heterogenous) culture
when comparing assay values.
Finally, the above discussion primarily confines it-
self to activity and detection of native ␤-gal. Based on
studies in prokaryotes, it is likely that additional is-
sues apply to studies performed with ␤-gal–protein
fusions, insofar as it is well documented that targeting
the protein to discrete cellular compartments can ei-
ther substantially alter the catalytic activity of the
molecule (73) or induce cellular toxicity (74), leading to
further indirect effects.
In sum, the expression and activity level of ␤-gal can be
impacted by heterogeneity at the cell and colony level,
which reflects the existence of transcriptional thresholds,
flux in levels of plasmids, and the relation between yeast
growth status and ␤-gal–protein stability.
3. Saturability and linearity: Optimizing enzyme
function. Finally, measurement of lacZ induction is
done in almost all cases by a measurement of ␤-gal
enzymatic activity. An important consideration in gen-
erating significant and reproducible results is whether
the assay can be performed in optimal (or at least
comparable) activity conditions, at a point falling in a
linear range for multiple samples. In common practice,
these elements are not very well standardized between
laboratories. For example, while the plurality of re-
ports describing quantitative liquid assay of ONPG
cleavage cite (75) for assay conditions and formulas to
calculate Miller units as measure of ␤-gal activity, the
recommended conditions for performing a ␤-gal assay
vary considerably. Assays are performed at 37°C, 30°C,
or at room temperature; pH is set between 7.0 and 7.5;
the composition of “Z buffer,” used for determination of
␤-gal activity, is described in diverse formulations. For
these reasons, it is generally most accurate only to
compare ratios of relative induction of ␤-gal activity for
a sample of interest against invariably included, con-
stant, positive and negative controls, rather than to
interpret activity based on absolute values of Miller
units obtained.
For alternative substrates, including CPRG (chloro-
phenol red-␤-D-galactopyranoside (76)), Galacton
(Tropix, Inc. (77)), and resorufin-␤-D-gal (78), the de-
tection limit of ␤-gal activity is greatly enhanced over
ONPG-based assays (Table 1). In one careful kinetic
analysis (76) comparing ONPG and CPRG as sub-
strates, the V max of ␤-gal for CPRG versus ONPG was
reduced 2-fold (from 41.1 to 21.4). However, the reduc-
tion in V max was more than offset by a greatly increased
9
extinction coefficient of the cleavage product chloro-
phenol red (for CPRG) versus o-nitrophenol (for
ONPG), resulting in a net 10-fold gain in sensitivity
using this compound. Extrapolating from these find-
ings, one implication is that linearity of reaction occurs
under a different range of assay conditions with alter-
nate substrates, making calibration particularly im-
portant if data utilizing multiple classes of substrate
are to be compared.
For filter or overlay assays, or growth on plates in-
corporating X-Gal, ␤-gal activity is standardly de-
scribed as high or low based on colonies being white,
light blue, or dark blue, a practice lacking any claim to
quantitation. We have found (79) that it is possible to
increase quantitation of such data by scanning plates
or filters with a flatbed scanner and analyzing gray
scale, and that the linear range of such analysis is
comparable to a number of other techniques (discussed
below). We note, of the different techniques utilizing
solid supports for growth of yeast, growth on plates
incorporating X-Gal involves one additional variable
affecting enzyme activity, beyond those presented in
previous sections. Yeast grow optimally on media of pH
5.5, and continue to acidify their media as they grow
(80). The ␤-gal optimal pH for enzymatic activity is at
least 7.0. While X-Gal plates are buffered to approach
pH 7.0 to enhance enzyme function, yeast grow subop-
timally under these conditions, potentially predispos-
ing them to toxicity effects such as those described
above (also discussed in (62)). Conversely, if yeast grow
over long periods to high density, the growth medium
is eventually acidified, impacting the effectiveness of
the assay (although the reduction in enzyme activity
should be general for all samples, globally reducing
obtained values, rather than contributing to sample-
to-sample heterogeneity).
For both solid- and liquid-based techniques, an im-
portant element to include if possible is determination
of relative ␤-gal activity at multiple points after com-
mencement of the assay. As shown (Fig. 4), such re-
peated analysis allows determination of baseline ver-
sus linear increase range for assay, enabling
calculation of a ratio of activity derived solely from the
linear component of the assay curve. This level of anal-
ysis is most readily accomplished for liquid samples
using a microplate-based system adapted for high-
throughput use, as discussed below. For plate-based
techniques, repeated scans of the plate on a flatbed
scanner at intervals after addition of substrate should
perform a similar function.
Comparative Ranking of Techniques for Assessment
of ␤-Gal Activity
The above sections addressed issues that generally
pertain to the dialog between ␤-gal and its host organ-
10 SEREBRIISKII AND GOLEMIS

different assessment strategies. The X-Gal overlay

plates were extremely sensitive, followed closely by
filters developed on nylon, with reactions into a satu-
rating range, with a strong signal, within 30 min (Fig.
5). Other classes of filter, such as Whatman paper,
were slower in development of color even though com-
parable amounts of yeast were retained. The X-Gal
plate assay was in distant last place, with results
shown reflecting the appearance of plates after 24 – 48
h of yeast growth. The difference in discriminatory

FIG. 4. Observed ratio of ␤-gal activity as a factor of time: contrast

between single-read and multiple-read techniques. Assessment of
␤-gal activity of a series of samples usually involves comparison of
samples of high and low activity. In a standard ONPG assay (left),
because the process of reading the individual samples by spectropho-
tometer takes considerable time, the reaction must be stopped prior
to reading to prevent changes in levels of cleaved ONPG. Because of
this constraint, only a single measurement can be made. The correct
timing for this measurement is generally determined “by eye,” based
on visual estimation that values are within linear range. Particu-
larly in samples with high activity, for which only a short time of
assay is desirable (⬍5 min), this can lead to inaccuracies, for in-
stance, if all samples are not started at the same time. In a micro-
plate assay performed with a plate reader (right), samples can be
repeatedly scanned without stopping the ␤-gal reaction. The starting
OD 420 value can be obtained as a baseline for each sample (t i), then
subtracted from reads taken at subsequent intervals (t 1 , t 2 , etc.),
until a signal is obtained at a point that falls within the linear limit
of the assay. For hypothetical clone 2 of high activity, an optimal
read would occur at t 1 , yielding point 2 1. For clone 1 of lower activity,
t 2 would yield point 1 2. Integration of plate readers with appropriate
database management software provides additional advantages, e.g.,
by allowing plotting of multiple read point to demonstrate chosen
points occur during linear increase in OD 420 values. FIG. 5. Comparison of different ␤-gal assay techniques based on
use of solid supports. Blue–white phenotypes of yeast expressing
␤-gal and cleaving X-Gal, assessed on nylon filters (nylon), Whatman
ism, that might cause differences in results obtained 3M paper (Whatman), by overlay assay (overlay), or on plates with
incorporated X-Gal (X-Gal plates) are shown. To demonstrate a
between different classes of assay. We were also inter- range of activity, yeast with very high (top row), high (second row),
ested in empirically comparing the effectiveness of moderate (third row), low (fourth row), or negative (bottom row)
closely related assays that are often used interchange- activities of ␤-gal, as determined by quantitation in ONPG liquid
ably. For example, for qualitative “blue–white” assays assay, were prepared. These yeast were resuspended in sterile wa-
on solid supports, investigators have used nitrocellu- ter, and OD 600 adjusted to ⬃24.5. Two subsequent 1:8 dilutions were
prepared, and all three suspensions were distributed into the wells of
lose (81), nylon (e.g., (82)) or Whatman 3M paper (83) microtiter plate, 100 ␮l/well. Two independent samples for each
to lift colonies for lysis and subsequent analysis for dilution point were used, in the order from highest concentration
cleavage of X-Gal. This is considered comparable to the (two left columns), through middle concentration (two middle col-
X-Gal overlay technique (84), which permeabilizes cells umns), to lowest concentration (two right hand columns). A replica-
tor was used to put drops of diluted yeast onto plates containing
on plates before adding substrate in an agarose matrix,
yeast growth medium, which for (nylon, Whatman, and overlay) was
or a standard X-Gal plate assay ((85), derived from simple defined minimal medium, and for (X-Gal plate) was defined
(75)), which incorporates X-Gal in the growth media. minimal medium supplemented with Z-buffer and X-Gal. After 18 h,
Using a series of well-behaved transcriptional activa- filters were placed on the yeast grown on the (nylon, Whatman)
tors (e.g., not prone to the effects described above), we plates, lifted, placed in empty petri dishes and subjected to two cycles
of freeze/thaw; then placed on Whatman filters presoaked in Z-buffer
assessed the comparable ability of these assays to qual- with 1 mg/ml of X-Gal. Incubation was stopped in 30 min by drying.
itatively resolve high, moderate, and low ␤-gal activity At an equivalent 18 h time point, the (overlay) plate was processed
(Fig. 5). with chloroform followed by X-Gal/agarose overlay, and incubated for
As shown, the apparent level of ␤-gal activity ob- 30 min. The (X-Gal plate) sample shown represents pattern observed
served is nonidentical in the tested set of experimental following 48 h continuous incubation: note, comparable effect could
be obtained in a shortened time period (⬃24 –30 h) by transfer of a
regimens. While most materials successfully allowed much heavier inoculum of starting yeast, i.e., by direct streaking of
detection of a graded profile of ␤-gal levels, the rate of colonies. Of the panels, the overlay and nylon results shown are into
saturation of the reaction differed markedly between the saturating range for the assay.
 ␤-GALACTOSIDASE ASSAYS IN STUDY OF GENE FUNCTION
range observed in with yeast lifted on nitrocellulose or
nylon membranes versus Whatman filters is more dif-
ficult to account for, but empirically is highly consis-
tent.
Overall, the variation in sensitivity may in part re-
flect the efficiency of lysis techniques employed. Cells
on X-Gal plates remain unlysed during the course of
the assay, while freeze–thaw cycles are standardly
used for lysis of yeast on filters, and addition of chlo-
roform for the lysis of yeast for the overlay assay. For
method of freezing used with the filters, both place-
ment in liquid nitrogen and placement in ⫺80°C freez-
ers for short periods of time (5–10 min) were compared:
no significant differences were observed. Of these tech-
niques, the X-Gal overlay technique provides the most
consistent results in our hands, in part because of the
avoidance of issues such as the sometimes uneven
transfer of colonies to filters.
There is much interest in developing high-through-
put assays that incorporate two-hybrid components,
for use in proteomic studies and drug analysis (re-
viewed in (61)). Much of the current thrust of such
work is to utilize vehicles such as microtiter plates to
enable rapid handling of multiple samples, as exempli-
fied in (86 – 88). These microwell plate-based ap-
proaches introduce new issues, including among them
the question of whether the growth properties of yeast
differ significantly (as reflected by ␤-gal induction) fol-
lowing culture in microwell versus on plate or in liquid,
and whether it is necessary to remove yeast prior to
assay, or whether they can be left in wells during read
of ␤-gal activity, improving streamlining. We have
compared a number of these parameters (Fig. 6, and
unpublished results), and additionally compared re-
sults with quantitation from scanned overlay plates (as
in (79)).
We find that all the techniques tested can effectively
discriminate low, medium, and high affinity interac-
tions. However, while rank order is efficiently main-
tained, the dynamic range over which variance is ob-
served is compressed in analysis of scanned overlay
plates or microtiter plates in which yeast are retained,
compared to standard ONPG assays. One key differ-
ence lays in the ability of the techniques to discrimi-
nate between very low levels of ␤-gal activity (e.g.,
between negative samples versus those with minimal
transcriptional activation capacity). Microplate-based
techniques are generally less able to do so than either
scanning of agar plates or spectrometer determination
of OD 420 in cuvettes (not shown). In microplate assays,
prior growth of yeast in tubes with subsequent transfer
to microplates does not significantly alter scored ␤-gal
activity, indicating potential changes in growth status
(e.g., due to differential oxygenation) is an insignificant
variable. Adapting the bacterial analysis procedure of
Menzel (63), in which yeast are removed from plates
11
FIG. 6. Dynamic range of different quantitative techniques. Yeast
expressing low, moderate, or high levels of ␤-gal were processed in
four ways, and fold-difference between low and high values is dis-
played. Dark gray, yeast were pregrown in culture tubes, transferred
to microwell plates for ONPG cleavage assay, and results read in
plate reader. Light gray, yeast were grown and assayed in microwell
plates. Hatched, yeast were grown on plates, X-Gal overlay assay
performed, and plates were scanned by flatbed scanner, with values
shown reflecting fold change in grayscale value. Medium gray, yeast
were pregrown in tubes, a standard ONPG cleavage assay per-
formed, and OD 420 determined by spectrophotometer.
prior to OD 420 determination, enables restoration of
dynamic range to levels comparable to a standard tube-
based ONPG assay, although at the cost of further
sample manipulation (V. Khazak, personal communi-
cation). This result indicates that the compression in
range from the high end is most likely attributable to
higher background deriving from absorbance of yeast
cellular debris at OD 420, as discussed (21). Notably,
removal of yeast does not improve the resolution of
microplate techniques at very low levels of ␤-gal activ-
ity, suggesting this might reflect an intrinsic limitation
of microwell plates. These points, as well as availabil-
ity of robotic sample handling equipment, and required
degree of resolution, should be considered before select-
ing an experimental strategy.
Additional Colorimetric Assays, Other ␤-Gal-Related
Issues, and Summary
Although lacZ has the greatest antiquity, there are
an increasing number of alternative reporters yielding
colorimetric readout. Some are in principle more con-
venient to use than lacZ. For example, repression of
expression of the ADE2 gene of S. cerevisiae leads to
production of a red pigment that can be directly scored
in the form of red versus white yeast colonies (89, 90).
Since its development as a reporter in 1994 (91), green
fluorescent protein (GFP) has been widely adapted as a
fluorescence reporter in multiple organisms including
yeasts (92), while other fluorescent reporter genes such
as cobA (93) have recently been described. Biolumines-
12 SEREBRIISKII AND GOLEMIS
cent proteins such as luciferase (94, 95) have also been
used for many years as reporters (e.g., (96)) and pro-
vide additional options. In contrast to these reporters,
which function quite differently than ␤-gal, gusA, en-
coding ␤-glucuronidase (␤-gluc) (97, 98) provides a
closely parallel reporter system. Cleavage by ␤-gluc of
the X-Gluc substrate produces a blue product whose
detection can be readily scored in parallel with ␤-gal
reporter systems, as we have demonstrated (99). Oth-
ers have reported the development of ␣-galactosidase
(MEL1) as a novel reporter for the yeast two-hybrid
system; it also promises to be a well matched parallel
system (100).
Many of the issues discussed above, as impacting
reliability and performance of ␤-gal reporter assays,
are known or likely to apply to assays using other
reporters. Because self-fluorescing alternative report-
ers do not require substrate, some of the issues dis-
cussed (such as mutations or gene expression changes
affecting permeability) are irrelevant, leading such re-
porters to be preferred if this is likely to be a trouble-
some variable. In some cases, the particular advantage
of substrate-free alternative reporters—that they func-
tion in vivo with no requirement for added substrate
for visualization—is also a partial disadvantage, in
that these alternative reporters are noncatalytic, and
hence signal cannot be magnified by continuing action
of the enzyme. In a side-by-side comparison with dual
lacZ and GFP reporters in an enhancer trap analysis
(101), ␤-gal was found to be marginally more effective
in providing signal in the context of weak enhancers
and to be particularly helpful in high-resolution histo-
chemical analysis. In conjunction with more sensitive
substrates, ␤-gal was also found to be comparable in
sensitivity to luciferase (77) and could be assessed si-
multaneously and effectively with reporters utilizing
␤-gluc (99) or luciferase (Tropix, Inc.). These results,
indicating an ability to combine and contrast ␤-gal
with other sensitive reporters, suggest a continuing
valuable role for the protein in studies of gene function.
In conclusion, lacZ/␤-gal have provided insights into
gene expression and function for more than half a
century, functioning both as a target of study and a
probe into the study of other genes. Such intensive use
has led to the delineation of assay parameters which
when uncontrolled can lead to artifactual results; how-
ever, with the elimination of such variables, it is clear
that lacZ remains one of the more valuable workhorses
in the stable of molecular reagents.
ACKNOWLEDGMENTS
Support for E.A.G. and I.S. is provided by Core Grant CA06927
from the NIH to Fox Chase Cancer Center, and an award to EAG
from the Merck Genome Research Institute.
Note added in proof. Supplementary information (including
FAQs, links to protocols and sources of substrates) is available on-
line at http://www.fccc.edu/research/labs/golemis/betagal.html.
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