Marker proteins for gene expression

Marker proteins for gene expression Keith V Wood Promega Corporation,

Reporter genes are widely molecular from

of the DNA

ensemble

The several genetic

USA

used as a rapid and convenient

genetic events. Their role in experimental

analysis

broader

Madison,

sequences

of molecular

linearity,

Current Opinion

RNA

events that define

reporters available

criteria to choose from, including

mediating

transcription

phenotype

to the

expression.

today impart a range of performance

assay convenience

simplicity

means of measuring

strategies has expanded

and reliability,

sensitivity,

and dynamics.

in Biotechnology 1995, 6:50-58

Introduction The rapid progress in molecular genetics over the past two decades has expanded our ability to manipulate genetic structure, necessitating the development of methods for detecting and quantifjling genetic activity. Methods for the direct measurement of gene expression include mRNA detection using oligonucleotide probes (northern blots) and protein detection using antibodies (western blots), but these methods are time consuming and thus costly. Furthermore, accurate quantification by these methods is typically limited as a result of technical restrictions. Reporter genes provide an alternative method of genetic analysis that in general, is more rapid and convenient. The central concept of a reporter gene is simple: it is a defined nucleotide sequence, which when introduced into a biological system, yields a readily measurable phenotype upon expression. This provides a convenient parameter that is correlated to the molecular events associated with genetic expression. The use of reporter genes today is so commonplace in molecular biology that they are now cited within virtually any journal issue in the current world literature. Although new potential reporter genes are introduced each year, only a few are used routinely. The most widely accepted reporter genes encode chloramphenicol acetyltransferase (CAT), p-galactosidase, &Jucuronidase, and firefly luciferase. Also commonly used are secreted alkaline phosphatase (SEAP) and bacterial luciferase. A new reporter introduced this year, green fluorescent protein (GFP), is attracting much interest because of its ability to autocatalytically generate a fluorophore without addition of exogenous substrate [lo*]. The measurable phenotype

of these reporters is assayed by radioisotopes, color, fluorescence, and luminescence; other reporters possess multiple assayable phenotypes. Because the range of reporters and their applications is so broad, a comprehensive review of this topic covering the past year is impractical. Instead, I focus on examples of the recently published research that are representative of the range of strategies employing reporter genes. I also briefly describe some of the distinguishing criteria used in selecting an appropriate genetic reporter, focusing especially on measurement sensitivity and reporter dynamics. Much of this discussion is drawn from my own experience in developing firefly luciferase as a genetic reporter.

Analyzing the broader genetic event using markers Early applications of reporter genes focused primarily on analysis of &acting genetic elements, usually promoters and enhancers. Today, this is still the most common application for genetic reporters; however, the scope of this research has expanded. Although, most of this research is carried out, as before, using cells grown in culture, an increasing amount of work is employing transgenic animals and plants. For example, Lee et al. [2*] have used transgenic mice to analyze the effects of mutations in different ®ulatory elements of a cardiac gene promoter. In another report, efficient regulation of expression of genes introduced in the heart muscle of adult rats has been demonstrated through the incorporation of tetracycline in their diet [3*]. Moreover, Fenerjian

Abbreviations CAT-chloramphenicol acetyltransferase; CCD-charge-coupled device; CRE-cAMP-responsive element; GFP-green fluorescent protein; HIV-human immunodeficiency virus; HTLV-2-human T-cell leukemia virus type 2; HMG-3-hydroxy-3-methylglutaryl-CoA; IRE-iron-responsive element; IRF-iron regulatory factor; LH/CG--luteinizing hormone/choriogonadotropin; TGF-&transforming growth factor 0; SEAP-secreted alkaline phosphatase; UTR-untranslated region.

50

0 Current Biology Ltd ISSN 0958-1669

Marker proteins for gene expression Wood

and Katatos [4*] have used two reporter genes to analyze a bidirectional promoter in transgenic Drosophila. The spatial organization of gene expression in plants and animals is now commonly analyzed using markers, most notably, ~glucuronidase or &$actosidase, which deposit a colored or fluorescent indicator in expressing tissues [4*-6*,7]. Bylund et al. [8**] have even been able to spatially analyze gene expression within Bacillus cells during spore formulation. Even so, these genetic staining methods in general, disrupt cellular physiology, and interest currently centres on measuring gene expression in intact living cells and organisms. The luciferases, and more recently GFP, provide a means of measuring reporter activity in living tissues without apparent stress on the cells. The luminescence from cells expressing luciferases can be measured non-invasively using sensitive charge-coupled device (CCD) cameras [9*,10*]. Luciferases in living cells have also been shown to provide dynamic measurements of gene expression [ 11,12**]; however, dynamic analysis has not yet been shown for GFl? Studies on gene expression conventionally emphasize the DNA sequences defining transcriptional regulation. But as our understanding of molecular genetics has expanded, our view of the ‘genetic event’ has broadened accordingly to include the entire process of physiology regulation and phenotype expression. As a result, our use of genetic reporters has expanded from the analysis of ci+ acting elements to the study of downstream events, such as RNA processing and protein synthesis, and upstream events, such as the biochemical mechanisms preceding DNA transcription. Reporter genes are capable of indicating events throughout the entire genetic process because their measurable parameter is an enzymatic phenotype. Usually, experimental conditions are established such that events other than transcriptional regulation are presumed to be constant. Changes in reporter expression are thus coupled to differences in transcriptional activity. Even so, alternative experimental strategies can reveal other stages within the broader genetic event. Oliveira et al. [13*] have used reporter genes to examine the role of stem-loop forming structures in the 5’ untranslated region (UTR) of mRNA in yeast cells. In similar work, the same group has examined translational regulation in yeast by human iron-regulatory factor (IRF) on the iron-responsive element (IRE) located in the 5’ UTR [14*]. The role of 3’ UTR structure in plant viral mRNA has been examined by Gallie and Kobayashi [15*] in carrot protoplasts. Messenger RNA processing has been studied by Norris et al. [16*] using the polyubiquitin genes in Arabidopris to examine the effect of intron splicing on gene expression. In the human T-cell leukemia virus type 2 (HTLV-2). the RNA sequences promoting ribosomal frameshifting to yield the garpro and gag-pro-pal fusion genes have been investigated by Kolhnus et al. [17**]. The use of reporter genes to study protein synthesis is focused mainly on the role of chaperones in protein folding. The luciferases are partic-

ularly suitable for this purpose because they are relatively unstable, and their activity is instantaneously measurable upon refolding. Schroder et al. [12**] have demonstrated the importance of chaperones for folding luciferase in Escherichia coli both in living cells and in cell extracts. One of the most rapidly growing areas of reporter applications is the analysis of transcription factors and intracellular signaling mechanisms that underlie the regulation of DNA transcription. Jones et al. [18**] have used luciferase reporter genes to show that transcriptional activation by the thyroid hormone receptor can be modulated by intracellular phosphatase and kinase inhibitors. Luciferase has also been exploited to monitor the activity of a temperature-sensitive mutant of p53 [19*]. Park et al. [20] have investigated the transactivation of virulence genes by PrfA in Lkteria monocytogenes using bacterial luciferase genes. Reporter genes may also be used to elucidate undefined regulatory mechanisms. For example, Mithin and Cohen [21*] have studied stress response in cells injected with denatured proteins using the B-galactosidase gene coupled to an hsp70 promoter. In another study, Gurvitz et al. [22*] h ave used a sporulation-specific promoter to identify mutants in the sporulation regulatory pathway. The effect of extracellular signals on gene regulation is also widely studied using reporter genes. In a recent example, Himmler et al. [23**] have carried out a functional analysis of human dopamine receptors using firefly luciferase. They found that dopamine analogs that interact with receptors in transgenic cells modulate intracellular CAMP, which in turn regulates luciferase expression through tandem CAMP-responsive elements (CREs). This research shows the utility of reporter system for monitoring agonist and antagonist effects on receptor activity in living cells. In analogous studies on human adenosine receptors, Castanon and Spevak [24**] have shown that the same genetic construct of luciferase is effective when applied to the analysis of different receptor types. Similar strategies have been employed to measure receptor interactions with luteinizing hormone/choriogonadotropin (LH/CG) [25*], transforming growth factor-b (TGF-B) [26*], antimineralocorticoids [27*], and a range of other steroid hormones [lo*]. Luciferase has also been used to assess androgen receptor function in clinical tissue samples in a study of abnormalities in male sexual development [28**]. The ability to couple reporter expression with extracellular factors has enabled the development of genetic biosensors. The studies above are examples of the use of markers as sensors for cellular growth factors, and in many cases, these sensors surpass the capabilities of alternative bioassay methods. In bacterial cells, this strategy is being used to measure environmental toxins. Heitzer et al. [29*] have used bacterial luciferase in Pseudomonas to measure environmental naphthalene and salicylate bioavailability. Van Dyk et al. [30*] have detected a range of environmental pollutants using heat-shock promoters in E. coli. Reporter genes can also be used as biosensors

51

52

Analytical biotechnology

of active viruses. For example, Olivo et al. [31**,32*] have developed indicator cell lines that specifically identifir infection by herpes simplex virus or by positive-strand RNA viruses. In addition to their use as indicators of genetic activity, reporter genes are also employed as genetic markers of specific tissues or organisms. A method termed enhancer trapping can be used in transgenic organisms to identifjr tissues with common genetic regulatory controls. Using this method, Callahan and, Thomas [33’] show how a tau-&galactosidase fusion reporter can be used to label di&erent classes of neuronal cells in Drosophila. The tau fusion allows visualization of cellular extension, particularly of neuronal axons, which are not usually detectable using fi-galactosidase alone. To measure the kinetics of human immunodeficiency virus (HIV) infection in cell culture, Chen et al. [34*] have constructed an HIV virus containing the firefly luciferase gene. Reporter genes are commonly used to measure the growth, ecology, and pathogenicity of bacteria [35.*]. For these applications, the most popular reporters are p-galactosidase and bacterial luciferase. Finally, reporter genes are widely used as markers in the development of genetic transformation methodologies. Examples include the development of transferrin-mediated transfection of mammalian cells [7], transfection of mammalian cells by particle bombardment [36*], and lipospermadine-based transfection of vertebrate embryos [6*]. An area of great interest is the use of markers to assess the effectiveness of gene-therapy technology. Gal et al. [37*] have studied myocardial transfection in rabbits and microswine by direct injection of the firefly luciferase gene into cardiac muscle. In another study, Mazur et al. [38*] have used adenovirus to introduce luciferase and fi-galactosidase into porcine coronary arteries.

Enzymatic activity of B-galactosidase, f%glucuronidase, and alkaline phosphatase can be measured by colorimetric [41*], fluorescent [42*], or luminescent assays [43*]; thus, quantitation may be achieved by several means. Luciferase activity ideally is quantitated using a luminometer [10*,14*,25*,27*,44**]; however, scintillation counters and sensitive fluorometers may also be used. GFP is quantitated using a fluorometer. For spatial analysis of gene activity, detection of color [4*,6*,33*] or fluorescence [8**] deposition by b-galactosidase or bglucuronidase is most common, although recently, much enthusiasm has surrounded the use of GFP [lo*]. Spatial detection of luciferases is usually achieved using sensitive CCD photon detectors [9*,10*,11,44**]. Photographic film may be used in some cases, but ofien it does not provide suficient sensitivity [44**].

High

Fluorescence microscopy

Proton flux

Photographic film

CCD detector Fluorometer Scintillation counter Luminometer Low

0 1995 Current

Which

reporter

gene for the job?

For most purposes, the primary consideration in selecting a reporter gene is convenience. Measurements of gene expression can often be performed through direct assay of specific mRNAs, but this involves substantially greater effort. Reporter genes provide analogous information much more efficiently. Even so, convenience is a matter of one’s own particular circumstances and includes a consideration of available equipment and reagents, specific experimental objectives, and experience with the reporter in the biological system. The acetylation reaction of CAT is usually measured using 14C-chloramphenicol or 3H-acetyl-CoA and thus requires a scintillation counter for quantitation [4*,39*,40*]. For most researchers, the use of these isotopes is becoming more problematic because of more rigid restrictions on waste disposal. A fluorescent assay of CAT activity is now available, but not widely used.

Opinion

in Bmtechnology

Fig.1. Schematic representation of luminescent and fluorescent reporter assays. The vertical dimension represents relative photon flux. The bars show the approximate range of photon flux supported by different reporter types in purified form. The shaded region of each bar represents the approximate range of interference expected from endogenous activities in biological systems. The approximate sensitivity limits for different photon detection methods are also illustrated. The diagram only roughly illustrates the relationship between different reporter technologies and detection methods; actual ranges of reporter performance and instrument sensitivities depend greatly on specific circumstances.

Sensitivity has always been an important criteria of reporter performance; however, it is not always properly evaluated. It is typically equated with signal strength, without proper regard for background and assay precision. The assay of reporter activity is a measure of signal (s) over background (b), (i.e; s-b). If the determinations of s and b were infinitely precise, then the reporter assay would be infinitely sensitive. In reality, all measurements have associated error and thus

Marker Droteins for eene extwession Wood

a more applicable expression is (s f error) - (b f error), or (s-b) +- (cumulative error). The limit in sensitivity is reached when the difference between signal and background is about equal to the assay precision. Because precision is typically proportional to signal magnitude (e.g. value &percent error), a greater background has a greater associated error, and thus a greater limit on assay sensitivity.

The difference between signal and background is dependent on the reporter chemistry, instrument sensitivity, and interfering chemical activities. The effect of these parameters can be illustrated by comparing reporter assays that are based on photon flux (Fig. 1). Because the enzymatic turnover of chemiluminescence-based reporters is typically greater than luciferases, they yield a greater signal strength (i.e. photon flux). Moreover, the

(a) Reporter expression

Rate of 1 .o transcription

600 500

0.8 400 0.6 300 0.4 200 0.2

100

0.0

0 3

Relative expression

4 Time (days) Rate of 1 .o transcription

1 .o

0.8

0.8

0.6

0.6

0.4

0.4

0.2

0.2

0.0

0.0 0

1

2

3

4 Time (days)

~-I

Relative expression

1 .o

1 .o

0.8

0.8

0.6

0.6

0.4

0.4

0.2

0.2

0.0

0.0

Rate of transcription

Time (days)

8

1995 Current Opmion in 8iotechnolog

Fig. 2. Model system showing the relationship between reporter stability and dynamic response. The levels of stable reporter (tt/z = 50 h) and unstable reporter (tt/z = 3 h) are shown, together with the relative rate of transcription. (a) Comparison of reporter expression (arbitrary units). (b) Comparison of relative reporter expression (normalized to maximum expression). (c) Relative reporter expression with circadian control of gene regulation.

53

54

Analytical biotechnology

Although these modifications bring benfits (varying degrees), the additional manipulations required increase the overall effort necessary to perform the reporter assays. Furthermore, more complex protocols probably also yield greater cumulative error, which can o&et gains made in the signal to background difference. In most research environments, assay simplicity is an important characteristic for achieving consistently reproducible results. Other important characteristics are linearity and reliability. All the commonly used reporters have been demonstrated to be generally reliable; firefly luciferase has a particularly large linear range (over log-fold) and the assay requires less than 1 min to complete.

signal strength of fluorescence, which is proportional to the photon flux of the excitation light source, is greater than all of the luminescence chemistries. Nevertheless, the background of luciferase assays is limited only by the sensitivity of the luminometer. Chemiluminescence yields a measurable photon flux in the absence of enzyme, and thus is limited by the chemical mechanism. When assaying purified enzymes, chemiluminescence and bioluminescence methods typically have comparable sensitivities. Endogenous enzymatic activities present in most biincrease the ological systems, however, significantly background of the chemiluminescent-based reporters. No analogous endogenous activity exists for the luciferases, so in reporter applications, the signal to background difference is generally greater for bioluminescence. This difference, though, also depends on the detection method. For instance, in photographic detection, where the background of all luminescence-based reporters is limited by the sensitivity of the film, the greater photon flux of chemiluminescence yields a greater signal over background. The limitation of.endogenous activity is even more limiting for fluorescence-based reporters because of the abundance of background fluorescence in biological systems. Even for GFP, which lacks endogenous homologs in most systems, the prevalence of other fluorescent molecules greatly limits sensitivity Nevertheless, the very high photon flux of fluorescence makes such reporters useful when using low-sensitivity detection methods. This explains why GFP is more useful than firefly luciferase in fluorescence microscopy, even though bioluminescence generally is much more sensitive than fluorescence.

The ability of a reporter to indicate changes in gene expression (i.e. the dynamic response of a reporter) is a property that is often taken for granted. For instance, enzymatic stability is commonly proclaimed beneficial to reporter performance without consideration for its inverse relationship to dynamics [44*]. To respond rapidly to changes in gene expression, the genetic reporter must have a short half-life within the biological system. This can be illustrated with a simple model where reporter synthesis is proportional to gene transcription (i.e. zeroorder kinetics, kl) and reporter degradation is proportional to its concentration (i.e. first-order kinetics, kz). Thus, changes in the reporter concentration (dC) can be described by dC =(kl + k2C)dt. It has been estimated that the half-life (hi[O.S]/k2) of firefly luciferase and CAT in mammalian cells is 3 h and 50 h, respectively, although these can vary significantly in different hosts [45]. These values can be used in the model to show how luciferase and CAT are expected to respond to a rapid change in gene expression (Fig. 2). At first appearance, CAT is apparently more responsive to a change in gene expression than luciferase (Fig. 2a). Reporter assays are, however, interpreted by the relative change in expression (Fig. 2b). Such an analysis reveals that luciferase actually responds much more rapidly than CAT. In an extreme case of genetic regulation through

Because of the widespread importance of genetic reporters in molecular genetics, every year witnesses the introduction of new improvements to the assay methods. Most of these refinements are directed at making the assays more sensitive, often through additional processing to minimize endogenous interferences [39*-42*].

O24 Q 1995

L

48

72

Current Opinion in Biotechnology

96

120

r

I

I

1

I

144

168

192

216

240

264

Time (hours)

Fig. 3. Circadian expression of luminescence in transgenic Arabidopsis. Firefly luciferase expression driven by the cab promoter (for the gene encoding chlorophyll a/b binding protein) is assayed by addition of luciferin to seedlings grown in microtiter dishes. The seedlings were measured repeatedly over approximately ten days in constant light.

Marker proteins for gene expressionWood a circadian

mechanism (Fig. 2c), the luciferase model is able to reveal the cyclic pattern of gene expression, whereas CAT exhibits an almost constant signal. This prediction is supported by data from an analysis of the circadian-controlled cub promoter in Ambidopsis (Fig. 3). Luciferase activity clearly shows cyclic regulation of this promoter over a nine day period; in contrast, this regulation pattern is not revealed using CAT Ill]. The choice of an appropriate reporter may depend on specific characteristics of the biological system under study. The greatest concern is, in general, the level of interfering endogenous activity. For this reason, p-glucuronidase is not commonly used in mammalian cells nor is p-galactosidase used in plant cells. Firefly luciferase is widely employed in most experimental systems because no endogenous bioluminescence is present. Because bacterial luciferase is a dimeric protein, it cannot be expressed from a single gene and thus is generally limited to bacterial systems. Fusion forms are available, but they do not perform as well as firefly luciferase in eukaryotic systems. Unexpected interactions may occur with any reporter in a complex biological system, and periodically, reports warn of potential limitations under specific conditions [5*,46**-480.1. To avoid potential interference caused by the native translocation of luciferase into peroxisomes, my colleagues and I have recently developed a cytoplasmic form of this enzyme. We have also engineered several other modifications to increase the general utility of this reporter [49**].

Conclusions Reporter genes are widely used in a diverse range of applications, and each year, the scope of this range broadens. In the p&t year, applications have continued to be reported in the analysis of genetic events upstream and downstream of DNA transcription, and further emphasis has been given to in vim analysis in transgenic animals and plants. Future trends will show an increased focus on the genetic analysis of individual cells and on non-invasive analysis methods. This will involve hrther refinement of both the chemistries and instrumentation of reporter analysis. The past year has also introduced new reporters and new improvements to reporter assays. The most promising new reporter is GFP, which is a non-invasive fluorescent indicator of gene expression [lo*]. This year has also witnessed the expansion of chemiluminescencebased reporter assays to include p-glucuronidase [43*]. The trend toward the adoption of luminescent and fluorescent reporter assays will probably continue into the fLture because of the sensitivity and range of these methods [44**,50**]. The choice of system, from an ever-broadening range of available reporter systems, also entails consideration of a greater number of performance criteria. Present technol-

ogy is sufficiently advanced that, for most applications, assay convenience is the major consideration. Assay reliability is also important, but in general, is not a problem for the most commonly used reporters, with the exception of idiosyncratic behavior in specific biological systems. Assay sensitivity must be sufficient to meet experimental objectives, and is determined by the assay chemistry, instrument sensitivity, and interfering chemical activities. Also relevant are assay linearity and simplicity and, in many circumstances, reporter dynamics. The advances made in reporter versatility and performance reflect the general importance of this technology to biological analysis.

Acknowledgement I thank Dr Steve Kay, of the University of Virginia, for use of his data to illustrate dynamic expression of firefly luciferase.

References and recommended Papers review, . ..

of particular interest, published have been highlighted as: of special interest of outstanding interest

reading within

the annual period of

1. ..

Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC: Green fluorescent protein as a marker for gene expression. Science 1994, 26X002-805. Describes the use of the Aequorea Victoria CFP as a marker. Expression of the cDNA for the GFP yields a fluorescent reporter molecule in bacteria and eukaryotic cells that does not require exogenous substrates and co-factors. Because the fluorescent chromophore of this protein is derived through an autocatalytic mechanism, CFP expression can be used to monitor genetic activity in living organisms. This represents a new type of reporter not previously available in molecular genetic analysis. 2. .

Lee KJ, Hickey R, Zhu H, Chien KR: Positive regulatory elements (HF-la and HF-lb) and a novel negative regulatory element (HF-3) mediate ventricular muscle specific expression of myosin light-chain 2-luciferase fusion genes in transgenic mice. MO/ Cell Bio/ 1994, 14:1220-1229. The cardiac myosin light-chain 2v gene, MLC-ZV, has served as a model system to identify the pathways that restrict the expression of cardiac muscle genes to particular chambers of the heart during cardiogenesis. In this paper, to elucidate the mechanisms of genetic regulation in the MLC-2v promoter, transgenic mice are generated that harbor mutations in five distinct c®ulatory elements. The effects of the mutations are ascertained by expression of firefly luciferase coupled to the promoter. 3. .

Fishman GI, Kaplan ML, Buttrick PM: Tetracycline-regulated cardiac gene expression in vivo. Am Sot C/in invest 1994, 93: 1864-l 868. A chimeric transactivator, designated tetracycline-controlled transactivator, is used to demonstrate gene regulation in vivo in rat cardiac muscle. The firefly luciferase gene coupled to a tet operator is injected directly into the cardiac muscle, and gene regulation is monitored with oral doses of tetracycline. Luciferase activity can be controlled over two orders of magnitude. 4. .

Fenerjian MC, Kafatos FC: Developmental specificity of a bidirectional moth chorion promoter in transgenic Drosophila. Dev No/ 1994, 161:37-47. B_galactosidase and CAT activity are used together to analyze a bidirectional promoter in Drosophilamelanogasrer. Mutations within the bidirectional promoter abolish expression from both genes. 5. .

Paldi A, Deltour L, Jami J: Cis effect of lacZ sequences in transgenic mice. 7iaansgenic Res 1993, 2:325-329.

55

56

Analytical biotechnology Transgenic mice containing the CAT gene coupled to promoter for 3hydroxy-3-methylglutaryl-CoA (HMC) reductase have previously been shown to ubiquitously express reporter activity. In this paper, a similar construct coupling the HMC promoter to the pgalactosidase gene fails to yield similar results. The authors suggest that the f%galactosidase gene may exert a cis effecl on expression in transgenic mice. 6. .

Demeneix BA, Abdel-Taweb H, Benoist C, Seugnet I, Behr JP: Temporal and spatial expression of lipospemdne-compacted genes transferred into chick embryos in viva. Biotechiques 1994, 16:49&501. These authors exploit kgalactosidase and firefly luciferase expression to develop a method for introducing genes into chick emb OS by lipospermadine-based transfection. The cationic lipid Transfectam%I was used to transfect the reporter gene generally, or to target the gene locally through microinjection. Quantitative analysis of the method is carried out using luciferase; kgalactosidase is used for determination of spatial expression. 7.

Zatloukal K, Wagner E, Cotten M, Phillips 5, Plank C, Steinlein P, Curie1 DT, Birnstiel ML: Transferrinfedion: a highly efficient way to express gene constructs in eukaryotic cells. Ann N Y Acad Sci 1992, 660:13&153.

8. ..

Bylund JE, Zhang L, Haines MA, Higgins ML, Piggot PJ: Analysis by fluorescence microscopy of the development of compartment-specific gene expression during sporulation of Bacillus subtilis. J Bacreriol 1994, 176:2898-2905. Compartmentalization of gene expression during sporulation in Bacillus subtiilis is studied using the bgalactosidase reporter gene. Expression of kgalactosidase activity is visualized by fluorescence microscopy using a fluorogenic substrate. Through the use of different promoters, gene expression can be clearly seen either throughout the cell, in the prespore region, and in the forespore. 9. .

Langridge W, Escher A, Wang C, Ayre B, Fodor I, Szalay A: Low-light image analysis of transgenic organisms using bacterial luciferase as a marker. / Biohmin Chemihmin 1994, 9:185-200. Several examples are given for imaging luminescence of firefly and bacterial luciferases in bacteria, yeast, plant cells, plant tissues and intact plant organs. 10. .

Cagne D, Balaguer P, Demirpence E, Chabret C, Trousse F, Nicolas J-C, Pons M: Stable luciferase transfected cells for studying steroid receptor biological activity. / Biolumin Chemilumin 1994, 9:201-209. Different stably transformed cell lines are constructed, containing the firefly luciferase gene coupled to an estrogen response element, a retinoid response element, or a 12-0-tetradecanoylphorbol-13.acetate-responsive element. Cell lines are selected by examining the induction of lumiliescence by the various effecters in individual cells using an intensified CCD camera to detect photon emission. After detection, the selected cells are repeatedly subcultured until a pure clonal culture is obtained. 11.

Millar AJ, Short R, Chua N-H, Kay SA: A novel circadian phenotype based on firefly luciferase expression in transgenic $ants. P/ant Cell 1992, 4:1075-1087.

12. ..

Schroder H, Langer T, Hard F-U, Bukau B: DnaK, DnaJ and CrpE form a cellular chaperone machinery capable of repairing heat-induced protein damage. EMBO J 1993, 12:4137-4144. The actions of DnaK (Hsp70), DnaJ, and GrpE in the molecular chap erone mechanism are investigated both in vivo and in vitro using firefly luciferase as a model protein. In E. co/i cells inhibited for protein synthesis by antibiotics, luciferase can be thermally denatured at 42-C and then renatured at 30-C (with a 50% recovery of activity). This renaturation requires expression of DnaK, DnaJ, and GrpE from host genes; however, the presence of these genes does not protect against the thermal denaturation. Analogous results are obtained in vitro using purified components. This paper is a good example of a dynamic analysis through reporter activity both in living cells and in a reconstituted cell-free system. Oliveira CC, Van den Heuvel JJ, McCarthy JEG: Inhibition of translational initiation in Saccharomyces cer&siae by secondary structure: the roles of the stability and position of stem-loops in the mRNA leader. MO/ Micro&o/ 1993, 9:521-532. To study the effects of stem-loop structures in mRNA translation, modular gene-expression systems are developed using the CAT or firefly luciferase reporter genes. After correction for changes in transcription, translational 13. .

efficiency in yeast cells is found to be related to the predicted stability of the stem-loop structures and by their position in the 5’ UTR. Results obtained using either of the reporter genes are similar. 14. .

Oliveira CC, Goossen B, Zanchin NIT, McCarthy JEG, Hentze MW, Stripecke R: Translational repression by the human ironregulatory factor (IRF) in Saccharomyces cerevisiae. Nucleic Acids Res 1993, 21:5316-5322. The regulation of the synthesis of ferritin and erythroid 5.aminolevulinate synthase in mammalian cells is mediated by the interaction of the IRF with a specific recognition site, the IRE, in the 5’ UTRs of the respective mRNAs. In this paper, this regulation mechanism is investigated both in yeast cells and in cell-free extracts using human IRF and firefly luciferase coupled to an IRE. Changes in expression of luminescence are correlated to changes in protein synthesis using antibodies to luciferase. 15. .

Gallie DR, Kobayashi M: The role of the 3’.untranslated re. gion of non-polyadenylated plant viral mRNAs in regulating translational efficiency. Gene 1994, 142:159-l 65. The genome of positive-sense RNA plant viruses functions as an mRNA but is not polyadenylated. In this paper, the role of the 3’ UTRs of several viral genomes is explored using fusions to reporter genes coding for pglucuronidase and firefly luciferase. Using electroporation of in vitro synthesized RNA constructs, differences in mRNA stability and translation efficiency are measured. Norris SR, Meyer SE, Callis J: The intron of Arabidqsis thaliana polyubiquitin genes is conserved in location and is a quantitative determinant of chimeric gene expression. Plant MO/ Biol 1993, 21:895-906. Polyubiquitin genes in Arabidopsiscontain an intron in the 5’ UTR immediately upstream of the initiator methionine codon. This report employs @glucuronidase and luciferase activity to analyze the effect of these introns on expression. 16. .

17. ..

Kolimus H, Honigman A, Panet A, Hauser H: The sequences of and distance between two &acting signals determine the efficiency of ribosomal frameshifting in human immunodeficiency virus type 1 and human T-cell leukemia virus type II in vivo. J viral 1994, 68:6087-6091. 5equences in HTLV-2 induce translational frameshifting to overcome the termination codon at the end of the gag gene. In this paper, these sequences are inserted between the bgalactosidase and firefly luciferase genes. Without frameshifting, only @galactosidase activity is detectable; with frameshifting, a kgalactosidase-luciferase fusion is synthesized that exhibits luminescence activity. The efficiency of frameshifting is determined as the ratio of luminescence to bgalactosidase activity. Expression of p-galactosidase and luciferase are correlated with immunoreactivity in western blots. 18. ..

Jones KE, Brubaker JH, Chin WW Evidence that phosphorylation events participate in thyroid hormone action. Endocrinology 1994, 134~543-548. The role of phosphorylation in signal transduction by thyroid hormone receptor is investigated using luciferase coupled to thyroid hormone response elements. Results show that stimulation of luminescence by thyroid hormone T3 is enhanced by addition of a protein phosphatase inhibitor, okadaic acid. The addition of okadaic acid without T3 has no effect on gene expression. Similarly, simulation by T3 is diminished by addition of a protein kinase inhibitor, H7. Again, without T3, the inhibitor has no effect. 19. .

Zhang W, Guo X-Y, Hu G-Y, Liu W-B, Shay JW, Deisseroth AB: A temperature-sensitive mutant of human ~53. EM60 J 1994, 13:2535-2544. The activity of a temperature-sensitive mutant of p53 is analyzed using firefly luciferase coupled to p53-binding elements. Mutations of p53 are commonly associated with human cancers. 20.

21. .

Park SF, Stewart for monitoring genes encoding Gen Micro&o/

GSAB, Kroll RG: The use of bacterial luciferase the environmental regulation of expression of virulence factors in Listeria monocyhgenes. / 1992, 138:2619-2627.

Mifflin LC, Cohen RE: Characterization of denatured protein inducers of the heat shock (stress) response in Xenopus laevis oocytes. / Biol Chem 1994, 269:15710-15717. Stress response in cells is believed to be induced by intracellular accumulation of denatured proteins. This paper studies the stress response in Xenopos kevis oocytes by injection of denatured protein derivatives. Cellular stress is measured by induction of B-galactosidase activity from a heat-shock promoter. The results show that stress response is dependent

Marker proteins for gene expression Wood on the mode of protein denaturation and the location of intracellular injection. Curvitz A, Coe JCS, Dawes IW: Use of reporter genes for the isolation and characterisation of different classes of sporulation mutants in the yeast Sacrharomyces cerevkiae. Curr Genet 1993, 24:451-454. Sporulation in yeast is not readily observable for lack of clear morphological changes, thus making mutants in the developmental pathway difficult to isolate. In this paper, the kgalactosidase reporter gene is coupled to a sporulation-specific promoter to provide a clear phenotype during the sporulation process. From this phenotype, three classes of sporulation mutations are isolated: those which overexpress the reporter gene under sporulation conditions, those which do not express the gene under any condition, and those which express the gene in vegetative cells not undergoing sporulation. 22. .

Himmler A, Stratowa C, Czernilofsky AP: Functional testing of human dopamine Dl and D5 receptors expressed in stable cAMP-responsive luciferase reporter cell lines. ) Recept Res 1993, 13:79-94. A good example showing the ability to assay receptor binding using reporter genes. The firefly luciferase gene is coupled to several CREs in a stably transformed cell line. Subsequent transfection with genes encoding human dopamine Dl and D5 receptors causes dose-dependent modulation of luminescence to dopamine agonists and antagonists. The rank of potency of the agonists and antagonists corresponds to published receptor-binding data. 23. ..

Castanon MJ, Spevak W: Functional coupling of human adenosine receptors to a ligand-dependent reporter gene system. Biochem 6iophys Res Commun 1994, 1 g&626-631. The CAMP-responsive cell line described in [23**1 for the analysis of dopamine receptors, is used for the analysis of human adenosine receptors (Al, A2a, and A2b). The intent and results of this study are analogous to those of [23..], demonstrating the ability to analyze different classes of G-coupled receptors using a common indicator cell line to reveal changes in intracellular CAMP. 24. ..

Jia X-C, Perlas E, Su J-C), Moran F, Lasley BL, Ny T, Hsueh AJW: luminescence luteinizing hormonekhoriogonadotropin (LH/CC) bioassay: measurement of serum bioactive LH/CC during early pregnancy in human and macaque. Biol Reprod 1993, 49:1310-1316. A bioluminescent LH/CG bioassay is developed using the activation of firefly luciferase expression in cells expressing a human LH/CC receptor cDNA. This assay indicates hormone bioactivity, which cannot be reliably ascertained by immunoreactivity. The luciferase gene, driven from a CAMP-dependent promoter construct, yields a dose-dependent response to human LH or CG. Treatment with follicle-stimulating hormone, thyroid-stimulating hormone, prolactin, growth hormone, adrenocorticotropin, insulin, prostaglandins, and several neurotransmitters has no effect. Stimulation of luminescence is observed, however, in the presence of basic fibroblast growth factor.

25. .

Abe M, Harpel JG, Metz CN, Nunes I, Loskutoff DJ, Rifkin DB: An assay for transforming growth factor-b using cells transfected with a plasminogen activator inhibitor-t promoter-luciferase construct. Anal Biochem 1994, 216276-284. TGF-p is a potent regulator of cellular differentiation, proliferation, migration, and protein expression. In this paper, a bioassay for TGF-p is developed using stably transformed cells containing the firefly luciferase gene coupled to a plasminogen activator inhibitor-l promoter. The cell line yields dose-dependent luminescence to TGF-b in the range of 0.2 mM to >30mM, providing greater sensitivity and specificity than widely used alternative bioassays. 26. .

27. .

Jausons-Loffreda N, Balaguer P, Auzou G, Pons M: Development of specific bioluminescent in vitro assays for selecting potential antimineralocorticoids. 1 Steroid Biochem MO/ Biol 1994, 49:31-38. A ‘minimal’ chimeric receptor is constructed to analyze the biological activity of various antimineralocorticoids. Transient and stable cell lines containing the firefly luciferase gene are used to indicate steroid binding to the receptor. McPhaul MJ, Deslypere J-P, Allman DR, Gerard RD: The adenovints-mediated delivery of a reporter gene permits the assessment of androgen receptor function in genital skin fib&last cultures. / Biol Chem 1993, 268:26063-26066. This is an interesting example of the use of a reporter gene to diagnose a clinical condition. Defects in the androgen receptor cause a spectrum 28. ..

of abnormalities in male carriers, ranging from feminized phenotype to minor defects in fertility. To evaluate receptor functionality, fibroblasts cultured from genital skin are infected with recombinant adenovirus to deliver an androgen-inducible firefly luciferase gene. In fibroblasts from normal individuals, androgen causes an 11- to 200-fold increase in Iuminescence, corresponding to the level of androgen receptor detected in ligand-binding assays. In contrast, only a negligible increase (about 1.2-fold) is evident in fibroblasts from men with testicular feminization. Heitzer A, Malachowsky K, Thonnard JE, Bienkowski PR, White DC, Sayler GS: Optical biosensor for environmental on-line monitoring of napbthalene and salicylate bioavailability with an immobilized bioluminescent catabolic reporter bacterium. Appl Environ Microbial 1994, 60: 1487-l 494. Sensors for continuous on-line monitoring of naphthalene and salicylate bioavailability are developed from Pseudomonas fluorescens containing a bacterial luciferase operon coupled to the nahG promoter. The engineered cells are immobilized onto an optical light guide, and the probe is then inserted into a measurement cell, which receives a waist stream mixed with maintenance medium. Reproducible luminescent signals are achieved with repeated exposures to naphthalene or salicylate. The sensor is also tested with jet fuel and leachate from contaminated soil, both of which contain naphthalene.

29. .

Van Dyk TK, Majarian WR, Konstantinov KB, Young RM, Dhurjati PS, LaRosa RA: Rapid and sensitive pollutant detection by induction of heat shock gene-bioluminescence gene fusions. App/ Environ Microbial 1994, 60:414-l 420. An example of the use of engineered bacteria as environmental sensors. An E. co/i strain is employed that contains a bacterial luciferase operon from Vibrio fischeri. The luciferase operon is coupled to two heat-shock promoters, dnaK and grpE, to indicate the presence of dissolved metals, solvents, crop-protection chemicals, and other organic compounds. Photon production is non-invasive because the entire luminescence operon is used. 30. .

Olive PD: Detection of herpes simplex virus by measurement of luciferase activity in an infected-cell lysate. / Wrol Methods 1994, 47:117-l 28. A stably transformed cell line is developed that expresses high levels of luciferase activity following infection with herpes simplex virus. The cell line contains a herpes simplex virus type 1 promoter-luciferase chimeric gene, which yields a greater than lOOOO-fold increase in luminescence upon virus infection. This paper shows the utility of firefly luciferase as an indicator of specific viral activity and also the wide assay range required in some reporter applications. 31. ..

Olivo PD, Frolov I, Schlesinger S: A cell line that expresses a reporter gene in response to infection by Sindbis virus: a prototype for detection of positive strand RNA viruses. Virology 1994, 198:381-384. Describes the development of a stably transformed cell line that contains a defective Sindbis virus genome under control of a Rous sarcoma virus promoter and the luciferase gene downstream of the viral subgenomic RNA promoter. The cell line expresses high levels of luciferase activity following infection with Sindbis virus and related variant viruses.

32. .

33. Callahan CA, Thomas JB: Tau-&galactosidase, an axon-targeted . fusion protein. Proc Nat/ Acad Sci USA 1994, 91:5972-5976. As a genetic marker of neuronal cells, &galactosidase is limited by its inability to readily diffuse into axons. In this paper, a modified form of the enzyme is constructed by fusing the cDNA encoding bovine microtubule-binding protein, tau, onto the bgalactosidase gene. Using an enhancer-trap transposon in Drosophila, this modified reporter is used to mark various neuronal cell types, as well as muscle fibers and glial cells. Chen BK, Saksela K, Andino R, Baltimore D: Distinct modes of human immunodeficiency virus type 1 proviral latency revealed by superinfection of nonproductively infected cell lines with recombinant luciferase-encoding viruses. / Wrol 1994, 68:654-660. To study mechanisms of cellular latency in HIV infection, a recombinant HIV is constructed with firefly luciferase replacing the nefgene. This recombinant virus is used to rapidly measure active viral growth on different latent cell lines. The study reveals both cis and tram mechanisms for latency. 34. .

35. ..

Prosser ]I: Molecular marker systems for detection of genetitally engineered micro-organisms in the environment. Microbiology 1994, 140:5-l 7.

57

58

Analytical biotechnology This review covers the use of reporter genes as markers for the detection of genetically engineered cells in the environment. Coverage is given to antibiotic resistance genes, kgalactosidase, catechol 2,3-dioxygenase (encoded by xy/D, 2,4dichlorophenoxyacetate monooxygenases, and bacterial luciferases. The use of bacterial luciferases constitutes the greatest part of the review. Detection methods for luminescence are also discusssed. Heiser WC: Gene transfer into mammalian cells by particle 36. . bombardment. Anal Biochem 1994, 217:185-l 96. Examines the factors affecting the efficient transfer of genes into mammalian cells by particle bombardment. Firefly luciferase is used for quantitative analysis of gene introduction, and kgalactosidase is used to visualize the spatial pattern of particles in cell-culture dishes. The particle bombardment method is compared with transformation by electroporation, lipofection, and diethylaminoethyl dextran. . Gal D, Weir L, Leclerc C, Pickering JC, Hogan J, lsner JM: Di37. . red myocafdial transfection in two animal models. Lab Invest 1993, 68:1&25. The effectiveness of gene therapy through direct injection of DNA into the myocardium is investigated using the firefly luciferase gene. The effect of the amount of DNA carried by the delivery vehicle and its volume were quantitatively analyzed. Also examined are the persistence of foreign gene expression and the feasibility of a percutaneous injection method. Mazur W, Ali NM, Raizner AE, French BA: Coronary restenosis 38. . and gene therapy. Texas Heart lnst / 1994, 21:104-l 11. The long-term effectiveness of coronary angioplasty is limited by the proliferative response of vascular smooth muscle cells to the site of vascular injury imposed by the technique. Currently, non-permanent gene therapy at the site of vascular injury is being investigated as a means of locally inhibiting the proliferative response. This paper evaluates the utility of an adenovirus vector for introducing DNA into vascular tissue, both in viva and in vitro, using the firefly luciferase and kgalactosidase reporter genes. Cassinotti P, Weitz M: Increasing the sensitivity of a common 39. . CAT assay. Bio@chniques 1994, 17136-39. An improvement on the standard phase-separation assay for CAT activity is described. The new method incorporates an additional extraction of the organic phase to reduce background from contaminating X-acetylcoenzyme A Chireux M, Raynal J-F, Weber MJ: Performance and limits of the mixed-phase assay for chloramphenicol acetyltransferase at low [3H]acetylCoA concentration. Anal Biochem 1994, 219:147-153. Describes a method for increasing the sensitivity of the two-phase CAT assay by omitting unlabeled 13Hlacetyl.CoA from the reaction. The new method yields several fold greater sensitivity than the conventional method, but it is still estimated to be 14-fold less sensitive than the assay for firefly luciferase. 40. .

Cannio R, dePascale D, Rossi M, Bartolucci 5: Gene expression of a thermostable B_galactosidase in mammalian cells and its application in assays of eukaryotic promoter activity. Biotechno/ App/ Biochem 1994, 19:233-244. Initial experiments in the use of thermostable bgalactosidase as a reporter gene are described. The thermostable reporter is cloned from the thermoacidophilic archaebacterium Sulfolobus solfataricus. The reporter exhibits very little activity at 37-C; enzyme activity is assayed by incubation at 75-C. A preliminary comparison with CAT is also given. 41. .

Young DC, Kingsley SD, Ryan KA, Dutko FJ: Selective inactivation of eukaryotic kgalactosidase in assays for inhibitors of HIV-1 TAT using bacterial kgalactosidase as a reporter enzyme. Anal Biochem 1993, 215:24-30. Describes a method for increasing the sensitivity of the 8_galactosidase assay by reducing interference from endogenous enzymatic activity. Heat treatment at 50-C for 1 h inactivates the B_galactosidase activity endogenous to several eukaryotic cell lines by as much as 40-fold without adversely affecting the activity of bacterial bgalactosidase. 42. .

Bronstein I, Fortin JJ, Voyta JC, Juo RR, Edwards B, Olesen CEM, Lijam N, Kricka LJ: Chemiluminescent reporter gene assays: sensitive detection of the GUS and SEAP gene products. Biotechniques 1994 17:172-l 77. Describes chemiluminescent reporter gene assays for human placental SEAP and bglucuronidase using adamantyl dioxetane derivatives.

44.

Bronstein I, Fortin J, Stanley PE, Stewart CSAB, Kricka LJ: Chemiluminescent and bioluminescent reporter gene assays. Anal Siochem 1994, 219: 169-l 81. This review describes the available reporter genes assayed by bioluminescence and chemiluminescence. It provides coverage of several luciferases and photoproteins; chemiluminescent methods are limited to adamantyl dioxetane chemistries. Some comparative information is given, together with a brief discussion of various detection methods.

..

45.

Thompson JF, Hayes LS, Lloyd DB: Modulation of firefly luciferase stability and impact on studies of gene regulation. Gene 1991, 103:171-177.

46. ..

Tillman 16, Crone DE, Kim H-S, Sprung CN, Spindler SR: Promoter independent down-regulation of the firefly luciferase gene by T3 and T3 receptor in CVl cells. MO/ Cell Endocrinol 1993, 95:101-109. Down-regulation of luciferase expression is demonstrated in CV-1 cells expressing T3 receptor in the presence of T3. Down-regulation is shown to be caused by the gene itself and not by other vector sequences. The mechanism underlying this effect is not identified and it may not be general to other cell types. Even so, the results show the general importance of confirming that reporters behave as expected in specific host cells under specific experimental conditions. A ‘baseline’ genetic construction should, thererfore, be included in most quantitative analyses using genetic reporters. 47. ..

Gonzalez-Flecha B, Demple B: Intracellular generation of superoxide as a by-product of Wbrfo harveyi luciferase expressed in Escherichia co/i. J Bacterial 1994, 176:2293-2299. In a study of the E. co/i soxRS regulon, which regulates various antioxidant defense enzymes, bacterial luciferase is found to cause gene induction in the absence of superoxide-generating agents. It is postulated that without aldehyde substrate the bacterial luciferase could generate superoxide through autoxidation of the flavin. This is supported by biochemical and genetic data. This observation may be general for all bacterial luciferases, but does not apply to other luciferases. 48. ..

Forsberg AJ, Pravitt GD, Higgins CF: Use of transcriptional fusions to monitor gene expression: a cautionary tale. / Bacterial 1994, 176:2182-2132. Bacterial luciferase yields anomalous results in E. co/i and Salmonella Iyphimurium when coupled to the leu-500 promoter. In contrast, coupling to CAT yields results consistent with those previously reported using galactokinase. Promoter activities are assayed from single-copy insertions into the host (5. typhimurium) chromosome and from low-copy and multiple-copy plasmids (in E. co/i and S. typhimurium). Anomalous results are also achieved when bacterial luciferase is coupled to the proU promoter in E. co/i, whereas kgalactosidase yields expected results. Coupling of bacterial luciferase to the lac promoter or the gyrB promoter does, however, report expected gene expression patterns. 49. Shed BA, Wood KV: Firefly luciferase engineered for improved .. genetic reporting. Promega Notes 1995, 49:14-21. This brief communication describes a new form of the firefly luciferase gene engineered for more reliable use as a genetic reporter. The primary modification yields a cytoplasmic form of the luciferase, which may be important to avoid the disruption of normal peroxisomal function in exogenous hosts. Other modifications make the gene more convenient to use and minimize potential interference in diverse biological systems. Campbell AK, Sala-Newby G: Bioluminescent and chemiluminescent indicators for molecular signalling and function in living cells. In Biological techniques. Nuorescent and luminescent probes for biological activity. Edited by Mason WT. London: Academic Press; 1993:5&82. This review provides a brief overview of bioluminescence and chemiluminescence and the use of bioluminescence genes for genetic reporting. Other topics include use of bioluminescent proteins expressed in exogenous hosts to evaluate various metabolites (e.g. ATP, NAD(P)H and Ca*+J. A short discussion is also given on the potential use of engineered luciferases to monitor intracellular kinase activity. 50. ..

43. .

KV Wood, Promega Corporation, Madison, Wisconsin 53711, USA.

2800

Wood

Hollow

Road,