Firefly luciferase as a tool in molecular and cell biology

ANALYTICAL

BIOCHEMISTRY

175,5- 13 ( 1988)

REVIEW Firefly Luciferase

as a Tool in Molecular and Cell Biology’

STEPHENJ.GOULDANDSURESHSUBRAMANI Department ofBiology, B-022, University of California at San Diego, La Joiia, California 92093 The unique properties of firefly luciferase and the cloning of the gene for this enzyme have spawned a number of novel applications of this protein. We summarize a few of these appfications including its use as a reporter gene, as a model for the study of protein import into peroxisomes, and as a component of a heterologous gene expression system. Q 1988 Academic mess, IN.

The firefly luciferase enzyme has had a long history of use in biology, especially for the detection of ATP. The cloning of the firefly luciferase gene (1) and its expression in cells from different organisms (1,2,17) has generated a great deal of interest in possible applications of the gene as a tool in biological studies. Its first use was as a reporter for monitoring promoter activity. Though available for only a short time, the gene has already been widely applied in this role, due to the great sensitivity, ease of use, and cost efficiency of the luciferase assay. Also, the observation that this protein is sorted to peroxisomes in several eukaryotes (3,4) has allowed the development of the luciferase gene as a model for elucidation of signals required for targeting proteins to peroxisomes. The purpose of this review is to highlight current applications of the luciferase gene and to discuss its more recent uses in the fields of molecular and cell biology. PROPERTIES

more detailed discussion see McElroy and DeLuca (5,7), and DeLuca and McElroy (6)]. Firefly luciferase (E) catalyzes the oxidation of D(--)luciferin (LHJ in the presence of ATP-Mg*+ and O2 to generate oxyluciferin (P) and light (hu) as shown below: Mgz’

1. LH,+ATP+E

-+ E.LHz--AMP + PPi

2.

E.LH2-AMP+O*+E.P* + CO2 + AMP

3. E.P*-tE.P+hv.

The quantum yield of the reaction (0.88) is the highest known for bioluminescent reactions. At room temperature and pH 7-8, the light emitted by the Photinus pyralis enzyme has a wavelength of 562 nm (yellow-green). The enzyme is composed of a single polypeptide 550 amino acids in length (62 kDa) and is active in the monomeric form. Most assays for the enzyme involve the measurement of light produced by the reaction. As few as lo-l9 mol of luciferase (2.4 X 10’ molecules) can be detected using currently available methods.

OF THE ENZYME

Only a brief overview of the enzymatic properties of luciferase is provided [for a ’ We dedicate thii article to Marlene DeLuca, who taught us much about luciferase and was a cherished collaborator and colleague.

5

0003-2697188 $3.00 Copyright0 1988 by Academic All

Press, Inc. rserved.

rights ofreproduction in any form

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GOULD

AND SUBRAMANI

CLONING AND EXPRESSION OF LUCIFERASE

The high quantum yield for the reaction, the ease with which the enzyme assay can be performed, and the fact that the active enzyme is composed of a single protein [unlike the two-protein bacterial luciferase system (S)] all indicated that the luciferase gene might prove useful as a reporter for monitoring gene expression. The cloning of the firefly luciferase gene (collaboratively in the laboratories of Marlene DeLuca and Don Helinski) allowed its development in just this capacity (1). In the initial study, not only were cDNA and genomic clones of luciferase isolated, but the gene was also engineered to function as a reporter for monitoring transcriptional activity. The use of luciferase as a reporter gene follows the same basic strategy developed for other reporter gene systems and can be summarized as follows: the DNA segment of interest, which controls expression of a gene, is inserted into a plasmid containing the luciferase cDNA so that it controls expression of luciferase. Upon introduction of the chimeric gene into the appropriate cell type, one obtains a measurement of transcriptional activity from the promoter by monitoring luciferase expression. Detection of luciferase can be accomplished either in vitro (after extraction of protein from expressing cells) or in vivo. The luciferase gene has been used to study promoter activity in bacteria (9), yeast (9), Dictyostelium (lo), plants (2,l l), viruses (12), cultured animal cells (l), and transgenic animals ( 13). An interesting variation on the use of luciferase as a reporter gene is its application for studying trans-acting transcription factors (14). By linking luciferase to a target promoter of a transcription factor, one can assess the effect(s) of mutations in the transcription factor by monitoring luciferase expression. Luciferase vectors suitable for assaying promoter and/or enhancer activities have been available for some time now ( 1). Re-

cently, modified versions of these vectors have been created that contain multiple cloning sites upstream of the luciferase coding region for more convenient insertion of promoters and enhancers (15). Such vectors should facilitate use of the luciferase gene for many researchers. The initial report on the expression of luciferase cDNAs described a simple in vitro assay for luciferase activity. Briefly, it consists of harvesting cells, extracting protein from them, and then assaying enzymatic activity using a luminometer (a machine capable of detecting light at 562 nm). The crude protein extract is mixed with Mg2+ and ATP and placed in a tube within the machine, and luciferin is injected into the tube to initiate the reaction. The luminometer measures the light output of the reaction. When luciferin and ATP are present in excess, the peak height of the light emitted reaches a maximum in 0.3 s and is proportional to the amount of luciferase in the reaction. Subsequently the emission decays to about 10% the value of the peak height and then decreases slowly with time. In our hands, the luciferase assay is 1OO- 1000 times more sensitive than that for bacterial chloramphenicol acetyltransferase and is considerably more rapid and less expensive. Also, the relationship between the enzyme concentration and the peak height of emitted light is linear over five orders of magnitude. Alternative methods for in vitro measurement include use of a scintillation counter (1,16) or exposure of the assay solution to X-ray or photographic films (9). The use of films is less quantitative than luminometer assays but the scintillation counter method can be made almost as sensitive as the luminometer assay. We have provided only a brief outline of the uses of luciferase as a reporter gene, as the topic has been reviewed previously ( 17). DETECTION OF LUCIFERASE in Viva

One disadvantage of the in vitro assay for luciferase activity is that the sample is de-

BIOLOGICAL

USE OF FIREFLY

strayed in the process (as is the case with most other reporter gene systems). Though in vitro assays are sufficient for many reporter gene applications, the development of in vivo assays for luciferase activity would expand the possible uses of this gene in biology. Toward this goal, a method was developed for detection of luciferase activity in vivo in bacteria (9) yeasts (9) and plants (2). Currently, the major limitation of the in vivo assay is the delivery of the substrate into the cells. The inefficient entry of luciferin into cells is not surprising since it is a carboxylic acid which is ionized at physiological pH. To optimize import of luciferin into cells, the assay utilizes a buffer at low pH ( 100 mM Na-citrate, pH 5.2) to protonate the substrate and with DMSO’ (10%) to permeabilize the cell membrane. Luciferin is present in excess, usually at 1 mM. For the film assay, cells are grown either on a nitrocellulose filter or in microtiter wells, incubated with the assay solution, and placed over a piece of OG-1 X-ray film in complete darkness. After development of the film, colonies of cells expressing luciferase are identified by the exposure pattern on the film. This detection system is very sensitive because it integrates light emission over a long period of time, but is difficult to use quantitatively. Thus it is most useful for identifying clones of cells expressing luciferase. Similar assay conditions can be used to obtain more quantitative measurements of luciferase activity in vivo. For this, permeabilized suspensions of bacterial, yeast, or plant cells (in the same buffer described above) are analyzed either in a luminometer or scintillation counter. This assay is less time-consuming than the standard in vitro assay for luciferase and seems to be the method of choice for luciferase detection in these organisms. Perhaps the most elegant use yet devised for the * Abbreviations used: DMSO, dimethyl sulfoxide; DME, Dulbecco’s Modified Eagle medium; FTS, peroxisomal targeting signals; DHFR, dihydrofolate reductase; ORF, open reading frame.

LUCIFERASE

7

in vivo assay has been in the elucidation of patterns of gene expression in plants. Transgenie plants expressing luciferase are fed a luciferin solution and subsequently placed over a photographic emulsion. After approximately 24 h the film is developed and an image is obtained that corresponds to the distribution of luciferase expression. Using this technique, it is possible to visualize the pattern of expression of a given gene in all parts of the plant simultaneously (2). Initially we hoped to use the in vivo assay described above to detect luciferase activity in mammalian cells. When the in vivo film assay was performed with a luciferase-producing animal cell line (L16, derived from CV-1 monkey kidney cells), some light was detected but the cells were also rapidly killed. Cell death was probably caused by the low pH of the assay solution. The addition of 1 mM luciferin to the growth media of cells expressing luciferase did not result in light production [though Rodriguez et al. have successfully used this approach to detect light in cells infected with a recombinant vaccinia virus carrying the luciferase gene (1 l)]. Faced with these results, alternative procedures were sought for the in vivo detection of luciferase in animal cells. We have devised two methods which allow the detection of luciferase in vivo without reducing the viability of the expressing cells. The first of these is actually a variant of the DMSO permeabilization protocol described earlier for use with bacteria and lower eukaryotes. Cells expressing luciferase are grown under normal conditions. The medium is removed and replaced with an assay mix that contains DME with NaHC03, pH 6.9, 1 mM Kf-luciferin, and 15% DMSO. The plate of cells is then placed over a sheet of preflashed ffi-1 X-ray film and left for a period of 8 h or less at room temperature. This method is also useful for detecting luciferase activity in a transiently transfected population of CV- 1 cells that normally do not express luciferase. Test results indicate that the light produced

8

GOULD

AND SUBRAMAM

by this method is a result of light emission from cells and not due to release of luciferase into the medium and subsequent reaction there (data not shown). The pH optimum for the assay was found to be 6.9, somewhat more acidic ,than physiological pH. Though this method successfully detected luciferase activity in a mammalian cell line in vivo, we felt that the assay could be improved. Because the major limitation to efficient luciferase detection lay in entry of the substrate into the cells, we decided to see if a more efficient permeabilization technique would increase the sensitivity of our assay. The ionophore nigericin has been shown to permeabilize mammalian cells to a variety of compounds without seriously affecting cell viability ( 18,19). We have used this ionophore to stimulate the entry of luciferin into mammalian cells by the following procedure. Cells are grown to near confluency in a 24-well microtiter dish. The medium is removed and 1 ml of the sterile assay mix (DME with NaI-IC03, pH 7.3 supplemented with an additional 3.5 mM KC1 and 60 mM NaCl, 1 PM nigericin and fresh K+-luciferin to 1 mM) is added to each well. The microtiter plate is then placed on preflashed Kodak OG-1 film and kept in a dark box for about 8 h at room temperature. The cells are still viable after this treatment (but not when assayed at 37°C). A comparison of this method with the DMSO procedure showed the nigericin technique to be more sensitive (Fig. 1). Maintaining the proper pH and supplementing the media with the prescribed amounts of KC1 and NaCl were critical for the success of this technique. It has been previously reported that the optimal permeabilization of HeLa cells by nigericin occurs with KC1 and NaCl concentrations slightly different from those we have specified (18,19). The amounts we added were those which resulted in optimal light emission and thus may not reflect optimal permeabilization, as some unknown variable may be affecting the assay. Alternatively, the differences between the ion con-

FIG. 1. A comparison of the film assaysfor in viva luciferase detection. L16 cells were grown to near confluency in a 24-well tissue culture plate. Cells in the well on the left were placed in &say solution A while cells in the well on the right were in assay solution B. Immediately after addition of the solutions, the tissue culture rack was placed over a preflashed piece of OC- 1 X-ray film in the dark. Afier 8 h of exposure, the film was processed in a standard X-my lilm processor. Solution A is the DMSOcontaining buffer; Solution B is the nigericin-containing buffer.

centrations we used and those arrived at earlier ( 18,19) may be due to differences between HeLa and CV-1 cells. In view of these differences, optimal conditions for this in vivo assay may differ from one cell type to another. Neither of the in vivo procedures we have described can detect mammalian cells expressing low levels of luciferase (i.e., less than about 2 X lo6 molecules/cell). However, it may be possible to refine the procedure through the use of uncharged luciferin analogs that enter cells readily, better permeabilization protocols or combinations of these improvements. Since’luciferase is peroxisomal, luciferin must cross two lipid bilayers (the plasma membrane and the peroxisomal membrane) before it can come in contact with the enzyme. The use of cytoplasmic variants of luciferase may increase the sensitivity of the assay by allowing luciferin to reach luciferase alter traversing only one membrane instead of two. Another aspect of the in vivo assay that can almost certainly be improved is the light de-

BIOLOGICAL

USE OF FIREFLY

tection mechanism. Currently we are using the film assay because it is relatively inexpensive and OG-1 X-ray film because it can be developed in most film processors. The use of films more sensitive to light in the yellowgreen range (560 nm) should dramatically increase the sensitivity of the system. Even more promising is the use of single-photon detecting instruments linked to microscopes (20). Tailoring the assay for use with such cameras, combined with the great sensitivity of such devices will hopefully lead to the ability to detect light emission from single cells in viva Such a capability could have a great impact in many fields, such as in the study of tissue-specific gene expression, cell lineages in development, and cell-cell interactions, to name just a few. LUCIFERASE AS A MODEL FOR THE TARGETING OF PROTEINS INTO PEROXISOMES

When luciferase was first expressed in mammalian cells we made the unexpected observation that the protein was localized to vesicles in the cytoplasm (1). Further investigation revealed that these organelles were peroxisomes (3). Subsequently, luciferase was also found to be peroxisomal in fireflies (3) as well as in plant and yeast cells expressing the protein (4). The fact that the signal(s) in luciferase responsible for its transport into peroxisomes is recognized in diverse organisms suggests that the mechanism of protein import into peroxisomes has been highly conserved through evolution. While the mechanisms involved in the targeting of proteins into the endoplasmic reticulum, mitochondrion, chloroplast, and the nucleus have been investigated extensively, less is known about the targeting of proteins to peroxisomes. Because luciferase was peroxisomal in a wide variety of organisms, it seemed to be an excellent model for the study of protein transport into peroxisomes. Mutant and wild-type luciferase genes were gen-

LUCIFERASE

9

erated and introduced into mammalian cells. The subcellular location of the encoded proteins was determined by indirect immunofluorescence. These experiments led to the observation that a region within the C-terminal 12 amino acids of luciferase was necessary for peroxisomal targeting. This same peptide was also found to be sufficient for targeting proteins to peroxisomes as it was capable of directing cytosolic proteins into the organelle when attached to their C-terminus (2 1). Like mitochondrial and secreted proteins, peroxisomal enzymes do not share substantial amino acid homologies that might identify putative peroxisomal targeting signals (PTSs) in these proteins. However, because the PTS in luciferase was at the C-terminus, we tested whether other peroxisomal proteins contained C-terminal PTSs. Four peroxisomal proteins-human catalase, rat hydratase:dehydrogenase, pig D-amino acid oxidase, and rat acyl CoA oxidase-were found to contain C-terminal PTSs which were sufficient, when fused onto genes for cytoplasmic proteins, to direct these proteins into peroxisomes (22). These five PTSs are the only C-terminal signals known to be involved in the transport of proteins across a lipid bilayer. This location of the PTS is consistent with the previously demonstrated post-translational import of proteins into the peroxisome (23). An examination of these PTSs has revealed the existence of a three amino acid sequence common to all of them. The tripeptide SerLys/His-Leu, or a conservative variant of it, is found in all peroxisomal proteins for which sequence information is available. The deletion of the Ser-Lys-Leu (last three amino acids) of luciferase results in the retention of the protein in the cytoplasm (24), as did the mutation of the penultimate Lys to Asn in the PTS from rat hydratase:dehydrogenase (22). We now know that the minimal peroxisomal targeting signal consists ofthis tripeptide (24). These data imply that one class of PTSs includes the Ser-Lys/His-Leu sequence but do

10

COULD

AND SUBRAMANI

PAN dD

--(LTR[

Luclsrsse

PAN

+--+

DHFR

v

PAN pLD7

-,LTR

1

Luclerr~

PAN DHFR

v PAN

pL-AD7-jLTR

1

Lucilerrre

/---j

DHFR

/--L--

FIG. 2. Structure of the luciferase-DHFR polycistronic constructs. The box marked LTR designates the Rous Sarcoma Virus long terminal repeat which provides a promoter for transcription; luciferase and DHFR coding sequences are represented by the additional boxed areas. Following the DHFR coding region in each of the constructs are the 3’ untranslated region of the DHFR gene and the splice and polyadenylation sequences from SV40, all derived from the plasmid pSV2DHFR (34). The region between the luciferase and DHFR coding regions consists of various lengths of both the luciferase cDNA 3’ untranslated region and the 5’ untranslated region from the DHFR cDNA. In both pLD and pLD7, the polyadenylation signal of the luciferase cDNA is present between the luciferase and DHFR cistrons. Also, each construct differs in the number of ORFs between the luciferase and DHFR coding regions (pLD, 5; pLD7,4; pGAD, 2; pL-AD7, I). pAn designates a polyadenylation signal.

not preclude the existence of other signals capable of directing proteins into peroxisomes. USE OF LUCIFERASE IN A HETEROLOGOUS GENE EXPRESSION SYSTEM

Another recent application of the luciferase gene is in an expression system designed for high level production of heterologous proteins in mammalian cells. Initially, the goal was to isolate cell lines expressing high levels of luciferase. To obtain such lines, we relied on the observations of Peabody et al. (25,26) that polycistronic messages can function in mammalian cells. Using essentially the same strategy described by Kaufman et al. (27), we constructed a plasmid in which the luciferase gene was placed downstream of a eukaryotic promoter and upstream of the mouse dihydrofolate reductase (DHFR) cDNA. A Chinese hamster ovary DHFR- cell line (DG44) was transfected with the plasmids and the cells were subsequently placed in medium se-

lective for DHFR+ cells. There is only one eukaryotic promoter on the plasmid and both the luciferase and DHFR open reading frames (ORFs) are present on the same mRNA. For DHFR to be expressed, translation must initiate at the AUG of luciferase, terminate at its stop codon, and then reinitiate at the AUG of the DHPR coding region. Since termination/reinitiation is inefficient in mammalian cells, much more protein is translated from the upstream coding region than from the downstream coding region. After DHFR+ selection, the expression of luciferase in the cell lines can be increased by selection for amplification of the polycistronic gene by culturing the cells in the presence of increasing concentrations of the drug methotrexate, a competitive inhibitor of DHFR. The plasmids used for this initial study are presented in Fig. 2. They vary in the number of additional ORFs between the luciferase and DHFR coding regions and in the presence or absence of a polyadenylation signal in this region, two variables which seem to affect expression of the downstream cistron. DHFR+ cell lines derived by transfection with these plasmids were assayed for luciferase activity. It is clear from Table 1 that plasmid

TABLE 1 Construct

Luciferase activity of DHFR+-derived cell lines

PLD pLD7 pL-AD pLaD7

1.5 x 106 6x 10’ 4x 10s 3x 10’

Note. Luciferase activity of DHFR+ cell lines derived from the polycistronic genes. Each of the polycistronic genes described in Fig. 2 were transfected into DG44 cells (a DHFR- line derived from CHO cells). Subsequently, cells were placed in medium selective for DHFR+ cells. DHFR+ colonies were pooled and assayed for luciferase activity. Activity is measured as lit-units per microgram protein. On the luminometer used for these Studies, 1000 ._ light-units represents the light output of 0.26 pg of luciferase.

BIOLOGICAL

USE OF FIREFLY

pGD was the most efficient in producing clones that express the highest levels of luciferase. Not surprisingly, this plasmid contains the greatest number of additional ORFs between the luciferase and DHFR coding regions and also has a polyadenylation signal present in the intergenic region. Though the polycistronic method of heterologous gene expression has been used quite successfully, we felt that one aspect of this technique could be improved by use of the luciferase gene. Currently, one must isolate several cell lines and test each for the production of the desired gene product. Depending on the available reagents and the gene product one wishes to over-express, each of the cell lines must be screened for either RNA or protein production, a task which can be difficult and time-consuming. By incorporating the luciferase gene between the gene one wishes to express and the selectable marker (usually DHFR for polycistronic messages) we thought that it should be possible to use the luciferase assay to screen for transfectiongenerated cell lines expressing the highest levels of the desired gene product. To determine the efficacy of this tricistronic system, we have used sis, the transforming gene of simian sarcoma virus (28), as a test case. The protein produced by this gene is secreted from the cell and can act as a growth factor for cells that express the PDGF receptor (29). The gene for sis was inserted into the plasmid pLD between the promoter and the luciferase coding region. After transfection of this tricistronic gene into DG44 cells, DHFR+ colonies were selected, isolated, and assayed independently for both luciferase and sis expression. The expression of sis correlated with the expression of luciferase (30). This indicated that it should be possible to screen cell lines initially for clones expressing the highest amounts of luciferase, then confirm the expression of the upstream coding region in the highest luciferase expressors. Eventually it should be possible to combine this system with the in vivo assay for luciferase, a method

LUCIFERASE

11

that would allow one to identify the highest expressing colonies while they are still on the original plate prior to isolation. Such a development would further reduce the labor required to isolate desired cell lines. This system should be of great use for many biologists interested in expressing high levels of a given gene product in mammalian cells. The major advantages of the system are its ability to efficiently produce cell lines that express high levels of a desired product and the ease with which the highest expressing cell lines can be identified. Currently the technique is limited to the DHFR- cell line but we are developing similar vectors using a combination of dominant selectable markers such as the G418 and hygromycin B resistance genes and the amplifiable DHFR and adenosine deaminase genes. The utility of this system for producing large quantities of anti-sense RNA in mammalian cells is also being investigated. CYTOPLASMIC MUTANTS OF LUCIFERASE

As we have mentioned earlier, the efficiency of the in vivo assay may be increased through the development of enzymatically active luciferase mutants that are cytoplasmic rather than peroxisomal. The hope is that by eliminating the need for luciferin to cross the peroxisomal membrane, more substrate will be able to interact with the enzyme, thus producing a greater light output. In the process of identifying the minimal peroxisomal targeting signal in luciferase, we have created many mutations, all of which have been assayed for their import into peroxisomes. In addition, many of these have also been assayed for their enzymatic activity. Several such mutants show activity close to that of wild-type luciferase (3 1). Differences in activity between these mutants may represent variations in specific activity of the enzyme but may also be due to changes in protein stability or mRNA turnover rates. Nevertheless,

12 it seems mutants wild-type in the in

GOULD

AND SUBRAMANJ

that there are several cytoplasmic that have retained approximately activity. The use of these mutants vivo assay is currently being tested.

ACKNOWLEDGMENTS This work was supported by Grants GM31253 and CA01062 and a March of Dimes grant (No. 108 1) to S.S. S.J.G. was supported by a fellowship from the Powell Foundation.

SUMMARY

In this review we have discussed several ap plications of the firefly luciferase gene including its use as a reporter gene, as a model for studying protein import into peroxisomes, and in a heterologous gene expression system. Nevertheless, there are several others we have failed to emphasize such as its use for the study of recombination in mammalian cells (32). Due to the rapidly expanding uses of the gene, it is doubtless there are other applications of which we are not aware. As this gene obtains wider use among biologists it is certain that new applications of it will be devised. One of the most exciting possibilities for the luciferase gene lies in the ability to detect luciferase activity in vivo. The synthesis of luciferin analogs that enter cells more readily, development of better assay conditions, and the creation of more sensitive light detection equipment should all lead to improvements in the current in vivo detection methods. Also, it should be remembered that P. pyralis is just one of over 1800 species of fireflies. It is quite possible that the luciferases from these other species may have different properties that make it more suitable for use as a tool in biology (broader pH optima, faster recycling time). One such enzyme, from the clip beetle Pyrophorus plagiophthalamus, has already been cloned (33). The kinetics of this enzyme suggest that it may recycle more rapidly than the P. pyralis luciferase, a property that would allow for greater light production over time. The characterization and cloning of this and other Iuciferases will undoubtedly lead to the development of additional applications for luciferases in molecular and cell biology.

REFERENCES 1. de Wet, J. R., Wood, K. V., DeLuca, M., Helinski, D. R., and Subramani, S. (1987) Mol. Cell. Biol. 7,725-737. 2. Gw, D., Wood, K. V., DeLuca, M., de Wet, J. R., Helinski, D. R., and Howell, S. H. (1986) Science 234,856-859. 3.

Keller, G.-A., Gould, S., DeLuca, M., and Subramani, S. (1987) Pm. Natl. Acad. Sci. USA 84,

4.

Keller, G.-A., Gould, S., Schneider, M., DeLuca, M., Howell, S., and Subramani, S. (1987)5. Cell. Biol.

3264-3268.

105,156a.

5. McElroy, W. D., and DeLuca, M. (I 985) in Chemiand Bioluminescence (Burr, J., Ed.), Vol. 16, Chap. 9, pp. 387-399, Dekker, New York. 6. De.Luca, M., and McElroy, W. D. (1978) in Methods in Enzymology (DeLuca, M., Ed.), Vol. 57, pp. 314, Academic Press, New York. 7. McElroy, W. D., and DeLuca, M. (198 1) in Bioluminescence and Chemiluminescence: Basic Chemis try and Analytical Applications (Deluca, M. A., and McElroy, W. D., Eds.), pp. 179-186, Academic Press, New York. 8. Engebrecht, J., Simon, M., and Silverman, M. (1985) Science227,1345-1347. 9. Wood, K. V., and DeLuca, M. (1987) Anal. Biothem. 161,501-507. 10. Howard, P. K., Ahem, K. G., and Firtel, R. A. ( 1988) Nucleic Acids Res. 16,26 13-2623. 11. Gw, D. W., Jacobs, J. D., and Howell, S. H. (1987) Proc. Natl. Acad. Sci. USA 84,4870-4874. 12. Rodriguez, J. F., Rodriguez, D., Rodriguez, J.-R., McGowan, E. B., and Esteban, M. (1988) Pm. Natl. Acad. Sci. USA 85,1667- 167 1. 13. DiLella, A. G., Hope, D. A., Chen, H., Trumbauer, M., Schwartz, J., and Smith, R. G. ( 1988) Nucleic Acids Res 16,4 159. 14. Waterman, M. L., Adler, S., Nelson, C., Greene, G. L., Evans, R. M., and Rosenfeld, M. G. (1988) Mol. Endocrinol. 2, 14-2 1. 15. Nordeen, S. K. (1988) BioTechnigues6,454-456. 16. Nguyen, V. T., Morange, M., and Bensaude, 0. (1988) Anal. Biochem. 171.404-408. 17. Submmani, S., and DeLuca, M. (1988) in Genetic Engineering (Setlow, J. K., and Hollender, A., Ed.%),Vol. 10, pp. 75-89, Plenum, New York.

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18. Alonso, M. A., and Carmsco, L. (1980) Eur. J. Bio-

chem.109,535-540. 19. Alonso, M. A., and Carrasco, L. ( 1982) FEBS Left.

h&567-569. 20. Hayakawa, T., Kinoshita, K., Miyaki, S., Fujiwake, H., and Oshuka, S. (1986) Phofochem. Phorobiol.

43,95-97. 2I. Gould, S. J., Keller, G.-A., and Subramani, S. ( 1987) J. Cell Biol. 105,2923-293 1. 22. Gould, S. J., Keller, G.-A., and Subramani, S. ( 1988) J. Cell. Biol. 107,897-905. 23. Lamrow, P., and Fujiki, Y. (1985) Annu. Rev. Cell Biol. 1,489-530. 24. Gould, S. J., Keller, G.-A., and Subramani, S. (1988) manuscript in preparation. 25. Peabody, D. S., and Berg, P. (1986) Mol. Cell. Biol.

7,2695-2703. 26. Peabody, D. S., Subramani, S., and Berg, P. (1986) Mol. Cell. Biol. 7,2704-27 11.

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27. Kaufman, R. J., Murtha, P., and Davies, M. V. (1987)EMBOJ. 6,187-193. 28. Devare, S. G., Reddy, E. P., Law, J. D., Robbins, K. C., Tronick, S. R., and Aaronson, S. A. ( 1983) Proc. Natl. Ad. Sci. USA 80,73 1-735. 29. Johnson, A., Heklin, C.-H., Wasteson, A., Westermark, B., Deuel, T. F., Huang, J. S., Seeburg, P. H., Gray, A., Ulhich, A., Scrace, G., Stroobant, P., and Waterlield, M. D. ( 1984) EMBO J. 3,92 l-

928. 30. Maher, D., Gould, S. J., Subramani, S., and Donoghue, D. (1988) unpublished observations. 3 I. Gould, S. J. and Subramani, S. (1988) manuscript in preparation. 32. Subramani, S., and Seaton, B. L. (1988) in Genetic Recombination (Kucherlapati, R., and Smith, G., Eds.), pp. 549-573, ASM press, Metals Park, OH. 33. Wood, K. (1988) unpublished observations. 34. Subramani, S., Mulligan, S. C., and Berg, P. (1981) Mol. Cell. Biol. 1,854-864.