A nonchromatographic assay for expression of the chloramphenicol acetyltransferase gene in eucaryotic cells

ANALYTICAL

BIOCHEMISTRY

156, 251-256

( 1986)

A Nonchromatographic Assay Acetyltransferase

for Expression of the Chloramphenicol Gene in Eucaryotic Cells

MERILYN J. SLEIGH

Received

January

2 I, 1986

A rapid procedure is described for assaying chloramphen~col acetyltransferase [CAT) enzyme activity following transfection of the CAT gene into eucaryotic cells. CAT enzyme activity in cell extracts catalyzes the transfer of [‘4C]acetyl groups from labeled acetyl coenzyme A to unlabeled chloramphenicol. Labeled reaction product is quantitated by liquid scintillation counting after extraction into ethyl acetate. The method is valid for use with transfected cell extracts only if the extracts are first heated to 65°C to remove a factor which degrades acetyl coenzyme A. The revised procedure offers considerable advantages in speed and ease of performance over the chromatographic assay in current use. ‘r’ 1986 Academic Pras, Inc. KEY WORDS: gene expression; transfection: transient expression; chloramphenicol acetyltransferase: gene regulation.

Transient expression systems are widely used to analyze the features of eucaryotic gene promoters and associated regulatory signals. The sequences of interest are fused to the coding sequences of a marker gene: the hybrid construct is then transfected into suitable recipient cells in culture. After 2-3 days, the level of marker protein in the transfected cells is assessed. Marker genes that have been used include those coding for herpes simplex virus thymidine kinase, &galactosidase (I). and the bacterial enzyme chloramphenicol acetyltransferase (CAT)’ (2). The last of these is particularly useful because eucaryotic cells have no equivalent enzyme to complicate the interpretation of assays for CAT activity. In the original CAT vectors, the CAT coding sequences were transcribed under the control of the SV40 early gene promoter (2). Subsequently, a range of vectors has been prepared with the SV40 promoter replaced by other promoter and gene regulatory regions of in-

terest. These have been widely used in transient expression experiments to study such factors as cell-type specificity of promoters, the role of enhancer sequences. and the mechanism of hormonal regulation of gene activity [see, for example, Refs. (3-6)]. The assay for CAT activity described in Ref. (2) and used in all of the above studies, measures the extent of acetylation of “C-labeled chloramphenicol using acetyl coenzyme A as the acetyl group donor. Labeled products are separated from the substrate by thin-layer chromatography and results are assessed by autoradiography. If quantitative assessment is required. radioactivity in the substrate and product spots must be determined by liquid scintillation counting after spots have been scraped from the thin-layer plate. The assay has. in our hands. been very reliable. but rather lengthy when many quantitative assays are to be performed. Including time for the assay. autoradiography, scraping, and counting, 3 days or more may elapse between cell harvest and availability of quantitated results. In a recent study on ColEI plasmid replication in EscAerichia c~li, Tomizawa (7) de-

’ Abbrevliations used: CAT, chloramphenicol acetyltransferase: DMEM, Dulbecco’s modified Eagle’s medium: PBS. phosphate-buffered saline. 151

0003-2697/X6

$3.00

Copyright ~GI1986 by Academic Press. Inc. All n&s of reproduction m any form rcxwed.

252

MERILYN

scribed the use of an assay for CAT. based on the donation of a “C-labeled acetyl group from acetyl coenzyme A to unlabeled chloramphenicol. In this paper the characteristics of such a procedure and its adaptation for use with extracts of transfected eucaryotic cells are described. The revised method offers considerable advantages over the previous chromatographic assay because of the reduced time and effort required. MATERIALS

AND

METHODS

Muterials. [‘4C]Acetyl coenzyme A (58 mCi/mM; Amersham Pty Ltd) was dissolved in water and dispensedinto 1-pCi (20 ~1) lots. Thesewere stored at -70°C. Each aliquot was diluted for use with 0.5 mM unlabeled acetyl CoA (P-L Biochemicals) to a concentration of 5 ,&i per ml. Chloramphenicol was obtained from the Sigma Chemical Company and CAT enzyme (400 units/ml) from P-L Biochemicals. One unit of CAT enzyme is the amount that transfers 1 nmol of acetyl groups from acetyl CoA to chloramphenicol in 1 min at 3O”C, pH 7.8. Ccl1 lines und truns/~ction procedures. The F9 cell line of mouse embryonal carcinoma (OTF963) was obtained from Dr. A. Levine. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) + 10% heat-inactivated fetal calf serum. The L-a mouse fibroblast line (thymidine kinase and aprt negative) was obtained from Columbia University, New York, via Dr. K. Raphael. Cellswere maintained in DMEM, 10% fetal calf serum, and diaminopimelic acid (50 yg/ml). Transfection of DNA into cells was carried out by calcium phosphate coprecipitation (8). The plasmid usedfor transfection waspSVZ CAT, in which the CAT coding sequencesare under the control of the SV40 early gene promoter (2). Preparation of &I e-xlracts. Forty-six hours after transfection, cells from each lo-cm dish were washed in phosphate-buffered saline (PBS) (NaCI 8.0 g. KC1 0.2 g, KH2P04 0.2 g,

J. SLEIGH

NazHP04 1.13 g per liter), harvested by scraping, and collected by centrifugation. Cells were resuspendedin 0.25 M Tris-HCl, pH 7.8 (0.2 ml per IO-cm dish of cells) (2). Cell extracts were prepared generally by sonication although freeze-thawing wasfound to be equally effective. In the former procedure. 0.2-ml cell suspensions in Eppendorf centrifuge tubes were sonicated individually for 3 X 10-s periods. with cooling in an ice-water bath, using an MSE Sonicator with a fine-tipped probe. Freeze-thawing wasachieved by three rounds of immersion in liquid Nz (4 min) followed by a 37°C water bath (4 min). Cell suspensions were vortexed thoroughly before each freezing step. In initial experiments, cell debris wasthen removed by centrifugation (10 min in an Eppendorf centrifuge at 12,OOOgand 4°C) and supernatants were used for determination of CAT enzyme activity (2). In subsequent experiments cell extracts were heated at 65°C for 10 min (6), centrifuged asdescribed above. and then used for CAT assay(seeResults). Revised ussay jiw CA T activity. The assay for CAT activity devised as a result of this study was as follows. Incubation mixtures contained chloramphenicol (0.02 ml of an 8 mM SOhtiOn), Cd CXtraCt (generally 0.03 ml), diluted acetyl CoA solution asdescribed above (0.02 ml. added to initiate the reaction), and 0.25 M Tris-HCI, pH 7.8. to bring the reaction volume to 0.1 ml. Final concentrations in the reaction mixture were chloramphenicol (I .6 mM), Tris (0.15 M) and acetyl CoA (90 pM and 1.0 pCi/ml). Incubation was at 37°C for I h. Samples were then transferred to an ice bath and labeled reaction products were extracted into 2 X 0. l-ml aliquots of cold ethyl acetate. The layers were mixed by vigorous vortexing and then separatedby centrifugation (12,OOOg) for 3 min at room temperature. Eighty microliters of the organic phase was removed after the first extraction and 0.1 ml after the second. This avoided any transfer of labeled substrate with the organic phase. To the combined organic extract in a 1.5-ml plastic tube was added 1 ml of Packard Instagel,

CHLORAMPHENICOL

and radioactivity was determined scintillation spectrometry.

ACETYLTRANSFERASE

IN

CELL

253

EXTRACTS

by liquid

RESULTS

As a preliminary step in developing the assay, the degree of extraction of the substrate, [‘4C]acetyl CoA, into organic solvents was measured. The two used were toluene, which was the solvent employed by Tomizawa (7). and ethyl acetate, which is used to extract chloramphenicol and its acetylated products in the chromatographic CAT assay(2) and in which chloramphenicol is reported to be highly soluble (9). In both caseslessthan 0.2% of the input counts were extracted from a model reaction mixture to the organic phase.

0.01 UNITS

Assays were carried out using a range of concentrations of purified CAT enzyme to determine the most effective solvent for acetylated chloramphenicol extraction. Reaction mixtures were used as outlined under Materials and Methods, but with a lower concentration of acet.yl CoA (Fig. 1). At the end of the incubation period, extraction was into either toluene or ethyl acetate (2 X 0.1 -ml aliquots). The counts extracted when different enzyme concentrations were usedfor the assay are shown in Fig. 1. Extraction of labeled reaction product was more efficient with ethyl acetate than with toluene, and this was used in subsequent assays. The consistency of the results and the low values in “zermoenzyme” assaysconfirm that there was no transfer of labeled substrate into the organic phaseduring extraction. Addition of a back extraction step, in which combined organic layers were extracted with an equal volume of 0.25 M Tris-HCl, pH 7.8, reduced background levels and improved the consistency of results only in assayswhere the entire organic layer was collected after each extrac-

OF PURIFIED

0.02 CAT ENZYME

FIG. I Revised CAT assay procedure using purified enzyme. (A) Incubation mixtures were set up as described under Materials and Methods, except that an acetyl CoA concentration of 50 ELM and 0.5 pCi/ml was used. Assays contained a range of concentrations of purified CAT enzyme. After incubation for 60 min at 37°C. [‘4C]acetyl chloramphenicol was separated from labeled substrate by extraction into 2 x 0.1.ml aliquots of toluene or ethyl acetate. (ES) Incubations with purified enzyme repeated to demonstrate the linear range for the assay. Labeled reaction product was extracted into ethyl acetate. For both panels. results relate the amount of enzyme used for the reaction (from the concentration in units/ml given by the suppliers) to 14C cpm extracted into the organic phase (lef-hand axis). The right-hand axis shows activity observed in terms of nanomoles of acetyl groups transferred by the enzyme. calculated by reference to the total radioactivity in [‘4C]acetyl coenzymc A (total 5 nmol) present in the reaction mixture. Each point shown represents a single determination.

tion (results not shown). However, when the extractions were carried out asdescribed under Materials and Methods. the back extraction step was unnecessaryand was not included in any of the experiments described here. The assayswere repeated, using the same incubation conditions, to determine the linear range of the assay (Fig. I B). This was found

254

MERILYN

to lie between 0.03 and at least 0.25 units of enzyme per milliliter of incubation mixture.

Cotnpari.son of’C4 T .-ls.w~~Mrf hod\ In the chromatographic CAT assay (2), the limiting substrate is chloramphenicol, present at 111 pM. while acetyl CoA is in excess at 440 PM. The two assay procedures were compared to determine whether the altered substrate concentrations of the revised procedure had substantially affected enzyme activity. Results in Fig. 1 show that 0.6 and 1.7 nmol of acetyl groups were transferred by 0.01 and 0.02 units of CAT enzyme, respectively. Results obtained by us using the chromatographic CAT assay as described in Ref. (2) and the same amounts of enzyme (although present at a lower concentration because of the 180~1 reaction volume), were 0.4 and 1.3 nmol of chloramphenicol present in the acetylated form after a similar incubation period. This comparison suggests that the kinetics of the reaction using purified enzyme have not been altered substantially by the alteration in relative substrate concentrations. However. we believed that a higher acetyl CoA concentration could be advantageous in assaying very active CAT samples. Thus in subsequent assays, and in the standard procedure given under Materials and Methods, we used 90 pM acetyl CoA, with a corresponding increase, to 1 &i/ml in the amount of labeled substrate.

Transj&Yeu’ Cells CAT assays using the method outlined above were carried out using extracts of L-acells transfected with various amounts of pSV2 CAT DNA. Ceil extracts were prepared using a procedure identical to that recommended for use with the chromatographic assay (2). The results obtained were highly reproducible when duplicate assays for a single extract were carried out. Thus the additional protein present in the assays did not affect the efficiency of transfer of the labeled reaction product dur-

J. SLEIGH

ing the organic extraction step. However, in these initial experiments the CAT levels measured were consistently lower than those obtained for the same cell samples by the chromatographic procedure.

.-In .ket~Y Co~~-l-(‘onsr~tning .dcfivi[!* Is Presettr in Cdl E.vtracts Table 1 shows that cell extracts contained a factor that inhibited the activity of purified CAT enzyme in the assay, and that the inhibition could be partially overcome by additional acetyl CoA. The results suggest that the low CAT values obtained for transfected cells are because the cell extracts themselves contain an acetyl CoA-consuming activity. This could present a serious difficulty in a procedure where acetyl CoA is the limiting substrate. Herbomel P/ u/. (3) noted that addition of extra acetyl CoA during the incubation period also improved the results obtained using the chromatographic procedure (2), even though acetyl CoA is in excess during this assay. The results in Table 2 show that cells washed extensively before extraction had a somewhat lower level of the inhibitory factor but the treatment was only partially effective in re-

TABLE CAT

INHIBITORY

Purllied CAT enrpne units

FACTOR

Cell

I

PRESENT

IN CELL

Extra acetyl co.4 h

extract”

-

F9-mock L-a--mock

0.004 0.004

L a--mock

cpm extracted into ethyl acetate

10 ~1 20 ~1

-

246 6156 27x2 1017

20 ~1

+ +

6249 2386

-

0.004 0.004 0.004

EXTRACTS

y F9 or L-a- cells, mock transfected. were harvested, washed. and sonicated as described under Materials and Methods. After centrifugation. aliquots of the cell extract were added to incubation mixtures for CAT assay, prepared as described under Materials and Methods. ’ In some assays, an additional aliquot of acetyl CoA (9 nmol and 0.01 pCi) was added after 30 min of incubation.

CHLORAMPHENICOL TABLE REMOVAL.

OF CAT

I-ROM

Puritied enzyme

CAT tInIts”

CELL

ACETYLTRANSFERASE

2

INHIBITORY EXTRACTS

ACTIVIT’I

cpm ewacted into ethyl acetate

Cell extracth -

307

mocb-untreated

305

0.004 0.004 0.004 0.004 0.004

8008 (hcated)c mock-untreated mock-washed mock-heated

8105 1288 1788 0026

pSV, pSVl pSVl

743 I IO8 3624

CAT-untreated CAT-washed CAT-heated

u CAT assays were’ carried out as described under Malcrlals and Methods. using 70 ~1 ofcell extract. with or without an ahquot of puntied CAT enr>me ’ Three dishes of I-9 cells were each transfected with IO ug pSVz CAT DNA, and three were mock transfected. At harvest 46 h later. cells from each group were pooled, then divided Into three shquots before collection by ~centnfugation. Aiier resuspension. one aliquot ofeach group was somcated. then centrifuged to remove cell debris (untreated sample). $ second aliquot was treated similarly. but the sonicate was heated at 65°C for IO m m before centrifugation (heated sample). In the thtrd aliquot. cells were washed by resuspension in PBS + 0.5 mM ethylene glycol bis(&aminoethyl ether),Y.X’tctraacetic acid, then twice m PBS before sonication (washed sample). ‘ In this sample. 65°C hefore way.

the purified

enLyme

was heated

for IO min

at

storing CAT activity to control levels. Thus if the inhibition is causedby a component of the cells’ growth medium, then this must be very tightly attached to the cell surface. Fromm Pi al. (6) reported that heating oftransfected plant cell extracts for 10 min at 65°C removed a CAT inhibitory factor. The results of Table 2 show that this treatment was also effective in removing the acetyl CoA-consuming activity present in animal cells. Heated extracts from mock-transfected cells no longer inhibited the production of [‘4C]acetyl chloramphenicol by purified CAT enzyme. The CAT enzyme itself appearsto be completely stable during the heat treatment. This was demonstrated for a range of enzyme con-

IN

CELL.

EXTRACTS

255

centrations of which one example is included in Table 2. As a result of removal of the inhibitory factor, extracts from pSV2 CAT-transfected cells showed a much higher level of CAT activity with heat treatment than without. CAT enzyme activities comparable with those predicted by the chromatographic procedure were now obtained. A factor reducing the activity of CAT enzyme during in dro assayis found in extracts of several cell types (e.g., L-a-, F9 mouse embryonal carcinoma. and MCF7 mammary carcinoma cells) as well as in plant cells, as described previously (6). It is possiblethat the factor is present at diflerent levels in different cell types. Thus its removal would be essential if CAT expression were being compared from one cell type to another. DISCUSSION

The method for assay of CAT activity described by Tomizawa (7) offers considerable advantages over the chromatographic assay (2), but hasnot so far been applied to the assay of CAT in extracts from transfected eucaryotic cells.We have found the method to be suitable for this purpose provided that modified cell extraction procedures to remove a CAT-inhibitory factor are adopted. In experinents of this type. the products of different marker genes (e.g., CAT and @-galactosidase)often need to be measured in the samecell extract. In this case, it may be necessary to subject only part of the extract to heating. We have found that fl-galactosidase, for example. is inactivated by 65°C treatment. Using the modifications described here, the CAT assayof Tomizawa (7) provides a quantitative measure of enzyme activity in transfected cell sampleswithin 3-4 h of cell harvest. As well, the number of manipulations required is much smaller than in the method previously used (7), reducing both the tedium and the opportunity for error. Becauseof its simplicity, the method has proved to be especially useful

256

MERILYN

when large numbers of transfected cell extracts are being examined. There are no obvious disadvantagesof this method compared with the chromatographic CAT assay. Duplicate assaysof the same cell extract or enzyme concentraton show variability which is generally lessthan 5% of the values obtained. Contamination of the extracted reaction product with labeled substrate appears to be minimal provided that the extraction steps are carried out with care. The specific activity of labeled acetyl CoA used in our procedure balances the need for an economical assayagainst sufficient sensitivity to measure low levels of CAT activity in cell extracts. However, the sensitivity of the assay can be increased by using higher specific activity substrate, such as was originally described by Gorman et al. (3). A possibleproblem could arise through instability of the limiting substrate, acetyl CoA. To avoid repeated freeze-thawing of the labeled substrate, the substrate was initially divided into aliquots suitable for single experiments and stored in the freezer. When substrate prepared under these conditions has been used some day to day variation hasbeen observed in the amount of acetylation by standard enzyme concentrations. However, there hasbeen no general decline over the period of use of a single batch of labeled substrate. The inclusion of purified enzyme standards in each assayhas proved useful, in that results

J. SLEIGH

obtained can be expressed as a percentage transfer of input acetyl groups. or in terms of units of CAT activity by reference to the standards. In this way results obtained on different days can be compared more easily, and any problems that might arise from acetyl CoA instability would be detected. ACKNOWLEDGMENTS The author is grateful to Dr. E. Dennis and Dr. T. Lockett for helpful discussions to Dr. P. Molloy for comments on the manuscript, and to Ms. D. Lewy for excellent technical assistance. Work in the author’s laboratory is supported in part by a grant from the Australian Meat Research Committee.

REFERENCES I. Hall. C. V.. Jacob, P. E., Ringold, G. M.. and Lee. F. (1983) J. .Uoi. .@pl. Gmrf. 2, 101-109. 2. Gorman. C. M., Moffat. L. F.. and Howard. B. H. ( 1982) nfo/. Cell. Bid 2, 1044-105 I. 3. Herbomel. P.. Bourachet. B., and Yaniv. M. ( 1984) Cell 39, 653-662. 4. Chepelinsky. A. B.. King, C. R.. Zelenka, P. S.. and Piatigorsky. J. (1985) Proc. N&l. Aud. Sci. C’S.4 82.2334-2338. 5. Alwine. J. C. (1985) n1o1. C&l. Bid. 5, 1034-1042. 6. Fromm. M.. Taylor, L. P., and Walbot. V. (1985) Proc. Natl. Acad. Sci. L!S.d 82, 5824-5828. 7. Tomizawa. J. (1985) C~ll40, 527-535. 8. Wigler. M., Silverstein. S.. Lee. L. S.. Pellicer. A.. Cheng. Y. C.. and Axel. R. (1977) Cull 11, 223232. 9. Windholz, M. (ed.) (1976) The Merck Index, 9th ed.. p, 2041, Merck & Co., Rahway. N.J.