APPLIED MICROBIOLOGY, Jan. 1969, p. 34-40Copyright © 1969 American Society for Microbiology

Vol. 17, No. 1Printed in U.S.A.

Micro Radiometric Method for Study of Acid-insolubleMaterials in Monolayer Cell Cultures

DONALD W. ZIEGLER, HARRIET D. HUTCHINSON, AND ERSKINE L. PALMERNational Communicable Disease Center, Atlanta, Georgia 30333

Received for publication 21 October 1968

A simple radiometric procedure for study of acid-insoluble products synthesizedin monolayer cell cultures is described. Cell cultures were produced directly on thebottom surface of scintillation vials or on glass cover slips (8 x 30 mm). The cellswere labeled and extracted; the radioactivity was determined while the cells re-

mained affixed to the glass surface upon which they were grown. This pro-cedure enabled rapid investigations of certain biosynthetic processes to becarried out by using many individual cell cultures. The method was applied to an

investigation of 1H-thymidine incorporation induced by vaccinia virus in a 5-bromo-deoxyuridine-resistant cell line. '4C-labeling was evaluated as an alternate pro-

cedure for cell quantitation.

Metabolic mechanisms in cell cultures are fre-quently investigated by observing the incorpora-tion and distribution of isotopically labeled com-pounds into cellular materials. The uptake anddistribution of labeled metabolites are generallystudied by disruption of the intact cells and frac-tionation of cellular materials prior to quantitativedetermination of the radioactivity in cell fractions.The use of radioisotopes has increased the sensi-tivity of many biochemical determinations, butthe complexity of the procedures limits the num-ber of replicate determinations which can beperformed. In the course of investigations con-cerned with the effects of virus infection on nu-cleic acid metabolism in tissue culture cells, alter-nate methods permitting greater facility wereperformed.

Several simplified procedures have permittedsample preparation and isotope determination tobe performed with cell fractions entrapped on afiltration matrix. Bollum (2) studied the incorpo-ration of radioisotope-labeled precursors intodeoxyribonucleic acid (DNA) by placing smallportions of reaction mixtures on filter paper discs.By use of similar techniques, Mans and Novelli(11) investigated amino acid incorporation intoproteins; Byfield and Scherbaum (3) isotopicallylabeled and quantitated proteins in intact ciliatedprotozoa. In each of these studies, radioisotope-labeled materials were entrapped on a filterpaper matrix and remained affixed during ex-traction procedures and radioactivity determi-

nations. Baltimore and Franklin (1) measured theincorporation of nucleic acid precursors into ribo-nucleic acid (RNA) and DNA in cells grown oncover slips using a windowless gas-flow counterfor radioactivity determinations.

In this study, a procedure was developed whichpermitted the culture of cell monolayers in anisotope-containing medium, extraction of cellularmaterials, and determination of radioactivity byliquid scintillation while the cells remainedaffixed to a glass surface upon which the mono-layer cultures were grown.

METHODS AND MATERIALSCells cultures. Two mammalian cell lines, HEp-2

and a pyrimidine analogue-resistant AV3, were usedin these studies; however, several additional cellcultures have been employed satisfactorily in re-lated investigations. Procedures concerned with de-termining optimal methods for incorporation ofprecursor metabolites into acid-insoluble materialsemployed HEp-2 cells. Physiological studies of theeffect of vaccinia virus upon tritiated thymidine(3H-TdR) incorporation into DNA employed AV3-5-bromodeoxyuridine-resistant cells. These cells weremade resistant to 5-bromodeoxyuridine (5-BUdR)in our laboratories by Palmer et al. (in press).

Stock cell cultures were propagated as monolayercultures using Eagle's medium (4) containing 10%calf serum, 50 jug of streptomycin per ml, 100 unitsof penicillin per ml, and, in the case of AV3-5-BUdR-resistant cells, 50 jug of 5-BUdR per ml.

AV3-5-BUdR-resistant cells were passaged at leasttwice in the absence of the pyrimidine analogue be-fore use. Cell cultures were planted either on coverslips (8 X 30 mm) contained in Leighton tubes or in20-ml borosilicate glass scintillation counting vials

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(Packard Instrument Co., Inc., Downers Grove, Ill.),in which monolayer cultures formed on the bottomsurface of the vessel.

Virus inoculum preparation. The Connaught strainof vaccinia virus was selected for use in these inves-tigations. It was received from Connaught Labora-tories, Toronto, Canada, as lyophilized calf lymphmaterials and was passaged in HEp-2 cells. Vacciniainoculum was prepared according to the procedureof A. W. Downie (personal communication). Thedetailed procedure for the preparation is describedby Ziegler et al. (14). Immediately before the virusstock was diluted for use in the test procedures, ameasured volume of the suspension was removed fromthe stock suspension and was sonically treated for 3min. This procedure assured uniform dispersion ofvirus particles in successive experiments.

Radioisotope-labeled compounds. 3H-TdR (6.7c/mmole), mixed amino acids labeled with 14C(14C-AA; -1 mc/mg), 3H-uridine (3H-UR; 2 c/mmole), and adenine-8-'4C (5 mc/mmole) were pur-chased from the New England Nuclear Corp., Bos-ton, Mass.

Labeling, fixation, and extraction of cell cultures.After the cell cultures produced monolayers, growthmedium was replaced by a maintenance mediumcontaining an appropriate isotopically labeled pre-cursor material. Adequate labeling of the cells wasattained with 16-hr incubation at 37 C in maintenancemedium containing 3H-TdR (1 to 2 jic/ml), 14C-AA(0.005 Mc/ml), or 3H-UR (1 Mc/ml). These amountsof labeled precursors and these conditions were usedin all experiments except where otherwise indicated.When the effect of virus upon the cell metabolismwas of interest, an appropriate virus inoculum wasintroduced into the maintenance medium. After theincubation period, cultures contained in the scintilla-tion vials or on cover slips were treated similarly.

Cultures in the counting vials were prepared whilethe cells remained affixed to the bottom surface; thecover slip cultures were transferred to test tubes(13 X 100 mm) for preparation. All cultures wererinsed twice with phosphate-buffered 0.85% NaCl(pH 7.4) and immediately fixed with glacial aceticacid-ethyl alcohol (1:3). During the fixation and ex-traction processes, the cultures were maintained in anice bath. Although most procedures for the extractionof acid-soluble materials from tissues use either coldperchloric acid (PCA) or cold trichloroacetic acid,it was observed that the cytological fixative also ex-tracted acid-soluble products. The efficacy of extract-ing monolayer cell cultures with 0.25 M perchloricacid, 0.31 M trichloroacetic acid, and glacial aceticacid-ethyl alcohol (1: 3) were evaluated.

Before the cell cultures were counted, they wererinsed and dehydrated by successive immersions intwo changes each of 95% ethyl alcohol and acetoneand were allowed to air-dry. The coverslip cultureswere then transferred to counting vials containing15 ml of scintillation fluid (0.5% 2,5-diphenyloxa-zole, 0.03% 1,4 bis [2-(4-methyl-5-phenyloxazolyl)]benzene in toluene), and 10 ml of scintillation fluidwas added to the counting vials in which the cellswere grown directly on the bottom surface.

Radioactivity was determined with a Packard3375 liquid scintillation spectrometer. Backgroundradioactivity was determined with cell cultures thathad been prepared for counting in the absence ofradioisotope-labeled precursors. Radioactivity wasrecorded as counts per minute.

Quantitation of cell material. Two procedures forquantitation of cell material were evaluated. In thefirst method, protein was determined by the methodof Lowry et al. (10). In the alternate procedure, cellcultures were grown in a medium containing 14C-AA.The '4C-radioactivity incorporated and remaining in

TABLE 1. Comparison of extraction methods for the removal of acid-soluble materials from fixed cells

RadioactivitybPrecursor Extraction method'

Extracted Acid extract Acid extract/cells total

14C-AA Glacial acetic acid-ethyl 26,990 753 0.026alcohol

14C-AA PCA 27,900 909 0.02914C-AA TCA 29,925 1,006 0.0323H-TdR Glacial acetic acid-ethyl 215,400 3,150 0.015

alcohol3H-TdR PCA 243,750 5,075 0.0203H-TdR TCA 202,000 4,250 0.0213H-UR Glacial acetic acid-ethyl 9,550 417 0.042

alcohol3H-UR PCA 12,175 1,122 0.0843H-UR TCA 10,035 667 0.063

a Monolayer cultures grown in scintillation vials, rinsed twice with PBS, histologically fixed withacetic arid-ethyl alcohol, and extracted as indicated.

b Results expressed as counts per minute per milligram of protein. Average of duplicate determina-tions. The duplicate values of the radioactivity ratio of acid extract to total varied from ±2 to ±11%for individual sets, with a mean variation of i6% for all sets of duplicate values.

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the cell material following extraction provided a meas-ure of the quantity of cells in the culture. Whencultures were labeled with 14C-AA, tritium-labeledprecursors were used for physiological studies.

Determination of acid-soluble materials. Extractedcell materials were isolated and prepared for countingby a method described by Tsuboi and Price (13).The glacial acetic acid-ethyl alcohol extracts, the PCAextracts, and the trichloroacetic acid extracts were ad-sorbed onto Norite A (20 mg/ml). The charcoal waswashed twice with distilled water, and finally the ad-sorbed acid-soluble materials were eluted by two 2.0-ml volumes of a mixture of 1% NH3, 60% acetone,and 39% water. The pooled eluate was reduced undervacuum to 0.5 ml. Eluate concentrates were quanti-tatively transferred to Whatman no. 1 filter paperstrips. Radioactivity was determined by immersingthe filter paper strips in scintillation fluid and bycounting as described by Geiger and Wright (6).

RESULTS AND DISCUSSIONSince the amount of radioactivity appearing in

a culture depends upon the metabolic activitiesof the living cells, standardization of the methodby deposition of known amounts of radioactivematerials on acoverslip is precluded. The quench-ing effect of the cellular material present on thecover slips and the orientation of the cover slipwith respect to the photomultiplier tubes wereevaluated. Conditions for the optimal extractionof acid-soluble materials from the cells and analternate method for quantitation of cell materialwere also investigated.

Extraction procedure. In conventional tech-niques, cold PCA or trichloroacetic acid extrac-tions are used to remove acid-soluble materialssuch as purines, pyrimidines, nucleosides, andnucleotides from tissues and cells. The cytologicalfixative, glacial acetic acid-ethyl alcohol, was ob-served to extract acid-soluble materials frommonolayer cultures during the fixation process.Therefore, the efficacy of the extraction by thehistological fixative was compared with conven-tional extraction methods. Three groups ofHEp-2 cultures were grown in the presence ofeach of three radioisotope-labeled precursorswhich resulted in the labeling of three differentclasses of macromolecular cell substances. Mixed14C-AA was primarily incorporated into proteins,3H-UR in the presence of excess thymine labeledRNA, and 3H-TdR was incorporated into DNA.The comparison of the efficacy of the threemethods for the removal of acid-soluble mate-rials (Table 1) shows that, generally, both PCAand trichloroacetic acid extractions removesomewhat more labeled material than glacialacetic acid-ethyl alcohol extraction alone.

Extraction of acid-soluble "4C-AA-derived com-ponents by PCA or trichloroacetic acid resulted

TABLE 2. Quenching effect of intact, extractedcell cultures

Precursor material Intact cells Digested cells Effi-counts/min counts/mina ciency

4C-amino acids... 3,596.4 8,092.0 44.43,870.0 8,769.0 44.1

3H-thymidine...... 13,541.0 444,706.4 3.0413,198.6 536,285.6 2.46

a Corrected by use of internal standard.

in only a 1.2-fold increase over glacial aceticacid-ethyl alcohol extraction. A somewhat largerproportion of 3H-TdR-derived acid-soluble ma-terials was removed by PCA and trichloroaceticacid. Of the total 3H-TdR-labeled materials in-corporated into the cells, glacial acetic acid-ethylalcohol extracted 1.4% of the label, whereas PCAand trichloroacetic acid extracted 2.0% and 2.1%,respectively. In the case of 3H-UR-derived ma-terials, glacial acetic acid-ethyl alcohol extracted4.2% ofthelabel, whereasPCA and trichloroaceticacid increased the extracted fraction to 8.5% and6.2%, respectively. The importance of the dif-ferences of efficiency among these methods willdepend upon the requirements of given experi-ments; however, in many comparative physio-logical studies, the somewhat more efficient ex-tractions with PCA or trichloroacetic acid wouldnot justify the added extraction procedure.

Quenching effect of extracted cells. The count-ing efficiency as determined by the addition ofstandard amounts of radioisotope-labeled tolueneto scintillation vials containing extracted cultureswas not affected by the presence of the cellularmaterial. Considerable quenching would havebeen expected if it had been possible to placestandard amounts of radioactivity within the cellmaterial. This quenching effect was indirectlyexamined with cells grown in media containingradioisotope-labeled precursors. The apparentradioactivity arising from the labeled and ex-tracted cells was measured first by adding scin-tillation fluid and counting. After the initial de-termination, the cell materials were rinsed free ofscintillation fluid, digested by NCS (Rohm &Haas, Philadelphia, Pa.) reagent, and recountedafter scintillation fluid was added. Finally, thecounting efficiency of the NCS-dissolved materialswas determined by the addition of internal stand-ards (Table 2). In the first column are counts ofradioactive materials contained within the intact,extracted cells. The data in the second columnrepresent the radioactivity of the same culturesdigested and dissolved in scintillation fluid, and

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then corrected for counting efficiency by the use ofinternal standards. The counting efficiency of'4C-labeled materials within intact cells affixed toa glass surface was 44%, whereas the countingefficiency of 3H-labeled materials was 2.7%. Inspite of the low counting efficiency of 3H-labeledmaterials, the high specific activity of most tri-tiated biological precursor compounds permitssufficient incorporation of radioactivity into cellsubstances.

Orientation of cover slip. Since the radioactivityof each sample is located in a thin film on a planarsurface, the effect of the position of the cover slipin the counting bottle was considered. When acover slip rests on the bottom of the bottle, theother end impinges on the wall of the bottle atapproximately a 450 angle. (Fig. 1). Although thismode of positioning the sample does not vary, theorientation of the planar surface in relation to theaxis of the photomultiplier tubes may varythrough 360° rotation of the bottle as it rests onthe elevator of the scintillation spectrometer. De-terminations were made with cover slip culturesoriented at different angles of rotation about thevertical axis of the counting bottle. This permitted

TABLE 3. Effect of orientation ofcover slip in rela-tion to the axis of the photomultiplier tubes-

Spectrometerchannel selection

3H(14C)b

Avg

l4CMH)c

Avg

Cover slip position

900

4,0934,1504,1384,1024,1514,127

984998954960971973

1800

4,1294,0744,1294,1034,1374,114

951946949945955949

2700

4,1224,1484,1044,1734,1624,142

1,006973967974969978

360°

4,1374,1074,1164,1604,1414,132

991969972963960971

,,Results expressed as counts per minute.Radioactivity determined with cover slip inindicated position.

b 8H(14C) Quickset Channel-Amplification Equi-valent, 100%/c Discriminator, 2keV-6keV.

¢ 14C(8H) Quickset Channel-Amplification Equi-valent, 5.7-7.5% Discriminator 8keV-155keV.

FIG. 1. Scintillation vial containing glass cover slipwith affixed cellular material.

an evaluation of the effect of positioning upon theapparent activity of the sample. The cells affixedto the cover slips in this study were labeled withboth 3H-thymidine and with adenine-8-4C (Table3). The orientation of the cover slip in relation tothe axis of the photomultiplier tube had littleeffect upon the apparent radioactivity. The varia-tion among tritium counts (Table 3) obtainedwith the cover slips placed at different points oforientation was nearly the same as the variationwithin replicate counts in which the cover slipremained in a fixed position. Somewhat greatervariation was observed with 14C counts (Table 3);however, repetition of the study yielded results inwhich the variation was not significant. The angleof rotation of the counting bottle, therefore, neednot be controlled. This made possible fully auto-matic counting of the samples.

Comparison of methods for cell quantitation.Metabolic activity of cell cultures must be relatedto the quantity of cells contained in each culture.Although equal volumes of cell suspension weredispensed into each culture vessel, variation in cellmultiplication existed; hence, quantitation of thecells was necessary. Estimation of the final num-ber of cells by trypsinization and counting in ahemocytometer chamber was not considered be-cause this method precludes metabolic assays onthe same culture.

Digestion of cells followed by determination ofcell protein and labeling of the cells during the

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growth period with 14C-AA were investigated asmethods for quantitation of cell material. Label-ing the cells with 14C-AA offered the most con-venient method because in many liquid scintilla-tion spectrometers 14C-radioactivity may bedetermined simultaneously with radioactivityresulting from tritium or phosphorus-32. Afurther advantage in employing the uptake of'4C-AA as a measure of cell material is that onlymetabolizing cells are labeled. On the other hand,both cell enumeration and protein determinationwill include dead or metabolically inactive cells;thus, the possibility of error is introduced when afunction of cell activity is expressed in terms oftotal number of cells or total protein.A comparison of the protein content of cell

cultures with the amount of '4C-AA radioactivityincorporated into acid-insoluble cell material wasmade by preparing cultures of AV3-BUdR-resistant cells in a growth medium containing0.01 ,uc of 14C-AA per ml. Two groups of replicatecultures were planted with 4.8 X 105 cells/culture and 9.6 X 105 cells/culture, respectively,and were incubated for 18 hr. The cultures werehistologically fixed and extracted with glacialacetic acid-ethyl alcohol, and the 14C-AA radio-activity was determined by liquid scintillation.After counting, the protein content of each cul-ture was determined by the method of Lowry etal. (10; Table 4). Cultures inoculated with 4.8 X105 cells contained an average of 0.388 mg of pro-tein and incorporated 'IC-AA, equivalent to1,774 +- l1% counts per min per mg of protein.

TABLE 5. Stability of radioactivity in cell materiallabeled with mixed 14C-AA

Radioactivity Protein Counts perTreatment of culture count per (mg", mg ofe

culture culture) protein

No additional in- 11,050 0.495 22,400cubation 10,403 0.447 23,200

10,899 0.472 23,200Incubated 16 hr; 4,218 0.314 19,500no 14C-AA 4,338 0.316 20,000

4,373 0.320 19,800Incubated 16 hr; 7,739 0.304 25,400

plus 14C-AA 8,383 0.324 25,8008,212 0.320 25,600

a Treatment ofgrowth period.

30 +

20 ±

10 -

8 -6-

4 -

(;PM (-H) 0/-Ratio CPM(14* P (' )

cultures after initial 24-hr

7i

2-

1:2000 1:1000 1:500 1:250 1:125 1:62VIRUS DIWTION

FIG. 2. Vaccinia virus-induced incorporation oftritiated thymidine into acid-insoluble cell fractions.

TABLE 4. Comparison ofincorporation of 4C-aminoacids with protein content of cell cultures

14C-AACell inoculum no. of couptake Protein Counts per

cells/culture c min per mg/culture mo per mgcultureb

4.8 X 105 602 0.380 1,587703 0.394 1,968663 0.388 1,710684 0.376 1,818722 0.404 1,788

Mean 655 0.388 1,774

9.6 X 105 1,292 0.728 1,7731,150 0.630 1,8251,138 0.622 1,8301,267 0.672 1,8851,328 0.712 1,865

Mean 1,235 0.673 1,836

a AV3-BUdR-resistant cells planted in 2-mlgrowth medium containing 0.01 Ac of 14C-AA.

b Culture fixed, extracted, and dehydrated;radioactivity determined by liquid scintillation.

Cultures planted with a twofold greater inoculumcontained an average protein content of 0.673mg/culture and incorporated 1,836 ± 3% countsper min per mg of protein. Variation amongreplicate cultures may reflect a lack of precisionin the analytical procedure; however, the relativemetabolic activity of the cells would also influencethe ratio of incorporated radioactivity to proteincontent. A culture containing dead or quiescentcells would exhibit a reduced 'IC-AA uptake, butprotein content would appear to be normal, thuscausing variation among cultures. These resultsshowed a relationship between the amount ofI4C-radioactivity incorporated into a culture andthe protein content of the culture which has madepossible the use of labeled amino acid incorpora-tion to measure cell material.

Stability of 14C-AA label in cell material. Inprocedures concerned with cell physiology orvirus-host cell relationships, labeling cell culturesduring an initial growth period was advantageous.The stability of the incorporated 14C-AA-labeledmaterials was, therefore, examined. Replicate5-BUdR-resistant AV3 cultures were planted in

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scintillation vials and incubated for 24 hr in amedium containing 0.020 ,uc of mixed IC-AAper ml. The cultures were divided into threegroups; protein content and "C-radioactivityincorporation were determined for each culture.Determinations were made on the first groupafter the initial 24-hr incubation period in growthmedium. A second group of cultures was incu-bated for an additional 16 hr in the absence of"C-labeled amino acids, whereas a third groupwas incubated an additional 16 hr in the presenceof "4C-AA (Table 5). In the groups incubated anadditional 16 hr, a decline in cell mass was noted.Washing procedures and incubation in mainte-nance medium may account for the loss.During the initial incubation period, the cells

incorporated radioactivity equivalent to 22,900counts per min per mg of protein; and during asubsequent 16-hr incubation in maintenancemedium containing no radioisotope-labeledmaterial, there was a 13% loss of radioactivity.Cultures which were incubated for an additional16 hr in "C-AA-containing maintenance mediumincorporated radioactivity equivalent to 25,500counts per min per mg of protein or an 11% in-crease over the initial 14C-AA uptake. Theseresults are consistent with results reported byEagle (5) in which radioactive amino acids wereshown to be incorporated at a rate of 1% perhour.

Effect of virus infection on 14C-AA label. Thisprocedure has been used in investigations ofnucleic acid metabolism in virus-infected cells;therefore, the effect of vaccinia virus infectionupon the stability of the incorporated 14C-AAlabel was investigated. A series of "4C-AA-labeled AVs cultures was infected with threedifferent concentrations of virus, and the proteincontent and radioactivity were determined afteran incubation period of 16 hr in maintenancemedium containing no 14C-AA. Uninfected cul-tures contained an average radioactivity of 980counts per min per mg of protein, and culturesinfected with 3.0 X 105, 1.5 x 105, and 0.75 X105 plaque-forming units of vaccinia per culturecontained 1,020, 990, and 1,020 counts per minper mg of protein, respectively. Virus-stimulatedprotein synthesis in the absence of 14C-AA wouldhave caused a decrease in the ratio of radioac-tivity to protein content. On the other hand,accelerated protein degradation would have beenrevealed by decreases in both total "C-radio-activity and protein content. During a 16-hrincubation period, neither accelerated synthesisnor degradation of protein was indicated. Thisprocedure has been used successfully for studiesconcerned with vaccinia-host cell physiology;however, the stability of the incorporated 14C-AA

should be evaluated if different conditions areimposed upon the system.

Application. The procedure described may beused in any metabolic reaction in which radio-active materials are incorporated within the cellin an acid-insoluble form. Thus, the conversionof isotope-labeled precursors into protein or theincorporation of nucleic acid precursors intoDNA and RNA are examples of metabolic proc-esses which may be studied by use of this method.Virus-induced incorporation of thymidine intoDNA is presented as a specific example of theapplication of this procedure.

Kit et al. (7, 8, 9) have shown that infection ofcell cultures with certain viruses induce theenzyme thymidine kinase. Furthermore, theydemonstrated that cells made resistant to 5-BUdR were resistant to the pyrimidine analoguebecause thymidine kinase was deleted and thatthe enzyme was induced by infection of the cellwith certain viruses. This suggested the use ofvirus induction of enzyme activity as an analyticaltool for measuring virus activity.The relationship of vaccinia virus concentra-

tion to 'H-TdR incorporation in infected AV3-5-BUdR-resistant cells is presented in Fig. 2. Since"4C-AA uptake was employed as a function of thetotal cellular protein, the amount of 3H-TdRincorporated into the acid-insoluble cell materialwas expressed as counts per minute of 'H-TdRdivided by counts per minute of 14C-AA. In Fig.2, the relationship of the 3H-TdR incorporationand the corresponding virus dilution was obtainedby plotting the log of the 3H-14C ratio on theordinate and the log of the virus dilution on theabscissa. The data demonstrate a dose-responserelationship in the induction of 3H-TdR incor-poration by vaccinia virus. The incorporation ofthymidine into DNA could be indicative ofstimulation of any of the synthetic steps leadingto DNA; however, the use of 5-BUdR-resistantcells makes the induction of thymidine kinase themost likely site of virus-induced activity.

LITERATURE CITED

1. Baltimore, D., and R. M. Franklin. 1962. The effect of men-govirus infection on the activity of the DNA-dependentRNA polymerase of L-cells. Proc. Natl. Acad. Sci. U.S.48:1383-1390.

2. Bollum, F. J. 1959. Thermal conversion of nonpriming DNAto primer. J. Biol. Chem. 234:2733-2734.

3. Byfield, J. E., and 0. H. Scherbaum. 1966. A rapid radioassaytechnique for cellular suspensions. Anal. Biochem. 17:434-443.

4. Eagle, H. 1959. Amino acid metabolism in mammalian cellcultures. Science 130:432-437.

5. Eagle, H. 1958. Amino acid metabolism in human cell cul-tures. Federation Proc. 17:985-986.

6. Geiger, J. W., and L. D. Wright. 1960. Liquid scintillationcounting of radioautograms. Biochem. Biophys. Res.Commun. 2:282-283.

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7. Kit, S., and D. R. Dubbs. 1963. Acquisition of thyrnidinekdnase activity by herpes simplex infected mouse fibroblastcells. Biochem. Biophys. Res. Commun. 11:55-59.

8. Kit, S., D. R. Dubbs, L. J. Piekarski, and T. C. Hsu. 1963.Deletion of thymidine kinase activity from L cells resistantto bromodeoxyuridine. Exptl. Cell Res. 31:297-312.

9. Kit, S., L. J. Piekarski, and D. R. Dubbs. 1963. Inductionof thymidine kinase by vaccinia infected mouse fibroblasts.J. Mol. Biol. 6:22-33.

10. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folin phenolreagent. J. Biol. Chem. 193:265-275.

APPL. MICROBIOL.

11. Mans, R. J., and G. D. Novelli. 1961. Measurement of theincorporation of radioactive amino acids into protein by a

filter paper disk method. Arch. Biochem. Biophys. 94:48-53.

12. McIlvaine, T. C. 1921. A buffer solution for colorimetriccomparison. J. Biol. Chem. 49:183-186.

13. Tsuboi, K. K., and T. D. Price. 1959. Isolation, detectionand measure of microgram quantities of labeled tissuenucleotides. Arch. Biochem. Biophys. 81:223-237.

14. Ziegler, D. W., and H. D. Hutchinson. 1969. Vaccinia neu-

tralization test using a radioisotope assay for virus-inducedenzyme activity. Appl. Microbiol. 17:41-47.

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