JOURNAL OF BACTERIOLOGY, May, 1966Copyright © 1966 American Society for Microbiology

Vol. 91, No. 5Printed in U.S.A.

Synthesis of Nucleic Acid and Protein in L CellsInfected with the Agent of Meningopneumonitis

ESTHER M. SCHECHTER

Department of Microbiology, The University of Chicago, Chicago, Illinois

Received for publication 27 December 1965

ABSTRACT

SCHECHTER, ESTHER M. (The University of Chicago, Chicago, Ill.). Synthesisof nucleic acid and protein in L cells infected with the agent of meningopneumonitis.J. Bacteriol. 91:2069-2080. 1966.-Synthesis of deoxyribonucleic acid (DNA),ribonucleic acid (RNA), and protein in uninfected L cells and in L cells infectedwith the meningopneumonitis agent was compared by measuring rates of incorpo-ration of H3-cytidine and C'4-lysine into nuclear, cytoplasmic, and agent fractionsin successive 5-hr periods during the meningopneumonitis growth cycle. Synthesisof meningopneumonitis DNA, RNA, and protein was first clearly evident in thelabeling period 15 to 20 hr after infection, soon after initiation of agent multipli-cation. The rates of synthesis of agent DNA, RNA, and protein increased loga-rithmically for a brief period and then declined. However, rates of isotope incorpo-ration into all three meningopneumonitis macromolecules were sustained at nearmaximal values throughout the remainder of the meningopneumonitis growthcycle. These data are most readily interpreted in terms of multiplication of themeningopneumonitis agent by binary fission. The L cell response to infection was

a decreased rate of DNA and RNA synthesis and an accelerated rate of cell death.Host protein synthesis was unaffected. The inhibition of nucleic acid synthesis ininfected L cells probably involved competition between host and parasite fornucleic acid precursors. Different sublines of L cells varied greatly in the degree towhich their nucleic acid-synthesizing mechanisms were damaged by infection. Thecytoplasm of infected L cells contained newly synthesized DNA and RNA thatcould not be accounted for as intact meningopneumonitis cells. This nucleic acidprobably arose from disintegration of the fragile intracellular forms of the meningo-pneumonitis agent.

Microorganisms of the psittacosis group con-tain both deoxyribonucleic acid (DNA) and ribo-nucleic acid (RNA) (20, 31, 32), multiply by bi-nary fission within membrane-bound vacuoles inthe cytoplasm of their host cells (1, 10), and de-grade glucose and pyruvate (37, 38) by enzymesthat are qualitatively different from those of thecorresponding enzymes of their host (22). Theseobservations, together with many others, as re-viewed by Moulder (21), indicate that the growthof a psittacosis agent within a host cell representsan intimate interaction of two organisms, eachwith its own set of macromolecule-synthesizingenzyme systems.

Schechter, Tribby, and Moulder (28) used p32_orthophosphate to follow the synthesis of nucleicacids in L cells infected with the agent of meningo-pneumonitis. Cell fractionation methods were em-ployed to distinguish between synthesis of men-

ingopneumonitis nucleic acids in the cytoplasmand host nucleic acids in the nucleus. This paperdescribes further investigations on macromoleculesynthesis in meningopneumonitis-infected L cellsin which nucleic acids were labeled with H3-cytidine and proteins with C'4-lysine. Particularattention was paid to the course of synthesis ofmeningopneumonitis DNA, RNA, and proteinand to the effect of meningopneumonitis multi-plication on the synthesis of macromolecules bythe L cell.

MATERIALS AND METHODS

Growth of L cells. Strain L cells (6, 27) obtainedfrom H. M. Jenkin (University of Washington,Seattle) were cloned in this laboratory by T. I. E.Tribby, and all the work reported here was done with asingle subline, designated clone 5b. L cells weregrown routinely in medium 199 (19; Grand IslandBiological Co., Grand Island, N.Y.) containing 16%o

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newborn calf serum (Grand Island Biological Co.),0.12% sodium bicarbonate, and 200 ,ug/ml of strepto-mycin. This medium will be referred to as 19984CaS16.The L cells were maintained at 37 C as monolayercultures in 4-oz (about 120 ml) prescription bottles inan atmosphere of 95% air-5% carbon dioxide or assuspension cultures in the apparatus described byMcLimans et al. (16) with the modifications of Mikaand Pirsch (17). Counts of viable cells in suspensioncultures were made by the trypan blue exclusion test asperformed by McLimans et al. (16). Suspensioncultures received complete changes of medium every2 or 3 days and were divided when the cell densityreached 1 million cells per milliliter.

Growth ofmeningopneumonitis agent in L cells. TheCal 10 strain of the meningopneumonitis agent,adapted to growth in L cells by Tribby (35), wasserially propagated in L cell monolayers. Theseexperiments were performed with the 109th to the163rd L cell passages. Monolayers (2 to 3 days old)were washed twice with medium 199 containing 10%newborn calf serum (l99goCaS1o) and infected with2 ml of a meningopneumonitis agent concentrateprepared as described below and containing 200meningopneumonitis cells per L cell (on the assump-tion that 1 chick embryo LD5o = 10 cells). After thebottles were gently shaken for 1 hr at 37 C, the super-natant fluid was decanted, the monolayers werewashed twice with 19990CaS10, 10 ml of 19984CaS16was added, and the infected monolayers were incu-bated for 72 hr at 37 C in an atmosphere of 95% air-5% carbon dioxide. Then the supernatant liquid wasdecanted from the almost completely destroyedmonolayer, centrifuged at 150 X g for 5 min toremove cell debris, and then centrifuged at 5,000 X gfor 30 min to sediment the meningopneumonitis agent.The resulting agent pellet was resuspended in 4 ml of19984CaS16 and used to infect monolayer or suspensioncultures.

Macromolecule synthesis in uninfected and infectedL cells was studied in suspension cultures preparedfrom monolayers about 1 week previously. The growthmedium was changed 24 hr before infection to insurelog-phase metabolism. The cells were recovered bycentrifugation at 150 X g, suspended in 19984CaSI6,and mixed with enough meningopneumonitis agent togive a multiplicity of infection of 200 and a volume of20 ml. The suspension was gently shaken for 1 hr at37 C, centrifuged for 5 min at 150 X g, washedonce in 19984CaSI6, and resuspended to a finalvolume of 50 ml and a final cell count of approximately5 X 105 cells per milliliter.To determine the percentage ofL cells infected with

the meningopneumonitis agent, duplicate Leightontubes containing flying cover slips were inoculatedwith 0.5 ml of the infected cell suspension and 0.5 mlof medium. The tubes were incubated at 37 C in 95%air-5% carbon dioxide for 24 hr, at which time thecover slips were fixed in absolute ethyl alcohol andstained with Giemsa; the percentage of infection wasdetermined by examining at least 200 cells for thepresence of meningopneumonitis inclusions. Infectionin the cultures used in these experiments was 85 to100%. No corrections for uninfected cells were applied

to any of the results. Most infected L cells containedmore than one inclusion.

Fractionation of L cells. The L cells in a 50-mlsuspension culture were centrifuged at 150 X g for5 min, the growth medium was discarded, and thecells were washed three times with a modification ofthe balanced salt solution of Hanks and Wallace (9)containing 0.15% methylcellulose (Methocel 15centipoises, reagent grade, Dow Chemical Co.,Midland, Mich.). Addition of the Methocel wasrequired to prevent leakage of L cell constituentsduring washing. The cells were suspended in 5 ml ofcold 20% citric acid and shaken vigorously for 1 hr at4 C. They were then centrifuged at 150 X g for 10 minat 0 C, washed once with 5 ml of 20% citric acid,resuspended in 5 ml of 30% citric acid, and shakenagain for 30 min. After this treatment, the liberatednuclei were sedimented by centrifugation for 10 min at150 X g. The supernatant liquid was added to theprevious two citric acid supernatant fractions to yieldthe cytoplasmic fraction, which was clarified by afinal centrifugation. The sediment was combined withthe main nuclear fraction in a volume of 4 ml. Thenuclei in each nuclear fraction were counted in ahemocytometer to an error of less than 5%. Thecalculations for each experiment were based on thenuclear count rather than the whole cell count.Of the cytoplasmic fraction, 4 ml was removed for

analysis, and the rest was centrifuged at 5,000 X gfor 30 min at 0 C to sediment the meningopneumonitisagent. The pellet was washed twice with balanced saltsolution containing Methocel, and was resuspended in2 ml of the same diluent containing 0.05 M MgSO4.Ribonuclease (Worthington Biochemical Corp.,Freehold, N.J.) and deoxyribonuclease (SigmaChemical Co., St. Louis, Mo.) were added to finalconcentrations of 0.25 mg/ml each, and the mixturewas incubated for 30 min at 37 C. Then the proteinasePronase (Calbiochem) was added to final concentra-tions of 0.50 mg/ml, and incubation was continued foranother 30 min at 37 C to yield a third fraction, calledthe cytoplasmic sediment, which consisted almost en-tirely of intact meningopneumonitis cells. Whenexamined with an electron microscope, a cytoplasmicsediment from L cells infected 24 hr previously con-tained large, small, and intermediate cell types of themeningopneumonitis agent (Fig. 1). There was no re-cognizable host cell debris. The meningopneumonitiscells in the cytoplasmic sediment were no longerinfectious for chick embryos.

Three milliliters each of the nuclear, cytoplasmic,and cytoplasmic sediment fractions were separated bythe Schneider-Schmidt-Thannhauser procedure (36)into acid-soluble, phospholipid, RNA, and DNAfractions and analyzed for radioisotope content asdescribed below.

Evidence that this L cell fractionation method wassuitable for the uses to which it was put in theseinvestigations will be presented in Results.

Radioisotopic labeling of L cells. Infected anduninfected L cell suspensions were exposed to radio-actively labeled precursors of nucleic acid and proteinat successive 5-hr intervals after infection. To measureradioisotope incorporation during a given interval, an

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Fic1. 1. Electron micrograph ofthe cytoplasmic sediment ofinfected L cellsfractionated by the citric acid method24 hr after infection. The preparation was air-dried and chromium-shadowed. See Litwin et al. (15) for a detaileddescription of sample preparation. The electron micrograph was taken by Robert R. Friis. X 13,000.

L cell suspension culture was divided into foursubcultures; two were infected and two were kept asuninfected controls. Eagle's (5) minimal essentialmedium was used during the 5-hr period of radio-isotope labeling, to keep dilution of the labeledprecursors to a minimum. However, it could not beused throughout the growth cycle because, althoughit is a complete medium for L cells, it does not supportmaximal growth of the meningopneumonitis agent.

H3-cytidine. At 5-hr intervals after infection, 50-mlsuspension cultures were centrifuged at 150 X g for5 min, and the cells were resuspended in 50 ml ofminimal essential medium with 16% newborn calfserum; 167 ,uc of H3-cytidine with a specific activity of1.0 c/mmole (Schwarz Bio Research Inc., Orangeburg,N.Y.) was added to each suspension culture. Exactly5 hr later, the cells were collected by centrifugation,washed with balanced salt solution-Methocel con-taining 10-' M cytidine, and fractionated. Samples(1 ml) of the Schneider-Schmidt-Thannhauser frac-tions of nucleus, cytoplasm, and cytoplasmic sedimentwere mixed with 9 ml of Bray's (2) scintillationmixture, and were counted in a Packard model 3324

Tri-Carb liquid spectrometer at a gain setting of 50%.The counting efficiency varied from 9 to 16%, de-pending on the nature of the sample.

C14-lysine. The same general procedure was followedas for H3-cytidine labeling; 5 ,uc of L-lysine-C'4, with aspecific activity of 180 mc/mmole (Schwarz BioResearch Inc.), was added to each suspension culture.The wash solution contained 10-4 M L-lysine. Chemicalfractionation was carried through the separation oftrichloroacetic acid-soluble and -insoluble fractions.Insoluble fractions were dissolved in 1.5 ml of a 1 Msolution of the hydroxide of Hyamine (PackardInstrument Co., LaGrange, Ill.) at 4 C overnight.Samples were counted at a gain setting of 9%; thecounting efficiency was only 15% because of theconsiderable quenching effect of the Hyamine solution.

P32-orthophosphate. Carrier-free sodium ortho-phosphate-P32 (Volk Radiochemical Co., Skokie, Ill.)was used to prepare labeled suspensions of themeningopneumonitis agent. To 100-ml L cell sus-pensions conitaining 106 cells per milliliter, 2.5 mc ofP'2-orthophosphate was added at the time of infection,and labeled meningopneumonitis agent was prepared

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as described in Results. P32-containing samples werecounted in Bray's scintillation mixture at a gainsetting of 2.9% and an efficiency of 73%.No corrections for quenching were made. Since

corresponding fractions from infected and uninfectedcultures seemed to be quenched to the same degree,the data were interpreted in direct comparison of theobserved counts. An external standardization systemhas become available since these data were collected.Its use with similar samples confirms the conclusionthat quenching is significant only in samples dissolvedin Hyamine and that it is equal in all correspondingfractions ofinfected and uninfected cells. Samples werecounted long enough to insure that the counting errorwould not exceed 0.9% in 96% of the observations.

RESULTs

Growth ofthe meningopneumonitis agent in cloneSb L cells. Figure 2 shows the growth curve ob-tained by infecting suspensions of clone 5b L cellswith enough L cell-adapted meningopneumonitisagent to infect virtually 100% of the L cells. Thetiter of both intracellular and extracellular infec-tions began to rise 10 hr after infection, andreached its peak at 40 hr. Growth of the meningo-pneumonitis agent in the cloned L cells differedfrom that in the uncloned L cells used previously(28) in that multiplication continued for about 10hr longer, reached a terminal titer more than 1 loghigher, and resulted in a slower rate of killing ofthe host cells.

H3-cytidine incorporation. Cytidine was a goodprecursor of both RNA and DNA in L cells andin the meningopneumonitis agent. Figure 3 is agraphic representation of cytidine incorporationinto the RNA and DNA of the cytoplasmic sedi-ment of infected L cells (see also Table 1). In Fig.3A, incorporation in each individual 5-hr labelingperiod was plotted on an arithmetic scale. Uptakeof cytidine into meningopneumonitis nucleic acidswas first clearly evident 15 to 20 hr after infection,reached its maximum at 25 to 30 hr, and declinedslightly after 30 to 35 hr. The rates of incorpora-tion of labeled cytidine into meningopneumonitisRNA and DNA rose and fell in unison. In Fig.3B, cumulative cytidine incorporation into men-ingopneumonitis nucleic acids was plotted on alogarithmic scale. Cytidine uptake into RNAand DNA increased logarithmically from 15 to30 hr after infection, but significantly declined inrate thereafter, indicating that the infected L cellcan support a maximum rate of synthesis ofmeningopneumonitis RNA and DNA for only abrief period.

Incorporation of labeled cytidine into the nu-clear RNA andDNA of infected L cells proceededat only half the uninfected rate from the 20 to 25hr labeling period onward (Table 1). In the earlierlabeling periods, hostDNA synthesiswas inhibited

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FIG. 2. Growth ofthe meningopneumonitis agent in asuspension of clone 5b L cells. The intracellular LD50was obtained by centrifuging down the cells in I ml ofsuspension, resuspending them in I ml of2% ammoniumacetate, and incubating at 4 C for 30 min to lyse the Lcells and release the meningopneumonitis agent (14).The lysate was then diluted at least 10-fold and im-mediately inoculated via the yolk sac into groups ofIS 6-day old embryos. The LD50 was calculated by thesingle-dilutionmethodofGolub (8). Thesupernatantfluidfrom each cellsample usedfor intracellular titration wasemployed to obtain the extracellular LD50. Each valueis the mean offour independent observations.

slightly, whereas host RNA synthesis was unaf-fected. Incorporation of cytidine into phospho-lipids of infected L cells was at no time inhibited.The effect of meningopneumonitis infection oncytidine uptake into nonsedimentable cytoplasmicfractions of the L cell will be discussed in a latersection.The good agreement among uninfected L cell

populations used as control for the different pe-riods of isotopic labeling is evidence of the repro-ducibility of the cell culture and fractionationmethods (Table 1). These cell samples shouldtheoretically be identical log-phase populations.The incorporation into the nuclear DNA in the5 to 10 hr labeling period was unaccountably low.Incorporation of cytidine into the nuclear RNA ofinfected L cells declined somewhat as the uptakeinto cytoplasmic RNA increased fourfold, indi-cating that the nuclear fraction obtained by citricacid fractionation was relatively free of cytoplas-mic contamination. The cytoplasmic sediment ofuninfected L cells contained only traces of incor-porated cytidine. These small amounts of radio-activity were in forms resistant to hydrolysis by

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15-20 25-30 35-40

10-15 20-25 30-3510 15 20 25 30 35 40

Interval of labeling (hours after infection) Time after infection (hrs)FIG. 3. Incorporation of H3-cytidine into DNA and RNA and of C'4-lysine into protein of the cytoplasmic sedi-

ment ofinfectedL cells. (A) Incorporation in each S-hr labelingperiod (arithmetic scale). (B) Cumulative incorpora-tion (logarithmic scale). The counts per minute for lysine incorporation were multiplied by a factor of 10 for easeof plotting.

either ribonuclease or deoxyribonuclease. This isfurther evidence for concluding that this fractionof infected L cells consisted almost entirely ofmeningopneumonitis cells.

H3-cytidine labeled the phospholipids of unin-fected and infected L cell cytoplasm and nucleusand the cytoplasmic sediment of infected L cells(Table 1). This observation suggests that boththe L cell and the meningopneumonitis agent con-tain cytidine diphosphate choline or other similarcompounds (13). Incorporation of the cytidinelabel into the meningopneumonitis agent phos-pholipid roughly paralleled its incorporation intoRNA and DNA.

C14-lysine incorporation. L-Lysine-C"4 was usedas the labeled precursor of L cell and meningo-pneumonitis proteins because it is an essentialamino acid for the L cell (4) and a major con-stituent of meningopneumonitis protein (23).Table 2 shows that lysine was incorporated intothe trichloroacetic acid-insoluble fractions (as-sumed to be protein) of both L cells and themeningopneumonitis agent. Uptake of lysine intothe proteins of the uninfected L cell populationsused as controls for the successive 5-hr labelingperiods did not vary significantly in the different

cell samples. The amount of label incorporatedinto the cytoplasmic sediment of uninfected Lcells was very small.

Incorporation of C14-lysine into the cytoplasmicsediment (meningopneumonitis agent) of infectedL cells was first detected in the 15 to 20 hr periodand reached a maximum 30 to 35 hr after infec-tion, 5 hr later than the period of maximal incor-poration into nucleic acid (Table 2, Fig. 3). Withthis exception, the rate of lysine incorporationinto the cytoplasmic sediment of infected L cellsclosely paralleled the rate of cytidine uptake intothe RNA and DNA of these fractions (Fig. 3A,3B). The failure of Starr et al. (30) to find specificpsittacosis group antigen with fluorescent labeledantibody until late in the growth cycle may havebeen due to the relative insensitivity of thismethod.

In contrast to the reduced rate of nucleic acidturnover observed in infected L cells (Table 1),there was no inhibition of lysine incorporationinto the proteins of the cytoplasmic and nuclearfractions of the host cells at any time in the growthcycle of the meningopneumonitis agent (Table 2).Nature of the nonsedimentable "extra" DNA

and RNA in the cytoplasmic fractions ofinfectedL

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MENINGOPNEUMONITIS NUCLEIC ACID SYNTHESIS

TABLE 2. Incorporation of C'4-lysine intotrichloroacetic acid-insoluble fractions ofuninfected L cells and L cells infectedwith the agent of meningopneumonitis

Uninfected cells Infected cellsPeriodof

Cytola -i ceu Cyto-labeling Nucleus pClasom psamiiNucleus Cyto- smeintpam

sediment plasmsedmicthr

5-10 141* 434 2 155 343 110-15 121 286 1 138 264 215-20 118 358 2 138 338 1720-25 130 398 2 119 318 4925-30 121 370 1 127 373 7430-35 143 374 2 130 292 9035-40 95 234 2 88 414 67

* Counts per minute per 105 L cells. Each valueis the mean of two cultures.

cells. Infection with the agent of meningopneu-monitis caused an increased incorporation of H3-cytidine into the cytoplasmic fractions of L cellsthat was greater than could be accounted for bythe presence of meningopneumonitis agent in thecorresponding cytoplasmic sediments (Tables 1

and 3). Cytoplasmic fractions from infected Lcells contained newly labeled DNA not sedi-mented by centrifugation at 5,000 X g for 30min (sufficient to sediment more than 99.9% ofintact meningopneumonitis cells) and partiallysensitive to deoxyribonuclease (intact cells arecompletely resistant). This extra DNA could havebeen synthesized by either the L cell or the men-ingopneumonitis agent. The cytoplasmic fractionsof infected L cells also contained increasedamounts of nonsedimentable and partially ribo-nuclease-sensitive RNA that could not be ac-counted for by the cytoplasmic sediments derivedfrom them. This extra RNA became particularlyprominent at the 20 to 25 hr labeling period. Itcould also have been made by either the L cell orthe meningopneumonitis agent. No increase innonsedimentable protein in infected L cell cyto-plasm was noted (Table 2). It may have been ob-scured by the high level of protein synthesis in theL cell ribosomes.The citric acid fractionation procedure was first

suspected as the source of these discrepancies.However, when infected L cells were separatedinto cytoplasmic and nuclear fractions by an en-tirely different procedure (7), which uses Tween80 to disrupt the L cells, essentially the sameamount of extra DNA and RNA was found aswith the citric acid method.

It was possible that intact meningopneumonitiscells were not being centrifuged down into the

TABLE 3. Nonsedimentable meningopneumonitisnucleic acids in the cytoplasmic fractions

of infected L cells

H3-cytidine H3-cytidinePOefriod incorporated incorporated RNA-DNA ratioofriad into DNA into RNAbeling . -

Sed.* Nonsed-t Sed. Nonsed. Sed. Nonsed.

hr

5-10 lot 72 51 1,39010-15 12 306 22 1,39015-20 57 629 80 1,42020-25 1,150 3,020 809 12,180 0.70 4.2125-30 1,740 1,870 1,300 8,130 0.75 4.3530-35 1,160 1,870 1,210 5,770 1.03 3.06

* Sedimentable DNA or RNA. These valueswere taken from the cytoplasmic sediment columnsof Table 1.

t Nonsedimentable DNA or RNA. Thesevalues were calculated from those in Table 1 bysubtracting the sum of the incorporation intouninfected cytoplasm and infected cytoplasmicsediment from that of the infected cytoplasm. Thehigh RNA values for the early labeling periodsprobably reflect the errors inherent in such anapproximation. For this reason RNA-DNA ratioswere calculated only during the intervals when theamount of agent present was appreciable.

t Counts per minute per 105 L cells.

cytoplasmic sediment but were remaining in thecytoplasmic supernatant fluid. Therefore, infectedand cytidine-labeled cytoplasmic fractions werecentrifuged for 60 min at 35,000 X g before andafter dilution to reduce the density of the citricacid solution in which they were contained. Suchcentrifugation left just as much extra labeledDNA in the cytoplasmic fraction as did the usualcentrifugation at 5,000 X g for 30 min.The extra nucleic acid could also have been

newly synthesized host cell DNA and RNA thathad leaked out of L cell nuclei damaged in thecourse of infection or of cell fractionation. Thishypothesis was tested by using the autoradio-graphic observation of Pelc and Crocker (24) thatH3-thymidine was incorporated into host cellDNA but not into the DNA of psittacosis groupagents. A 50-ml suspension of infected L cells wasexposed to 250 luc of H3-thymidine (specific activ-ity, 0.36 c/mmole; Schwarz BioResearch Inc.)during the 20 to 25 hr period after infection, andwas fractionated as in the experiments with H3-cytidine (Table 4). Thymidine was incorporatedinto the DNA of both infected and uninfected Lcell nuclei, although the inhibitory effect of infec-tion on DNA synthesis was readily apparent.However, the thymidine label did not appear ineither the whole cytoplasmic fraction or in the

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TABLE 4. Incorporation of H3-thymidine into thenucleic acids and phospholipids of uninfectedL cells and of L cells infected with the

agent of meningopneumonitis*

DNA RNA Phospholipid

FractionUnin- In- Unin- In- Unin- In-fected fected fected fected fected fected

Nucleus 8,900t 3,920 109 39 149 73Cytoplasm 20 40 11 14 8 3Cytoplasmic 1 13 1 9 1 1sediment

* The experiment was carried out in exactly thesame way as those with H3-cytidine. The labelingperiod was 20 to 25 hr after infection.

t Counts per minute per 105 L cells. Each valueis the mean of two cultures.

cytoplasmic sediment, thus confirming the ob-servations of Pelc and Crocker and indicating thatthe extra nonsedimentable DNA in the cytoplas-mic fractions of infected L cells was not made inthe L cell nucleus. This experiment also showedthat cytoplasmic fractions obtained by the citricacid method were not contaminated with hostDNA.The rate of accumulation of extra nonsedimen-

table RNA and DNA in infected L cell cytoplas-mic fractions (Table 3) closely paralleled meningo-pneumonitis multiplication and maturation, asjudged by infectivity titers (Fig. 2) and appearanceof labeled RNA and DNA in cytoplasmic sedi-ments (Fig. 3). Accumulation of extra cytoplasmicDNA and RNA reached its highest rate during the20 to 25 hr labeling period, a time when themeningopneumonitis population consisted chieflyof the large and fragile immature cell type, andsubsequently declined in rate as the large cellmatured into the more resistant small cell type[see Moulder (21) for discussion of psittacosisgroup cell types]. This correlation suggested thatthe large meningopneumonitis cells were beingdisrupted during L cell fractionation and werereleasing their DNA and RNA into the nonsedi-mentable portion of infected cytoplasmic frac-tions.To test this hypothesis, two experiments were

performed with P32-labeled meningopneumonitisagent. In the first, infected L cells were allowedto incorporate P32-orthophosphate for 25 hr, andthen were lysed by shaking in 2% ammoniumacetate at 0 C for 60 min (14). This released theintracellular meningopneumonitis agent which, at25 hr after infection, consisted mainly of the im-mature large cell type. L cell debris was removedby centrifuging for 10 min at 150 X g. The men-ingopneumonitis agent was sedimented by centri-

fugation at 5,000 X g for 30 min, and was washedtwice in balanced salt solution-Methocel; a por-tion was then removed to test its initial sensitivityto ribonuclease and deoxyribonuclease. The re-maining crude labeled meningopneumonitis agentwas mixed with unlabeled L cells, and was carriedthrough the usual citric acid cell fractionation.Portions of whole cytoplasmic and cytoplasmicsupernatant fractions were dialyzed against phos-phate-buffered saline for 48 hr to remove citricacid, and half of each preparation was thentreated with the two nucleases. The cytoplasmicsediment was also divided in two portions, andone was treated with nuclease and one not. Nu-clease digestions were carried out, and sensitivityto ribonuclease and deoxyribonuclease was es-timated by comparing the p32 content of RNAand DNA fractions obtained with and withoutnuclease digestion. An uninfected L cell culturewas carried through the same procedure, so thatthe p32 counts obtained for each infected samplecould be corrected for the contribution of anylabeled L cell debris that might be present. Suchcorrections were less than 13% of the infected cellRNA values, and less than 5% of the infected cellDNA values.

In the second experiment, mature extracellularagent, consisting chiefly of the small maturemeningopneumonitis cell type, was obtained fromthe supernatant fraction of an infected L cell sus-pension that had been allowed to incorporate p32_orthophosphate for 44 hr. The supernatant frac-tion was clarified by centrifugation at 150 X g for10 min, and the meningopneumonitis cells weresedimented at 5,000 X g for 30 min. They were re-suspended in balanced salt solution-Methocel,taken through another cycle of high-and low-speedcentrifugation, and treated in the same manner asthe 25-hr meningopneumonitis preparation.The mature extracellular agent was insensitive

to both ribonuclease and deoxyribonuclease be-fore and after citric acid treatment (Table 5). Itwas almost quantitatively recovered in the cyto-plasmic sediment. The immature intracellularmeningopneumonitis population was initially re-sistant to deoxyribonuclease but partially sensi-tive to ribonuclease. After citric acid fractiona-tion, a significant amount of the p32 originallyassociated with the immature meningopneumoni-tis cells appeared in the cytoplasmic supernatantfraction, and the labeled DNA and RNA in thecytoplasmic sediment had become partially sus-ceptible to the respective nucleases. These experi-ments suggest that the extra nonsedimentableRNA and DNA in the infected cytoplasmic frac-tions may have come from the breakdown of thelarge immature forms of the meningopneumonitisagent.

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TABLE 5. Stability of the meningopneumonitis agentduring fractionation ofL cells

Kind of P82-labeledmeningopneumoni-tis agent added tounlabeled L cells

Fraction

Intra- Extra-cellular, cellular,

25 hr 44 hr

% %oLabeled agent before cell frac-

tionationSensitivity to deoxyribo-

nuclease................. 0 0Sensitivity to ribonuclease ..... 46 0

Cytoplasmic fraction after cellfractionation

Nucleic acid in sedimentDNA recovered.............. 85 93

Sensitivity to deoxyribo-nuclease................. 70 0

RNA recovered.............. 77 94Sensitivity to ribonuclease. 33 0

Nucleic acid in supernatant fluidDNA recovered.............. 15 7

Sensitivity to deoxyribo-nuclease................. 42 0

RNA recovered.............. 33 6Sensitivity to ribo-nuclease.40 0

DIscussIoN

Macromolecular synthesis in the meningopneu-monitis agent in relation to its growth cycle. Theresults of Higashi, Tamura, and Iwanaga (11) andof Tamura (31) showed that absolute increases inthe amount of RNA and DNA in various frac-tions of L cells infected with the meningopneu-monitis agent cannot be demonstrated until rela-tively late in the growth cycle, after extensivemultiplication of the agent has occurred. The tech-niques used in the present investigation allow thesynthesis of new macromolecules in the meningo-pneumonitis agent to be detected much earlier,but they in turn suffer from the disadvantages ofmeasuring only relative rates of synthesis, not ab-solute amounts, and of failing to distinguish be-tween net synthesis and turnover. However, in thediscussion to follow it will be assumed that incor-poration of isotopic labels was the result of netsynthesis and that the observed relative rates ofincorporation are valid measures of the rates ofsynthesis of the macromolecules of uninfected andinfected L cells.

In a previous study of meningopneumonitisagent growth in uncloned L cells, RNA and DNAsynthesis was first observed 10 to 15 hr after infec-

tion; in this investigation on the same agent grow-ing in a cloned L cell line, nucleic acid synthesiswas first seen 15 to 20 hr after infection. Moore(18), working with the same host-parasite systememployed here, and Crocker et al. (3), using anornithosis agent in HeLa cells, made similar ob-servations by means of autoradiography. On thebasis of thin-section electron microscopy, Higashi(10) concluded that members of the psittacosisgroup begin to multiply by binary fission no laterthan 10 hr after infection of their host cells. Thisis the time when intracellular infectivity began torise in L cell suspensions infected with the menin-gopneumonitis agent (Fig. 1). Podolyan et al. (25)used phase-contrast microcinematography to es-tablish the generation time of psittacosis groupagents as 2 to 3 hr. Thus, when DNA synthesis inthe meningopneumonitis agent was first unmis-takably evident, approximately 15 hr after infec-tion, each invading meningopneumonitis cell hadprobably undergone only two or three divisions.As measured by increase in the intracellular in-fectivity titer, the meningopneumonitis agent con-tinued to multiply until 40 hr after infection.Synthesis of DNA, RNA, protein, and phospho-lipid was maintained throughout this period, al-though the rates of incorporation of labeled pre-cursors decreased in the later labeling periods. Theautoradiographic studies of Crocker et al. (3) andMoore (18) also showed that the DNA of theornithosis and meningopneumonitis agents wassynthesized throughout the growth cycle.The nearly simultaneous appearance of new

intracellular infectivity and of newly synthesizedmeningopneumonitis DNA, RNA, protein, andphospholipid, the ensuing period of logarithmicincrease in macromolecules, and their sustainedsynthesis throughout the growth cycle are all con-sistent with the concept of multiplication by bi-nary fission. Growth of the meningopneumonitisagent in the L cell may be analogized to thebacterial growth cycle. The lag phase is the timerequired for reorganization of the invading smallcells into the large intracellular ones; the logarith-mic phase is the period during which the large cellsundergo binary fission at a constant rate; the de-creased logarithmic phase is the time when theconcentration of agent precursors in the L cell be-comes limiting, and the large cells divide at de-creased rates and begin to reorganize into smallcells; and the stationary phase coincides with thetermination of the growth cycle when the L cellcan no longer support meningopneumonitis mul-tiplication, and mature small cells are releasedinto the medium.

Because 5'-fluorodeoxyuridine (34), aminop-terin (26), and 5'-fluorodeoxycytidine (29) in-

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hibit multiplication of psittacosis group agentswhen added to infected cells early in the growthcycle but are unable to do so when added laterthan 15 to 20 hr after infection, it was concludedthat most of the DNA of these organisms is madeearly in the growth cycle and that there is a lag be-tween synthesis of DNA and appearance of newinfectious units. The results of these investiga-tions, together with those of Crocker et al. (3) andMoore (18), show that this interpretation cannotbe correct. DNA synthesis in psittacosis groupagents continues throughout the growth cycle andis most rapid at a time when their multiplicationis no longer affected by these drugs. Another ex-planation must be sought for this interestingchange in drug susceptibility during the course ofthe growth cycle. Such a change is not limited toinhibitors of DNA synthesis. Meningopneumoni-tis multiplication in L cells is first susceptible andthen resistant to inhibition by actinomycin D,which prevents DNA-directed RNA synthesis(33), and psittacosis multiplication in McCoy cellsis similarly affected by p-fluorophenylalanine, aninhibitor of protein synthesis (26). It is possiblethat all these observations have a common ex-planation.

Significance ofnonsedimentable forms ofmenin-gopneumonitis DNA and RNA in the cytoplasmicfractions of infected L cells. The experiment withH3-thymidine, which labels L cell DNA but notmeningopneumonitis DNA (24), conclusivelydemonstrated that the nonsedimentable DNAwas made by the meningopneumonitis agent andnot by the L cell. No such specific label was availa-ble for either L cell or meningopneumonitis RNA,and so the origin of the extra cytoplasmic RNAcould not be settled with certainty. However, ac-cording to Tamura and Iwanaga (33), the RNAmade in L cells 10 to 20 hr after infection with themeningopneumonitis agent was localized in theagent inclusions and had the base composition ofagent RNA. Therefore, it is highly probable thatthe extra cytoplasmic RNA was also made by themeningopneumonitis agent.These nonsedimentable nucleic acids of men-

ingopneumonitis origin could have come fromthe destruction of intact meningopneumonitis cellsduring L cell fractionation, or they could haveoriginated by a viruslike reproductive mechanismand never have been a part of the intact men-ingopneumonitis cell. The experiments in whichcrude preparations of P32-labeled meningopneu-monitis agent were added to unlabeled L cells andthe mixtures fractionated as usual favor the firstpossibility but do not rule out the second. How-ever, in light of present knowledge of the natureof microorganisms of the psittacosis group, the

possibility of the meningopneumonitis agent re-producing in the form of its naked nucleic acidsseems unlikely.The ratio of RNA to DNA is 1 :1 in prepara-

tions of small mature meningopneumonitis cells(32) and 4:1 in large immature cells (31). Table 3compares the RNA-DNA ratio in cytoplasmicsediments and supernatant fractions of infected Lcells. These ratios suggest that the meningopneu-monitis agents isolated in cytoplasmic sedimentsrepresented mainly mature or nearly mature cells,whereas the nonsedimentable nucleic acids of thecytoplasmic supernatant fractions represented theyield from destruction of large immature cellsduring the isolation procedure. If this is true, thenthe sum of the incorporation of labeled precursorinto the sedimentable and nonsedimentable frac-tions is an estimate of the true extent of synthesisof meningopneumonitis macromolecules. How-ever, changes in the rate of this combined incor-poration closely paralleled changes in the rate ofincorporation into the cytoplasmic sedimentalone, and its use as a measure of the rate of syn-thesis of meningopneumonitis macromoleculeswould not change significantly any of the inter-pretations of the relation of macromolecular syn-thesis to the agent's growth cycle offered in theproceeding section.

Effect ofmeningopneumonitis infection on macro-molecular synthesis in the L cell host. Table 6 com-pares the effect of meningopneumonitis infectionon incorporation of labeled precursors into theRNA and DNA of the nuclei of uncloned andcloned L cells. The data on uncloned cells wereobtained with a P32-orthophosphate label (28),but more recent use of a H3-cytidine label hasyielded identical results. The effect of infection onnucleic acid synthesis in the two closely related Lcell lines differed greatly. DNA synthesis in theuncloned cells was almost completely stopped 10to 15 hr after infection, but was never reduced bymore than 56% in the cloned cells. RNA synthesiswas similarly more severely inhibited in the un-cloned cells. These differences are in accord withthe observation that meningopneumonitis multi-plication is less rapidly lethal and is longer sus-tained in the cloned L cell line.

Thus, the profound inhibition of nuclear DNAsynthesis and rapid death of the host cell, firstfound in the uncloned L cells by Schechter,Tribby, and Moulder (28), has turned out to be anextreme case of the effect of infection on the hostcell. I. I. E. Tribby (personal communication) hasisolated from the original L cell line received inthis laboratory several clones differing in sus-ceptibility to infection with the meningopneu-monitis agent, generation time, and pattern of

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MENINGOPNEUMONITIS NUCLEIC ACID SYNTHESIS

TABLE 6. Inhibition of synthesis of DNA and RNAin nuclear fractions of L cells infected with

the meningopneumonitis agent

Inhibition of uncloned Inhibition of clone 5bcells, P32-orthophosphate cells, H3-cytidine as

Period of as label* labellabeling

DNA RNA Dead DNA RNA Deadcells cells

hr % % % % % %5-10 14 0 6 0 6 4

10-15 93 32 10 35 3 515-20 93 16 16 15 4 720-25 89 76 30 56 33 1525-30 99 45 52 52 42 2430-35 53 53 43

* Data from Schechter, Tribby, and Moulder(28).

growth in suspension cultures. DNA synthesis inone of these clones is as severely inhibited bymeningopneumonitis infection as in the unclonedpopulation. Apparently, the dominant cell type atthe time the uncloned L cells were studied was onewhose nucleic acid synthesis was highly suscepti-ble to inhibition by meningopneumonitis growth.

In their autoradiographic studies, Crocker et al.(3) noted a moderate inhibition ofDNA synthesisin HeLa cells infected with an ornithosis agent.By use of the same technique, a partial inhibitionof DNA synthesis in cloned 5b L cells infectedwith the meningopneumonitis agent has also beenobserved in this laboratory (18). Crocker et al.(3) gave two possible explanations for this inhibi-tion: (i) production during cytoplasmic growth ofthe psittacosis agent of a factor that blocks DNAsynthesis in the host cell nucleus, and (ii) a com-petition between host cell nucleus and psittacosisagent for cytoplasmic energy sources and precur-sors needed for DNA synthesis. They favor thelatter explanation and suggest that the competi-tion between host and parasite is for nucleosidetriphosphates, which Moulder (20) postulated asbeing required for nucleic acid synthesis in psitta-cosis group agents. However, recent work in thislaboratory (I. I. E. Tribby and J. W. Moulder,personal communication) suggests that the menin-gopneumonitis agent can utilize certain host cellnucleosides for DNA synthesis but that it cannotuse the host nucleotides for this purpose. There-fore, the postulated competition may be for nu-cleoside precursors or for the energy required tophosphorylate them to the nucleoside triphos-phates.

In clone 5b L cells infected with the meningo-pneumonitis agent, it may be calculated that thesum of the host and agent DNA and RNA syn-

thesized in any 5-hr labeling period was equal tothat made in uninfected cells. This suggests that,in this system, resources for nucleic acid synthesiswere limiting and that the second explanation ofCrocker et al. was correct. However, it is unlikelythat a simple competition forDNA precursors canexplain the profound early inhibition of DNAsynthesis observed in uncloned L cells by Schech-ter, Tribby, and Moulder (68). The amount ofmeningopneumonitis agent present 10 to 15 hrafter infection was much too small for it to robthe host nucleus ofDNA precursors. In addition,the total DNA synthesized by the infected un-cloned cells fell far below that made by the unin-fected cells, indicating that there was no shortageof synthetic resources. Here, the first explanationof Crocker et al. seems more plausible, and theunlikely possibility that the meningopneumonitisagent synthesizes an inhibitory histone such as theone produced in cells infected with ME virus (12)cannot be completely disregarded. It may be signi-ficant that the meningopneumonitis agent con-tains the basic amino acids lysine, arginine, andhistidine in nonprotein peptide form (23).The absence of any detectable inhibition of pro-

tein synthesis in infected cells at any time duringthe meningopneumonitis growth cycle suggeststhat the observed inhibitions of nucleic acid syn-thesis were relatively specific and not due to gen-eralized depression of host cell metabolism.

ACKNOWLEDGMENTS

This investigation was supported by Public HealthService grant AI-O1594 from the National Institute ofAllergy and Infectious Diseases, and by grants fromthe Dr. Wallace C. and Clara A. Abbott MemorialFund of The University of Chicago and from AbbottLaboratories, North Chicago, Ill. The author was apredoctoral fellow of the Upjohn Co., 1960-1963, anda predoctoral trainee of the U.S. Public Health Service(graduate traininggrant 1 Tl AI-238 fromthe NationalInstitute of Allergy and Infectious Diseases), 1963-1965.

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