Proc. Nat. Acad. Sci. USAVol. 72, No. 1, pp. 182-186, January 1975

Interfel-on Action 1I. Membrane-Bound Alkaline Ribonuclease Activityin Chick Embryo Cells Manifesting Interferon-Mediated Interference*

(viral interference/vesicular stomatitis virus)

PHILIP I. MARCUSt, THOMAS M. TERRYt, AND SEYMOUR LEVINET

t Microbiology Section, U-44, University of Connecticut, Storrs, Conn. 06268; and $ Department of Immunology and Microbiology,Wayne State University School of Medicine, Detroit, Mich. 48201

Communicated by Theodore T. Puck, October 21, 1974

ABSTRACT Membrane fractions from thick embryocells manifesting,vifai interference mediated by interferonor poly(I). poly(C) contain high levels. of an alkaline ribo-nuclease. Enhanced RN se activity is not observed wheninhibitors of cell protein or RNA synthesis are presentduring interferon treatment, or when heterologous inter-reron is used. The RNase associated with comparable mem-brane fractions from cells treated with mock-interferonis about 1/10 as active, and shows qualitative differences.In principle, divergent views of interferon action may bereconciled to a common mode of action by Postulatingthat viral interference results from a newly induced oractivated RNase of cellular origin and proper specificitythat acts to reduce the accumulation and functionalcapacity of newly synthesized viral RNAs, particularlyniRNA. Previous data in support of interferon's acting toinhibit virion-derived transcription in vivo are now in-terpretedas demonstrating enhanced degradation of viraltranscripts (mRNA).

Interferon-mediated interference with viral replication hasbeen reported to act at the level of translation (1-5) andtranscription (6-11). These seemingly divergent views ofinterferon action may be reconciled to a single mechanism bypostulating that a cellular ribonuclease with proper specificityacts to reduce the intracellular accumulation of newly syn-thesized viral mRNA, or to alter its capacity for translation(6, 12, 13). Modulation of such an RNase by cellular (14-16)or viral inhibitors, or shifts in the location or orientation of theenzyhie in relation to the sites of transcription or translationmight provide still a higher order of restriction on the ac-cumulation or function of viral RNAs, and perhaps some kindsof cellular RNA critical for viral mRNA translation (17),thereby achieving precise control over messenger expression.This communication presents preliminary evidence to supportsuch a concept by demonstrating that a membrane-associatedalkaline RNase appears in chick embryo cells that manifestviral interference induced either by homologous interferon orby poly(I) * poly(C).

MATERIALS AND METHODS

Cells and Virus. Primary chick embryo cell monolayersaged in vitro for 5-7 days as described previously were usedthroughout this study (18). These cells produce high levels ofinterferon upon induction, and are more responsive to theaction of interferon (18). Stock cultures of vesicular stomatitis

virus (VSV)-HR, Indiana serotype, were prepared as re-ported previously. (19).

Interferon and Interferon-Mediated Viral Interference.Chick interferon was prepared as has been reported (20), andproduced no adverse effects on cell macromolecule synthesisor growth. Mock-interferon preparations represented growthmedium from plates of chick cells that were not exposed to in-ducers of interferon but otherwise weke treated identically.All preparations produced comparable results when equatedto interferon activity as measured by a 50% reduction assayof VSV plaque-forming particles [PRso (VSV) uhits]. Viralresistance was induced by interferon preparations over a 24-hr period at 370, at which time mock-treated cells also wereprocessed. Mouse interferob (50,000 U/ml) was obtained byinfecting mouse L-cells with ultraviolet-irradiated Newcastledisease virus, and processing through the zinc acetate stage ofpurification (20). No reduction in the yield of VSV plaque-forming particles was observed on chick embryo cell mono-layers treated with 5000 PR50 (VSV) units/ml of mouse cell-derived interferon. Viral interference induced by poly(I)-poly(C) (P-L Biochemicals) was accomplished by exposingaged chick cell monolayers for 24 hr at 370 to fresh mediumthat contained DEAE-dextran (10 Mug/ml) and various con-centrations of the polyribohucleotide. All experiments in-cluded bioassays for viral interference.

Membrane Fractionation. After treatment with interferon,mock-interferon, or poly(I) * poly(C), cell monolayers were re-moved virtually intact by incubating for about 5 min at 370in saline D (21) containing 5 mM EDTA. The sheets of cellswere chilled to 40, washed twice with cold saline 1), swelled inRSB-K (22), and Dounce homogenized to disrupt the cells.Subsequent steps to secure membrane fractions followed theprocedure of Caliguiri and Tamm (22). Visible membranebands were collected from the 25 to 30% sucrose interface(fraction 3) and from the 30 to 40% sucrose interface (frac-tion 4), diluted with RSB-K buffer and collected by centrif-ugation at 40,000 X g for 4 hr and resuspension in RSB-K.

VSV In Vitro Transcription. Preparation of transcribingnucleoprotein (TNP) from VSV followed the procedure ofSzil~gyi and Urvayev (23). Transcriptase activity was alsoassayed by their procedure. Reaction mixtures (50 ul) con-tained: 5 ,.d of TNP; 3.5 mM dithiothreitol; 20 mM Tris- HC1(pH 8.0); 0.1 M NaCl; actinomycin D (0.8 ,ug); 0.64 mM eachof ATP, CTP, and GTP; 6.4 MAM UTP and about 25,000 cpmof [5'-3H]UTP (specific activity 14 Ci/mmol, Schwarz/

182

Abbreviations: VSV; vesicular stomatitis virus; PR,o, 50% reduc-tion in viral plaques; TNP, transcribing nuclteoprotein.* Paper I in this series is ref. 6.

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Interferon Action and Membrane-Ribonuclease 183

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FIG. 1. Effect of fraction 4 membranes from variously treatedchick embryo cells on the accumulation of acid-precipitable viralRNA in the standard reaction mixture for VSV TNP transcrip-tion in vitro. "cpm" represent product labeled with [3H]UTPas precursor, and are cumulative to the time of sampling (ab-scissa). All reaction mixtures except the control (TNP) receivedmembrane protein at a final concentration of 6 g/i100 Al, fromcells previously treated as noted in the text. Interferon treatmentproduced a 104-fold decrease in virus yield and full protectionagainst cell killing (19).

Mann), and included up to 15 Al of membrane fractions inRSB-K (<10 ,ug of protein). The reaction was started byadding 1 Mul of 440mM MgCl2 at 310.

Ribonuclease Assay. VSV RNA was transcribed from TNPtemplates for 90 min, using the reaction mixture describedabove, without added membrane. After heating at 500 for 5min to inactivate transcriptase (24, 26), the mixture waschilled and used to test VSV-specific RNA product as a sub-strate for RNase. Reaction mixtures (80 MAl) for the RNaseassay contained: 0.5 M Tris HCl (pH 8.0); [3H]RNA, gen-erated as TNP product (5 ul); and membrane fractions(< 15M1A, representing < 10jOg of protein) in RSB-K. The assayswere carried out at 310 by adding membrane fractions to-theheated reaction mixture and testing for the loss of acid-pre-cipitable viral [3H]RNA as a function of time.

RESULTS

Effect of hiembrane Fractions from Interferon-Treated Cellson VSV Transcript Accumulation In Vitro. Our basic observa-tion is illustrated in Fig. 1, and demonstrates that membranesfrom interferon-treated cells adversely affect the in vitro ac-cumulation of VSV mRNA. The uppermost curve representsradioactivity in acid-insoluble VSV RNA synthesized as pri-mary transcripts from the transcribing ribohiucleoproteincomplex (TNP) reaction mixture as described by Szildgyi andUrvayev (23). No cytoplasmic membranes were added to thisreaction mixture. The curve labeled mock was generated byadding fraction4 membranes from mock-treated cells to theregular TNP reaction mixture at time zero. The curve illus-trated is typical although the mock and TNP curves fre-quently were superposable. Occasionally, the mock mem-branes slightly enhanced the apparent rate of transcription.Cells harvested without benefit of a mock regimen producedmembrane fractions that behaved like mock membranes.Membranes obtained from cells exposed to cycloheximideduring mock treatment behaved like mock membranes (com-pare mock and mock + cycloheximide) in that the rate of ac-

3H / - ~ ~ °- -4

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FIG. 2. Effect of interferon concentration on the activity offraction 4 membranes in the VSV TNP in vitro transcriptionreaction. Conditions were as described in the text. Membrane pro-tein (6 lug/100 Al) added to the in vitro reaction mixture was fromchick embryo cell monolayers previously treated with 2000, 200,20, or 2 PRjo (VSV) units/ml of chick interferon, or mock treated.Yields of VSV plaque-forming particles from these monolayerswere 4 X 104, 6 X 104, 10 X 104, 3 X 107, and 1 X 108/ml, re-spectively. The three highest doses of interferon protected againstcell killing (19). 3H cpm as in Fig. 1 legend.

cumulation of VSV RNA was indistinguishable from that incontrol preparations (TNP) for the first 90 min of incubation,then slowed, approached zero, and, finally, RNA already in anacid-insoluble form was slowly solubilized. Half-lives for thedecline varied from 5 to 10 hr in different experiments. Thebottom curve (interferon) indicates that adding membranefraction-4 (or fraction-3) from cells treated with interferon[500 PR50 (VSV) units/ml in this experiment] to the in vitrotranscribing mixture initially caused a marked decrease in therate of acid-insoluble RNA accumulation, a rapid leveling offwithin the first hour, and a subsequent slow decline in acid-precipitable RNA over the ensuing 4 hr.

Prevention of Interferon-Induced Membrane Alteration by theInhibition of Cellular Protein or RNA Synthesis. Character-istically, viral interference is not induced in cells treated withinterferon if inhibitors of cellular protein or RNA synthesis arepresent during the induction period (1). The curve labeledinterferon + cycloheximide in Fig. 1 indicates that the pres-ence of this inhibitor of protein synthesis during interferontreatment prevented the effect labeled interferon in Fig. 1.Since actinomycin D produced the same result, it appearsthat cellular synthesis of both protein and RNA is required toalter the membranes of interferon-treated cells so that theyact to reduce the accumulation of VSV transcripts in vitro.

Dose-Dependent Eyect of Interferon on Membrane FractionsAffecting VSV Transcription In Vitro. Characteristically,interferon reduces virus yield in a dose-dependent manner,with the effectiveness of increasing doses decreasing at highconcentrations (25). Such an effect is recorded in Fig. 2 formembrane fraction-4 from cells treated with interferon over a1000-fold range of concentrations. Membranes from cellstreated with high doses of interferon [>200 PR5o (VSV)units] initially produce a measurable decline in the rate ofVSV RNA accumulation, and an earlier plateauing and sol-ubilization of previously synthesized RNA, compared to allcontrols. For lower doses the effect is less. The curves gen-

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FIG. 3. Effect of poly(I)-poly(C)-induced interference on theactivity of fraction 3 membranes in the VSV TNP in vitro tran-scription reaction. Membrane protein (6 ,Ag/100 Ml) added to thein vitro reaction mixture was from cells previously treated withDEAE-dextran (10 ,g/ml) and poly(I) - poly(C) at concentra-tions of 2, 0.2, or 0.02 ,g/ml. All three doses of inducer producedbackground levels of virus upon challenge with VSV, and pro-tected against cell killing (19). Controls consisted of membranesfrom mock-treated cells and poly(I) - poly(C)-treated cells ex-posed simultaneously to cycloheximide (50 ,sg/ml). 3H cpm as inFig. 1 legend.

erated in Figs. 1-3 represent cumulative radioactivity inRNA.

Homologous Interferon Is Required to Produce MembraneFractions Effective Against Transcript Accumulation In Vitro.Chick-cell derived interferon was used to induce the mem-brane changes responsible for the effects on in vitro transcrip-tion recorded in Figs. 1 and 2. In contrast, membrane frac-tions prepared from chick embryo cells treated with 5000PR5o (VSV) units of mouse interferon derived from L cellsbehaved like membranes from cells treated with mock inter-

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FIG. 4. Production of VSV mRNA as substrate through heatinactivation (500, 5 min) of virion transcriptase. The time course

of VSV TNP RNA synthesis was followed at 310 by measuringthe incorporation of 3H ([3H1UTP as precursor) into acid-in-soluble material in the standard reaction mixture (0). After 2 hrthe mixture was held at 500 for 5 min, cooled, and returned to 310for subsequent measurements of acid-insoluble product (0).The stable product represents single-stranded primary tran-scripts of VSV, i.e., mRNA (23, 32).

feron, thus defining a requirement for homologous interferonto produce the effects recorded in Figs. 1 and 2.

Perhaps the chick-derived interferon preparation itself con-tained a nonspecific factor with a high affinity for membranes.To test this possibility we prepared membrane fractions fromthe Vero line of green monkey kidney cells treated with 5000PR5o (VSV) units/ml of chick interferon. None of these frac-tions reduced in vitro transcription by VSV TNP.

Effect of Membrane Fractions from Poly(I) -Poly(C)-treatedCells on In Vitro Transcription. Cell monolayers were exposedto poly(I) -poly(C) in the presence of DEAE-dextran (10 ,ug/ml) to induce high levels of interference with VSV replication.Fig. 3 demonstrates that all three concentrations of poly(I)-poly(C) used in this experiment (0.02, 0.2, and 2.0 ,Ag/ml)produced membrane fractions that were highly active in pre-venting the accumulation of VSV transcripts. Diluting poly-(I) * poly(C) beyond 0.02 ug/ml revealed a dose-responseeffect similar to that illustrated in Fig. 2 for interferon (datanot shown). When cycloheximide was added to cells simul-taneously with poly(J) - poly(C) and DEAE-dextran, themembrane fractions were inactive as tested in the in vitrotranscribing system and behaved much like the preparationsfrom mock (not shown) or mock + cycloheximide-treated cells(Fig. 3).

Inactivation of Transcriptase and Stabilization of VSV RNAby Heat Treatment (500, 5 min). Adding active membranesfrom cells treated with interferon or poly(J) -poly(C) to thereaction mixture after the in vitro transcription product hadaccumulated for 1 or 2 hr produced a relatively rapid loss ofpreviously synthesized VSV RNA, suggesting the presence ofan active RNase. Curves generated by this experimentalmanipulation represent the vector resulting from at least tworeactions, the synthetic process of transcription, and the con-comitant or subsequent degradation of its product. To sim-plify analysis, we generated isotopically labeled VSV tran-scripts over a linear portion of the reaction, usually for 90min, and then stopped further transcription by inactivatingthe heat-labile virion transcriptase by subjecting the reactionmixture to 500 for 5 min (24, 26). Results typical of this treat-ment, shown in Fig. 4, demonstrate that heating stops tran-scription immediately, and that the resultant transcripts re-main stable. Preparations of heat-treated VSV RNA werestable after freezing and storage at -70°. All subsequent ex-periments were performed with these 8H-labeled VSV tran-scripts as substrate.Enhanced Ribonuclease-Like Activity Associated with Mem-

branes from Cells Treated with Interferon or Poly(I) * Poly(C).Membrane fractions 3 or 4 from cells treated with interferon,interferon + actinomycin D, or mock-interferon were ad-justed to equal concentrations of protein, and measuredamounts were added to a heat-stabilized reaction mixturecontaining 2500 cpm of 3H-labeled VSV primary transcripts.Samples were removed at different times and assayed foracid-insoluble radioactive material. Fig. 5a represents a typ-ical assay. All the membrane preparations produced a loss ofacid-insoluble VSV RNA at linear rates, indicating that theyhad some RNase-like activity. However, membrane fractionsfrom cells treated with interferon solubilized the viral RNA ata significantly faster rate than that produced by membranesfrom the various control cells. In the experiment illustrated inFig. 5a, 6 ug of membranes from interferon-treated cells per

VSV TNP

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Interferon Action and Membrane-Ribonuclease 185

100 M1 of reaction mixture solubilized 50% of the VSV RNAin 16 mim at 310, whereas an equivalent amount of membranefrom cells treated with mock interferon required 76 min, orabout five times longer. A similar experiment with mem-branes from poly(J) -poly(C)-treated cells (Fig. 5b) revealed asolubilization half-time of 7.5 min, with mock-treated mem-branes requiring 66 min to reach that same level, i.e., ninetimes longer. As shown in Fig. 5, treatment with actinomycinD or cycloheximide during induction with either interferon orthe polyribonucleotide reduced the RNase-like activity to lessthan that of the mock preparations. In a series of such assays,membrane fractions 3 or 4 from cells showing high levels ofviral interference consistently solubilized VSV RNA at 5 to20 times the rate of comparable mock preparations. Mixingmock- and interferon-membranes had no adverse effect on thenuclease activity of the latter.

Preliminary Characterization of the Ribonuclease ActivityAssociated with Membranes from Interferon or Poly(I) - Poly(C)-Treated Cells. Detailed characterization of the membrane-associated RNase is not complete; however, we can report thatthe nuclease activity from cells showing viral interference isoptimal at about pH 8.2 and 600, and requires Mg++. WhileEDTA at 3 mM, in the presence of 0.75 mM MgCI2, inhibitsthe activity completely (it can be recovered upon addition ofexcess Mg++), the chelating agent has little effect on anequivalent activity of crystalline bovine pancreatic RNaseassayed under similar conditions. The nuclease activity asso-ciated with 1 ,ug of membrane protein per 100 Ml of assay mix-ture at 310 is equivalent to 0.01 Mg/100 Mil of pancreaticRNase, and is derived from about 106 chick embryo cells. Freesulfhydryl groups may not be required for the activity of themembrane-bound RNase, since it is not affected by 1 mM p-chloromercuribenzene sulfonic acid. Sonication, or the addi-tion of the neutral detergent Triton N-101 (0.1%) had nodeleterious effect, nor did it enhance the nuclease activity, butit was inactivated at 700 after 10 min.We also observed that heat-stable (700, 5 min), activity in

standard and commercial (Searle Co.) preparations of an alka-line RNase inhibitor from mammalian cells (14-16) inactivatesessentially all the nuclease activity of interferon- or poly(I).poly(C)-membranes while leaving unaffected about one-halfof the activity on mock-membranes. For example, 1 unit ofnuclease inhibitor (14) added to 1 ug of interferon- or mock-membrane protein per 100 MuI of assay mixture produced a 24-and 1.6-fold reduction, respectively, in the rate of viral RNAsolubilization. The RNase activity can be eluted and recov-ered from membranes by exposing them to 1 M KCl for 30min. We have not yet tested the membrane preparations forDNase activity, or on other RNAs as substrates.

DISCUSSIONOur data demonstrate that the induction of interferon-medi-ated interference in chick embryo cells by interferon orpoly(I) -poly(C) is accompanied by a marked increase in theactivity of a membrane-associated alkaline RNase. ThisRNase is active against the RNA transcripts synthesized invitro from the genome strand of VSV by virion-bound tran-scriptase, is expressed at 5 to 20 times the ribonuclease ac-tivity per weight of protein of comparable fractions from cellstreated with mock interferon, and is qualitatively differentfrom that nuclease activity. The nuclease associated with

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FIG. 5. Degradation of VSV primary transcripts (mRNA) bymembrane-bound ribonuclease from cells manifesting viral inter-ference induced by chick interferon (a) or poly(I)-poly(C) (b).Membrane protein (6 ,g/100 ,1 of reaction mixture) was derivedfrom cells treated with interferon (500 U/ml), poly(I)-poly(C)(0.2 Mg/ml), or mock treated in the presence or absence of actino-mycin D (ACD) (1 Mg/ml) (a), or cycloheximide (50 ,g/ml) (b).Each reaction mixture contained 2500 cpm of VSV mRNA pre-pared as shown in Fig. 4. Samples were removed as a function oftime after addition of membrane and assayed for acid-insolubleradioactivity.

interferon-membranes is totally sensitive to a heat-stable,Pronase-resistant inhibitory factor found in standard prepara-tions of an alkaline RNase inhibitor from mammalian cells,and has optimal activity at about pH 8.2, with a significantdecrease at pH 7.2. The comparable activity on membranesfrom the mock-treated cells declines little if at all from 8.2 to7.2, and is relatively resistant to this same inhibitory factor.Thus, cytoplasmic membranes, the cell organelles most likelyto become involved with viral synthesizing processes (22, 27),acquire a highly enhanced capacity to degrade mRNA. In-creased RNase activity in topologically propitious points inthe cell may profoundly affect viral replication, or, for thatmatter, cellular events reflecting nonantiviral effects of inter-feron (28).

Syntheses of cellular RNA and protein are required for theexpression of enhanced RNase activity, although it is not yetclear whether new molecules are synthesized or membranechanges resulting from interferon action provide new or morefavorable sites for binding preexisting or activated nuclease.

Degradation of viral nucleic acid by the release or activa-tion of cellular nucleases is usually attributed to lysosomalenzymes (29). Our demonstration of an alkaline RNase tendsto eliminate lysosomes and their acid nucleases as a source ofthis enzyme.The membrane-bound RNase may represent an endo-

nucleolytic activity similar to that present in highly purifiedmouse interferon (12). However, our preliminary studies sug-gest that, for chick cells, the membrane-bound nucleasefound upon interferon treatment and that found free in both

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186 Microbiology: Marcus et al.

interferon and mock-interferon preparations representdifferent molecular entities. For example, the heat-stablecomponent of the inhibitor from mammalian cells does notinhibit the free nuclease-in contrast to its action on themembrane-bound enzyme.Although definition of the exact relationship between the

interferon [or poly(I) -poly(C)] dose and membrane-boundRNase activity is incomplete, our preliminary results alreadyshow two effects characteristic of the interferon system: first,increasing the dose of interferon [or poly(I) -poly(C) ] resultsin an increase in response, i.e., in membrane-bound RNase ac-tivity, and second, high doses of either inducer tend to ap-proach saturation in their effect-producing almost equalamounts of membrane-RNase activity. These results arestrikingly similar to the dose-response curves reported earlierfor the effects of these two inducers of interferon action on theaccumulation in chick embryo cells of VSV RNA from virion-derived transcription (ref. 6; Figs. 3 and 4).The present data provide evidence that *a membrane-

bound alkaline RNase may constitute one facet of interferonaction. From this vantage, we can interpret more accuratelyour previous report of inhibition of virion-derived primarytranscription in chick embryo cells as reflecting an increase inthe rate of degradation of transcription product; Enhancednuclease activity associated with membranes might also ac-count for some recent results (30), which provide evidence forprimary transcription in interferon-treated cells-seeminglyin conflict with other reports (6, 8). Thus, selective nucleolyticaction might permit detection of primary transcripts throughmolecular hybridization (31, 11), the technique used, eventhough the transcripts were nonfunctional as mRNA. Con-tinued exposure of transcripts to nuclease action would leadeventually to a net decrease in the rate of viral mRNA ac-cumulation, as we and others have observed (6, 8, 11). Pre-sumably, the efficiency at which primary transcripts functionwould depend upon the extent to which the transcripts in-teract, during the initial stages of viral replication with cyto-membranes laden with proper nuclease.As we noted earlier (6), there is no a priori reason for sup-

posing that the interferon system consists of a single molecularspecies with a single mode of action. In fact, the myriad non-antiviral effects attributed to interferon treatment (28) castdoubt on such thinking. Nonetheless, it is tempting to con-sider that properly selective and situated RNase(s) could actto effect many of the intracellular changes attributed to inter-feron action, as astutely pointed out by Graziadei and co-workers (12, 13). Add to this the possibility of inducing in-hibitors of RNase action, and there emerges a finely tunedregulatory system, aimed at a key molecule in the syntheticcapacity of the virus, or cell-mRNA.

Certainly these experiments do not preclude translation ortranscription processes per se as sites of interferon action, butraise the possibility that increased levels of membrane-boundcellular RNase may control the extent to which viral mRNAreaches the translation system intact. In principle, then, viralmRNA, as both the product of transcription and the mes-

senger for translation, provides a common target for properlyselective and situated RNase molecules. Further experimen-tation is required to determine whether the membrane-boundalkaline RNase described here constitutes such a molecule.

This research was supported by USPHS Grant AI-09312 fromthe National Institute of Allergy and Infectious Diseases, andbenefited from the use of the Cell Culture Facility supported byGrant CA-14733 from the National Cancer Institute. We thankMargaret J. Sekellick for a critical reading and discussionof themanuscript, and John J. Papale for excellent assistance in somerecent experiments.

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