The in vitro synthesis of T2 bacteriophage-induced deoxycytidylate deaminase and its regulation by allosteric effectors

.I~CHIVI:S

OF

UfOCHKMIHTltY

The

.LND

in Vitro

163,

I~IOI’HYSICS

Synthesis

Deoxycytidylate

515-525

of T2 Bacteriophage-Induced Deaminase

and

by Allosteric

Division

of Laboratories

aud Research,

New

(1972)

Iyork

Received

Its Regulation

Eff ectors’

State

Department

June

20, 1972

of Health,

dlba~y,

Xew

1.ork

12601

Conditions are described for t,he it/ vitro synthesis of the T-even bacteriophageinduced deoxycytidylate deaminase using either T2, T4, or T6: phage I)NA or mlZNA as template. The deaminase appears after dihydrofolate reductase, but before thymidylate synthetase in this syst’em. The T-odd phage I)NA were not active in the synthesis of deox,vcytidylate deaminase, a fact consistent with the inability of these phages to induce deoxycytidylate deaminase on infection of Escherichia coli. Proof that the activity being measured was from the deaminase was obtained with antibody to this enzyme and t,hrough the use of a specific ilrhibit.c)r, tetrahydrodeoxyuridylate. Attempts to demonstrate the feedback regulation of the deoxycaytidylate deaminase svnt hesized i/h vitro were unsuccessful iuitially due to the presence of a phosphot,rausferase capable of converting deoxycytidylate to deoxycnytidine t riphosphate with thymidine triphosphate and other nucleoside triphosphates as donors. Removal of this activity, however, enabled a definitive pH dependence of activatiotl by deoxycytidine triphosphate to be shown. In addit,ion, other properties associated with the pure deaminase, such as the irrhibit,ion by thymidine triphosphate atId its reversal by deoxycytidine triphosphate, became evident.

In our initial studies on the induction of dCJIP deaminase (EC X5.4.12) in Bschericluia coli by T2 bact,criophage,2 the level of dcaminasc induwd I\-as found to vary inversely with tht> wll titer (1). More rccctnt s:tudics rcvealcd t)hat’ of six phagc-induced c~nzymcs measured (a), only the deaminase activit,y increased on dilution of highdensity cell cult’ures after phagc infcct’ion. Through the USC of inhibitors of transcription and translat8ion, the cause for the variability in deaminasc expression was placed at, the level of translation. To define more ckarly t’hose factors involved in rcgulatjing 1 This work was supported in part by (irant (;I%27598 from the National Science Foutldation and a Grant,-ill-Aid from the American Heart Association. 2 The T-even phages used in these experiments were the lysis-inhibiting (r+) variety. T&d1 phagc was a generorls gift of Dr. I. Tessman.

doaminasc: synthesis, a system capable of synthwizing dCi\Il’ deaminase ilz ~ittw wah dcveloprd. The utility of wll-fret prot,cinsynthesizing systems has been documented amply, as in the case of p-galactosidasc (3), phage yapsid prot#eins, and enzymes (4-lo), hemoglobin (11)) and mow rewntly, animal virus proteins (12-15). Howvcr, t)he adcquat.e in Gfro synthesis of an allosttvically regulated enzyme and the influence of its effecters on the activity of the final product have not brcn described. Whik thcsc studies wcw in progress, a report, on thn i/l. vitro synthesis of T4 phagc dCMP deaminase appcbarcd (16)) but the enzymtl activit’y was low and the allostcric cffcrts attributed to dCT1’ and dTTP, thcwfrw, wrc questionable. A rcdwming fcat,urc, howcvcr, was the apparent, absence of trmplate activit,?; nit)h DKA from or-

516

TRIMBLE,

MALEY,

ganisms unable to produce dCMP deaminase in tivo. While our att,empts at reproducing these results were unsuccessful, the use of a system similar to that described by DeVries and Zubay (17) with T2 phage DNA as template led to a several hundred-fold improvement in measurable deaminase activities above those reported (16). In addition, phage mRNA was found to direct deaminase synthesis. That the enzyme synthesized from the T2 phage DNA was deoxycytidylate deaminase was verified with antibody to highly purified T2 deaminase. Attempts to demonstrate feedback regulation by dCTP and dTTP, however, a characteristic property of the T-even phage-induced enzyme, led to inconclusive results. This finding suggested that newly synthesized dCMP deaminase may not be regulated by its allosteric effecters. Some of the reasons for this apparent anomaly have been uncovered now and will be discussed in this paper. A preliminary report on these studies has been presented (18). EXPERIMENTAL Phage preparation and conditions of injection. Escherichia coli B, maintained on nutrient agar slants, was used throughout this study. Bacteriophage T2, T4, and T6 were propagated in E. coli as described previously (1) with added cofactors as necessary. The T3, T5, and T7 were prepared by similar procedures. The E. coli were infected at a multiplicity of 5 (phage/bacterium) after growth to a density of 4 X lo8 cells/ml at 37°C in MS medium (19) containing 0.4% glucose. The infected cells were aerated vigorously on a rotary shaker (200 rpm) and at specific times the infection was terminated by pouring 300-ml aliquots of the culture over equal volumes of chipped ice. After centrifugation, the cells were resuspended at a concentration of 2.5 X lOr@/ml in a solution of 10 rnM Tris-HCl (pH 7.5), 10 mM sodium chloride, and 5 rnM magnesium chloride. A l-ml aliquot of each suspension was frozen at -56°C and assayed subsequently for enzyme activities. Enzyme assays. To determine phage-induced enzyme activities in vivo, the frozen cell suspensions were thawed and diluted with 1 ml of 10 mM Tris-HCl (pH 7.5) containing 80 pM dCTP as a stabilizing agent for dCMP deaminase (20). These suspensions were disrupted by a 1-min sonic treatment (Biosonik II microprobe at 90% full intensity) followed by centrifugation at 30,OOOg for 30 min. The supernatant fraction from this cen-

AND

MALEY

trifugation was assayed for the enzymes described below. S-100 fractions were prepared by centrifuging the E. coli sonicate at 31,000 rpm for 2 hr in an SW 39L Spinco centrifuge head and retaining the upper four-fifths of the supernatant fraction. Hsjolate reductase (EC 1.5.1.5). The spectrophotometric procedure of Mathews (21) was used, with the assay mixture containing 100 rnM KCI. Trimethoprim (a gift from the Burroughs Wellcome Co.) at 3.5 X lo-* M was included in the assay to suppress the level of the E. coli reductase. At the concentration of drug employed, the T2 phageinduced enzyme is not impaired (21,22). DTMP synthetase (EC 2.1.1 .b). This enzyme was assayed by a modification (23) of the procedure of Wahba and Friedkin (24). DCMP deaminase. The amount of enzyme present in the extracts from phage-infected cells was assayed spectrophotometrically (25). The enzyme synthesized in vitro was assayed according to the procedure of Schweiger and Gold (16) but with the following modifications. The residual ATP in a 25-~1 aliquot of the cell-free system was removed by addition of 5 ~1 of a solution containing 1.0 rmole of glucose, 0.4 unit of hexokinase (Sigma Chemical Co.), and 6.0rg of chloramphenicol. This mixture was incubated for 3 min at 37”C, followed by the addition of a substrate solution containing 25 nmoles [2-14C]dCMP (6 cpm/pmole) (SchwaraMann Bioresearch) and 0.13 pmole of dithiothreitol. After a 30-min incubation, the reaction was terminated with heat (100% for 2 min). Precipitated protein and nucleic acid were removed by centrifugation and duplicate 2+1 aliquots of the supernatant fraction were chromatographed on thin-layer sheets of polyethyleneimine cellulose (Brinkmann) with 0.2 N sodium formate (pH 3.2) as the developer (16). The region corresponding to dUMP, as determined with marker, was cut out and counted in a scintillation counter in 10 ml of Liquifluor (New England Nuclear Corp.). The counting efficiency was 667e. Specific activities are reported in millior punits/mg of protein, where a milliunit is equivalent to the conversion of a nmole of substrate to product in 1 min under the conditions of assay. In vitro protein synthesis with DNA as template. Preincubated dialyzed cell-free extracts (S-30 fractions) were prepared from uninfected E. coli B grown to 3 X lo8 cells/ml (17). Reaction mixtures for DNA-directed protein synthesis contained in 0.2 ml: 50 mM Tris-acetate (pH 8.0); 1.25 mM dithiothreitol; 11 mM magnesium acetate; 50 mM potassium acetate; 100 mM ammonium chloride; 5 mm calcium chloride; 0.2 mM of each of 20 amino acids; 2 mM ATP; 0.5 mM of each of CTP, UTP, and GTP; 20 rnM P-enolpyruvate; 0.2 rnM N-5-

DEOXYCYTIDYLATE

DEAMINASE

CHO-Hdfolate (folinic acid); 17 /Ig of phage DNA; and 1.35-1.65 mg of S-30 protein. In vitro protein synthek with mRNA as template. The reaction mixtures for RNA-directed protein synthesis were the same as those for the DNA-directed system, except for the concentrations of magnesium acetate, ammonium chloride, GTP, and P-enolpyruvate which were reduced to 9, 50, 0.2, and 5 mM, respectively; CaClz , CTP, and UTP were omitted. The DNA was replaced with 200 rg of RNA. For both the RNA and DNA systems, all components with the exception of the S-30 fraction and N-5-CHO-H4folate were mixed together and preincubated at 37°C for 3 min (17). Protein synt.hesis was initiated by the addition of a 50-11 mixture of S-30 and N-5-CHO-Hlfolate at the concentration presented above. At specified intervals, protein synthesis was terminated by pipetting samples into an ice-cold ATP trap (discussed above) containing chloramphenicol (200 pg/ml, final concentration). For determining the rate of protein synthesis in the cell-free protein-synthesizing system, L[4,5-3H]leucine (6 cpm/pmole), 0.2 mM final concentration, was included in parallel reactions to t*hose used to measure enzyme synthesis. At appropriate times, 25-~1 aliquots were removed and pipetted into 50 ~1 of ice-cold 7.5y0 trichloroacetic acid. The latter mixture was heated for 20 min at 90°C and then transferred in 5 ml of 5% trichloroacetic acid t.o 2.4-cm GF/A glass filter papers (Reeve Angel). The filters were washed with two lo-ml aliquots of 5’j; trichloroacetic acid followed by 15 ml of 95% ethanol. After drying, the filters were counted in 10 ml of Liquifluor at an efficiency of 207,. Partial purijication of dCMP deaminase synthesized in vitro. The deaminase synthesized in 10 ml of a proteiri-synthesis mixture was partially purified about, 5-fold by ammonium sulfate fractionation from 0.30-0.65 saturation, after centrifugation of the ribosomes at 100,OOOg for 2 hr. The ammonium sulfate precipitate was centrifuged and dissolved in 10 mM potassium phosphate (pH 7.5) and 20 mM 2-mercaptoethanol, and dialyzed against two l-liter changes of this solution. Nucleic acid extraction. RNA was isolated by a modification (7) of the hot phenol extraction procedure of Bolle et al. (26) and adjusted to a final concentration of 5 mg/ml based on a AzWonm for RNA at 1 mg/ml equal to 25. DNA was prepared from the T-even and t,he T-odd bacteriophages by the procedure of Thomas and Abelson (27). The final DNA concentration was 300 pg/ml. Both RNA and DNA were stored unfrozen in ice. Other methods. Protein was determined by t,he

SYNTHESIS

in

vitro

517

method of Lowry et al. (28). Hzfolate and Hdfolate were prepared as described earlier (23). RESULTS

Temporal order of expression of specific genesin viva. The genetic mapping experiments of Yeh et al. (29,30) indicated that the genes for Hzfolate reductase, dTMP synthetase, ribonucleotide reductase, and dCMP deaminase are closely linked, in the order indicated, on the T4 genetic map. Because of the proximity of t,hese genes to one another, it was anticipated that they might be expressed as a coordinate unit, either in the above temporal sequenceor in the reverse order. However, as indicated in Fig. 1, the kinetic order of expression was Hzfolate reductase, dCMP deaminase, and dTMP synthetase. Although these results were obtained aft,er infection with T2 phage, identical results were obtained with T4. The above findings are presented mainly to indicate the location of the deaminase relative to other closely linked phage genesand are discussedin more detail in a recent publication (31). Phage DNA as a template for dCMP deaminasesynthesis.While the system for the in vitro synthesis of dCMP deaminase as described by Schweiger and Gold (16) was only marginally active in our hands, one similar to that of DeVries and Zubay (17) provided far more satisfactory results. As indicated in Fig. 2, the enzyme appeared after a brief lag, although protein synthesis was initiated almost immediately. The level of enzyme made, in addition to protein synthesized, was dependent on the concentration of DNA template employed (Fig. 3). The appearance of enzyme was markedly blocked by inhibitors of RNA and protein synthesis (Table I) indicating a direct relationship between these processes.Evidence that the enzyme activity measured was actually due to dCMP deaminase was verified with specific inhibitors of this enzyme. Thus, as shown in Table II, antibody prepared against the purified T2 deaminase (32) inhibited the activity attributed to this enzyme in the in vitro protein-synthesizing system. Similarly, Hb-dUMP, a potent specific competitive inhibitor of dCMP deaminase (33)) effectively impaired the

518

TRIMBLE,

MALEY,

AND

MALEY

,

4

8

12

16

20

MINUTES FIG. 1. Early enzyme induction after T2 phage infection of E. coli at 30°C. A 2.5.liter culture of E. coli B was infected with T2 phage and, at the times indicated, 300.ml portions were poured over an equal volume of chipped ice. Extracts were prepared from the disrupted cells and assayed for HZ folate reductase (0), dCMP deaminase (o), and dTMP synthetase (A) activities, as described in Experimental. Maximum activities in milliunits/milligram of protein were: reductase, 37; deaminase, 170; and synthetase, 2.2. Background activities for reductase and synthetase (4.3 and 0.5 munits/mg of protein, respectively) present in uninfected cells were subt,racted from the corresponding act,ivities in infected cells.

of the newly synthesized in, vitro enzyme. Of additional significance was the inhibition effected by ethylenediamine tetraacetate, also an inhibitor of dCMP deaminase (20). The significantly diminished inhibition by ethylenediamine tetraacetate at 0°C is also characteristic of this enzyme. The apparent lack of inhibition by dTTP, however, is not. The specific requirement for T-even phage DNA as a template for enzyme synthesis Gevitro, presented in Table III, confirms the presence of the deaminase gene in T-even phage DNA. Consistent with these findings is the diminished activity associatedwith the DNA isolated from T4cd1, a phage mutant that induces about 30 7%of the deaminaseof the wild typt: T4 (Tcssman, personal communication).

FIG. 2. Kinetics of DNA-directed dCMP deaminase synthesis ilz vitro. Incubations for protein synthesis were expanded to 0.5 ml and contained 3.75 mg of S-30 protein and 37 kg of T2 DNA. At the times indicated, 25.~1 aliquots were withdrawn and pipetted into tubes on ice containing 5 ~1 of an ATP trapping mixture (see Experimental). The deaminase activity (0) and [3H]leucine incorporation into protein (0) were determined as described in Experimental. z

250

+z .-

activity

FIG. 3. T2 DNA saturation curve for the in vitro synthesis of dCMP deaminase and protein. Incubations for enzyme synthesis were as described in Experimental with each 0.2-ml reaction containing 1.5 mg of S-30 protein and T2 DNA as indicated. After 30 min of synthesis at 37”C, 25-~1 aliquots were removed and dCMP deaminase activit,y (0) and [3H]leucine incorporation (0) were measured as described in F,xperimental.

DEOXYCYTIDYLATE TABLE

1)EAMINASR

I

l';FFI,;W OF RNA .\NI) PIE.\MIN.\SE SYNTHESIS" ‘Template

None T2 l)NA

Inhibitor

None None Actinomycain Aurintricarboxylic acid Chloramphenicol Puromyrin

Leucine incorporation (nmolesi mg,l30 min)

1)

dCMP deaminase activity (punits/ w)

0.04 2.10 0.08

0 280 7

0.05 0.04 0.02

0 2 4

i/l

319

vitro

but consistent, requirement, for SIgZ+ in t)he DNA-directed system (11 mAI) as compared with the RNA-directed system (9 mhr) (Fig. 6). In addition, no lag was encountered in the synthesis of deaminasc wit’h R?;A as template (unpublished dat’a), which was not the case with DNA (Fig. 2). Comparison of Icigs. 3 and 5 rcvcals that mor(l enzyme is made p(‘r milligram of protein synthtsizcld with R-VA as tcmplatc than with DNA. The reason for this effect relates probably to the uw of R?;A isolated at a sptlcific time aftc>r phagcl infc&on, whereas a broadt>r spectrum of RKA is transcrib(>d from th(> DKA ,I/). vitro. The dcaminasct mc’ssagcl lvould roprthsclnt, therefore, a smaller p~~n’(~ntagc~ of th(x 11SA

a Incubations for l)NA-directed protein synthesis contained 1.35 mg of S-30 protein in each 0.2-ml reaction, and where indicated 17 /.~g of T2 DNA. Act,inomyc*in I), aurintricarboxylic arid, chloramphenicol, and puromycin were added before the initiation of protein synthesis to final concent.rations of 42 pg/ml, 200 fig/ml, 200 pg:‘ml, and 55 &ml, respectively. Aft,er 30 min of prot,ein synthesis at 37”C, 25-~1 aliquots were assayed for dCMP deaminase act,ivity. [3H]Leucine incorporation into protein was determined in parallel inrubations. (See li:xperimental for details.)

nlRNA as a template for dC3IP deaminase act&y. Figure 4 reveals that the RXA isolated at various times after infection of E. coli with T2 phage contained transcripts \vith information for dCMP deaminase synthesis. The enzyme-specific mRNA preceded, as expected, the appearance of intrac~ellular deaminase by l-2 min, peaked at about ;i min, and decayed with tIiz of about 3 min. Similar tljz’s for other phage-induced messengers have bec>n documented (8, 3436). In contrast to the rclativc instability of deaminase mRNA, the enzyme protein appeared to bc quite stable (Fig. 4). The deaminasc mRXA represents only a small fraction of tho extracted RNA used as templat,c, for, as indicated in Fig. 5, 200 pg of RXA were required for maximum enzyme synthesis in the ill. vitro protein-synthesizing system, while only ;i pg of DNA were rcquired (Fig. 3). Another difference between the two tcmplatcls was the slightly higher,

SYNTHESIS

TABLE

Experiment

I

II

Xdditions

None ITGdUMl’ (0.2 m.M) Antibody (1 mg ’ ml) Prebleed serum (2 mg/ml) Versene (2 mM)* dTTP (0.8 mM) None Versene (2 mM)

II

dCM1’ deaminase activit? &units/mg)

Inhibition t-T 1

272

0

17

94

13

95

240 18 270 425 354

12 93 0 0 17

a Each 50.~1 assay contained 0.5 crmole potassium phosphate (pH 7.5), 0.5 pmole 2.mercaptoethanol, 25 nmoles magnesium acetate, 25 nmoles [2-l%]dCMP (0.0 cpm/pmole), 0.27 mg of concentrated enzyme protein, and inhibitor as specified. The antibody and prebleed serum were inrubated wit,h enzyme for 10 min at 37°C before reactions were initiated by addition of substrate. After incubation for 30 min at, 37°C in likpt I and for 270 min at 6°C in Expt II, the reactions were terminated by heating at 100°C for 2 min. The dUMP formed was det,ermined by PEIcellulose c*hromat.ography as described in li;xperimental. * Abbreviation l’or pthylenediamirre tetraacetate.

TRIMBLE, TABLE In

Vitro DIRECTED

Template

None T2 DNA T3 DNA T4cdl DNA T4 DNA T5 DNA T6 DNA T7 DNA

0.04 1.80 1.20 2.06 2.20 1.50 1.92 1.30

AND

MALEY

III

SYNTHESIS OF dCMP BY DNA FROM THE T BACTERIOPHAGES Leucine incorporation (nmoles/mg/30 min)

MALEY,

DEAMINASIG CLASS OF

dCMP deaminase activity (punits/mg) 0 240 0 52b 260 0 220 0

= Protein synthesis incubations contained 1.65 mg of S-30 protein and 17 pg of the indicated phage DNA, except for T3 where 9 pg of template were used. After 30 min of protein synthesis at 37”C, 25-~1 aliquots of the reactions were assayed for dCMP deaminase activity. [3H]Leucine incorporation was measured in parallel incubations. (See Experimental for details.) 6 Wild-type T4 DNA yielded 195 runits/mg of deaminase/mg of protein in this experiment. The ratio of deaminase induced in viva by T4cdl to that induced by wild-type T4 was 30:112 munita/mg.

synthesized from the latter than present in the former. Substrate level required for maximum activity. One apparent reason for the relatively low deaminase levels obtained previously (16) became evident on determining the level of substrate required for maximum deaminaseactivity. The dCMP concentration used in the earlier studies was 80 PM, and as shown in Fig. 7, the deaminaxe is only about one-tenth as active at this concentration as at 5 mM. To verify that the newly synthesized deaminase possessedthe characteristic properties of an enzyme, the kinetics of deaminase activity versus time and versus protein concentration were determined. The reaction was linear for at least 60 min and was directly proportional to the amount of i?z vitro synthesis mixture added to each assay, over at least a IO-fold range. Phosphotransferase activity. A complicating side reaction was encountered during attempts to demonstrate the feedback regula-

2

5

8

II

14

17 -

o

MINUTES

FIG. 4. Synthesis of dCMP deaminase and dCMP deaminase mRNA after T2 phage infection. Two liters of E. coli B grown to 4 X lo* cells/ml at 37°C were infected with T2 phage and 300.ml portions of the culture were poured over ice at the times indicated. The chilled cells were collected by centrifugation and resuspended in 5 ml of a solution of 10 mM Tris-HCl (pH 7.5), 10 mM sodium chloride, and 5 mxu magnesium chloride. A portion of this suspension was processed for the determination of the phage-induced dCMP deaminase activity (o), while RNA was extracted from the remaining suspension, as described in Experimental. After 20 min of incubation at 37°C for RNA-directed cell-free synthesis, 25-~1 aliquots of the reactions were withdrawn for the determination of in vitro dCMP deaminase activity (0) with the standard 30-min assay (see Experimental for details).

i5

0

02

mg RNA

04

1 3;

m

FIG. 5. T2 RNA saturation curve for in vitro dCMP deaminase synthesis. Incubations for RNAdirected enzyme synthesis were as in Experimental, except that each 0.2-ml reaction mixture contained the indicated amount of T2 S-min RNA as template. After 20 min of incubation at 37”C, dCMP deaminase activity (O ) and [3H]leucine incorporation into protein (0) were determined as described in Experimental.

DEOXYCYTIDYLATE

6

12

DEAMINASE

18

mM Mg++ FIG. 6. Magnesium ion requirement for T2 DNAand RNA-directed in vitro dCMP deaminase synthesis. Incubations for enzyme synthesis were as described in Experimental, using 80 pg of T2 S-min RNA (0) or 17 pg of T2 DNA (0) as template. After incubation at 37°C for 20 min with RNA as template or 30 min with DNA as template, dCMP deaminase activity was assayed as described in Experimental. Mg2+ was added as magnesium acetate.

SYNTHESIS

in

vitro

521

known, but may be a consequence of the latter nucleotide’s ability to inhibit thymidine kinase (37). No effort was made to separate dUDP and dUTP from dCDP and dCTP as the dURD nucleotides are rapidly dephosphorylated relative to the dCyd nuclcot’ides. Additional proof for the potential involvemcnt of dTTP in dCTP formation was obtained through the uw of partially purified T2 dcaminaso from phage-infected cells (Fig. 8). The spcctrophotometric continuous recording assay shows clrarlp that while dCTP is necessary for the demonstration of deaminaseactivity (curves 1, 2, 3), preincubation of dTT1’ with dCAIP in the presence of the S-100 fraction (curve 4) accomplishes the same net effect. In the absence of the E. coli S-100 fraction (curve 6), no deamination was obtained, but when dTTP was added at the arrow to the cuvctte containing the S-100 protein (curve 5), a slow but’ steady increment in activity was observed. The latter finding could be explained by an accumulation of dCTP at the expense of dTTP, with the nctt result, being a reversal of the inhibition dur to the dTT1’.

6001

of dCMP deaminasc by dTTP. The results from such experiments were conGstently negative until it was found that dTTP when added to S-100 extracts was capable of transferring its terminal phosphate to other nucleotide acceptors, in particular dCMP. dTTP is not alone in this property for, as shown in Table IV, deoxyribonucleoside di- and triphosphates are formed from dCMP in the presence of other phosphate donors. The dUMP formed by the E. coli extracts results most probably from the following pathway:

tion

dCMP -+ dCyd -+ dUrd + dUMP as dCMP deaminaseis not present normally in E. coli. Evidence in support of this reaction sequence was provided by the finding that HI-dUMP did not impair the conversion of dCMP to dUMP, while HI-dUrd, an inhibitor of cytidine deaminase, did. The reason for the low level of dUMP present when dTTP was used as a donor is not

mM dCMP FIG. 7. Effect

of substrate concentration on the activity of dCMP deaminase synthesized in vitro. Conditions for DNA-directed protein synthesis were as described in Experimental using 1.5 mg of S-30 protein in the 0.2-ml reaction. After 30 min of incubation at 37”C, 25~~1 aliquots were assayed for dCMP deaminase activity as a function of substrate concentration (see Experimental for details).

522

TIIIMBLE, TABLE

EFFECT ON

V.\mous

OF THE

Ra~roMmvrT~

AFTER

AND

MALEY

IV PHOSPHITE

L)ISTHIIIU’I’ION

MALEY,

OF INCUILLTION

DONORS [2-“C]dCMP ~VITH

ON

E. Cob

B EX’~R.WIW Phosphate donor

None dTTP ATP GTP UTP CTP PEP

Distribution

of radioactivity

at 30 min

.dCMP (%)

dUMP (r/o)

dCDP (%)

dCTP (%I

75 34 25.3 41.4 39.7 65.9 41.5

0.6 1.3 18.6 14.6 11.9 2.6 9 7

0.9 30 25.C 19.3 21.3 12.4 21.2

0.2 12.6 12.6 4.9 4.3 0.4 4.3

a Each 50-hl reaction contained 0.1 /Imole Trisacetate (pH 7.8); 0.6 pmole potassium acetate; 0.14 pmole magnesium acetate; 0.1 pmole dithiothreitol; 25 nmoles [2-‘%]dCMP (6.0 cpm/ pmole); 140 rg of E. coli B S-100 protein; and 40 nmoles phosphat,e donor as indicated. After a 30-min incubation at 37”C, reactions were terminated by heating at 100°C for 2 min. The distribution of radioactivity was measured by chromatographing 2-~1 aliquots on PEI-cellulose thin-layer sheets. The separation of dUMP and dCMP was effert.ed with 0.2 N sodium formate (pH 3.2); dCDP and dCTP were separat,ed with 1.5 M sodium chloride. ln each case, the combined uracil-uridine content was determined to be about 18% of the total radioactivity by chromatography on Cel-300 DEAE thin-layer sheets (Brinkmann) with He0 as developer.

Evideme that the irk vitro-synthesized clear&use is subject to feedback regulation. Since data similar to those in Table IV and Fig. 8 indicated that contaminating nucleotides may have prevented the clear-cut demonstration of the feedback regulation of newly synthesized dCMP deaminase, the latter was separated from the contaminants by dialysis or partial purification. As shown in Fig. 9, the difficulty in demonstrating deaminase regulation could be obviated, at least in part, by 5-fold ammonium sulfate purification of the newly synthesized enzyme. Another lo-fold purification of this fraction could be effected by DEAE cellulose chromatography. The results obtained were not much different and the purified enzyme was highly unstable. Although the activation

0

IO

20

30

40

MINUTES Fro. 8. The effect of E. co/z’ B S-100 protein on the activity of partially purified, T2 bacteriophage induced dCMP deaminase in the presence of dCTP and dTTP. An aliquot of the phosphocellulose fraction of dCMP deaminase (0.8 unit) (32) was added to 1.0 ml of a solution con taining 5 mg bovine serum albumin, 10 pmoles Tris-HCl (pH 8.0), 5 rmoles dithiothreitol, and 2 pmoles magnesium chloride. Aliquots of 50 ~1 were assayed by the spect,rophotometric assay (25) wit,h the following additions: (1) 0.04 rnM dCTP at zero time; (2) 0.04 mrw dCTP at, arrow; (3) 0.4 mM dTTP at zero time, then 0.16 mix dCTP at arrow; (4) 0.4 mM dTTP + 0.18 mg S-100 protein preincubated for 30 min at 30°C followed by the dCMP deaminase at, zero time; (5) 0.18 mg S-100 protein at zero time, then 0.4 rnM dTTP at, arrow; (6) 0.4 mM dTTP at arrow.

of dCTP was not as dramatic as that with highly purified deaminase from phageinfected cells (32), the enzyme activity was significantly higher in the presence than in the absence of dCTP, and similar to the homogeneous enzyme (32), the extent of activation was pH dependent (Fig. 10). While the inhibition of the deaminase by dTTP was impossible to demonstrate in the in vitro protein synthesis mixture for the reasons discussed above, the inhibitory qualities of this nucleotide became more pronounced on partial purification of the deaminase. Here, too, the inhibition was not as effective as with the pure enzyme, but the

I)IK)XYCYTII)YLATE

DEANINASE

SYNTHESIS

ire Z&O

523

have separate promoters and that, their temporal order of expression may be related direct,lv to the d&awe of t,hese genes from their rkpectivc promoters (31).

0.25

0.5

0.75

1.0

mM dCMP Fru. 9. Stimulation of partially purified ijz r@lro-synthesized dCMP deaminase (see Experimental) by dCTP at various substrate levels. The assay mixtures were essentially the same as those described in Fig. 10, with pH 8.0 wide-range buffer (32) and varying dChfP conrentrntions as indicated. The reactions were terminated after 15 min of inc~ubation at 37°C and assayed as descsribed in k:xperiment,al.

results reported in Tabk V dispel someof the ambiguous findings obtained with dTT1’ in thcl original protein synt,hrsizing mixture (Id’ig. 8, Tablo II). It may seemincongruous that morr inhibition was obtained in t’he prcwnw of 0.2 rnlr dCTI’ than in its abwnce, but if viewed in thr pcrspcctive that dTTI’ rtwrscs the activation by dCTP, the rwult,s obtained aw not implaukblr.

0.1

0.2

0.3

mM dCTP FIG. 10. ERect of pH on the dCTP requirement. of in vitro-synthesized dChlP deaminase. Each 50+1 assay mixture contained 1.25 @moles widerange buffer (32) at the pH values indicated, 0.25 pmole 2.mercaptoethanol, 50 nmoles magnesium chloride, 25 nmoles [2-%]dCMP (5.1 cpmjpmole), 5 nmoles dCTP where indicated, and 0.12 mg of partially purified enzyme protein (see Experimental). After incubation for 30 min at 37”C, the reac’tions were terminat,ed t)y heating at, 100°C for 2 min and assayed for dlJM1’ as described in Experimental.

1)ISCUSSION

Although numerous wgulatory agents responsible for the appropriat’e order of gene transcription and subscqurnt translat#ion into protein haw bwn described, there remain many imp&antj gaps in the undcrstanding of thcw processw. An indication of such a gap is pnwntcd in Fig. 1 where it is shorn-nthat, although t’hc genes concerned with the expression of H2folate wduct,ase, dTM1’ synthetase, and dCJI1’ deaminase ckxist as a closely linked cluster, they arc n&her expressed in thi,q order nor the IFverse, but as Hafolatc reductase, dCM1’ dcaminase, and dThIP spnthetase. Evidence \\‘a~ provided recently that, these genesmay

Additions

NOW?

+4 mbl dTTP

%TTM (pm&s/l5 Nom? +O.l mM dCTP +0.2 miu dCTP

610 814 1240

440 (28) 3% (51) 52C1 (58)

min) 274 348 348

(55) (57) (72)

cf The assay mixtures were the same as described in Fig. 10, escaept that the pH of t.he buffer was 8.0. The concentrations of dCTP and dTTP used are presented in the table. After a reaction period of 15 min, t,he assays were t.erminated with heat (100°C for 2 min). The figures in parentheses represent percentage of inhibitiorl.

524

TRIMBLE,

MALEY,

Another anomaly involves the relative quantity of enzyme found after phage induction as compared with that made using RNA or DNA as templat’es. Thus, the ratio of Hzfolate reductase, dTMP synthetase, and dCMP deaminase induced after T2 phage infection was 0.67 to 0.07 to 1.0, respectively, but this ratio became 62 to 0.62 to 1.0 with T2 DNA as template in the cell-free protein-synthesizing system. With RNA as template, the ratio was 24 to 0.10 to 1.0. While the synthesis of enzymes in vitro with reconstructed systems has been successful, an adequate explanation for the altered rates of transcription and translation in vitro relative to those in tivo is still to be provided. Similarly, an explanation for the titer effect involving dCMP deaminase, a finding that initiated these in vitro studies, is open to conjecture. Studies to date have shown that the restriction in deaminasesynthesis in the high-titer cells is most probably localized in translation and not in transcription. This assumption is based on the finding that rifampin did not impair the elevation in deaminase synthesis after a 4-fold dilution of phage-infected cells, but did prevent mRNA synthesis (2). The use of the in vitrcl system provided similar results in that ribosome-free extracts from high-titer cells (8 X 108 cells/ml) coupled with ribosomes from low-titer cells (2 X lOa cells/ml) were severely impaired in their capacity to synthesize dCMP deaminase or any other proteins. This was not the case with ribosome-free extracts from low-titer cells and ribosomes from high-titer cells. It appears, therefore, that the limiting factor in the translation of proteins resides in the ribosome-free cell extract, possibly at initiation or elongation. It is of interest to note that Initiation Factor f, was shown to possessthe property of selectivity toward different messengers(3840). Whether elevated levels of a specific initiation factor for dCMP deaminase mRNA are responsible for the enhanced synthesis observed in low-titer phage-infected cells is a question that should not be ignored. The initial inability to demonstrate the feedback regulation of dCMP deaminase synthesized in vitro suggested the possi-

AND

MALEY

bility that this enzyme was not subject to feedback regulation, or at least not as sensitive to this phenomenon in crude extracts. Evidence was obtained earlier (1) indicating that the deaminase in unpurified extracts is not as sensitive t,o feedback regulation as the homogeneousprotein. The finding of an active phosphotransferase capable of converting dCMP to dCTP in the presence of a variety of high-energy phosphate donors provided in part an explanation for the inability of dTTP to inhibit the deaminase in crude extracts. A similar phosphotransferase was shown to be associated with Rous sarcoma virions (41). Removal of most of the phosphotransferase activity by partial purification of the newly synthesized deaminase enabled a definitive response to the allosteric effecters to be demonstrated (Figs. 9, 10; Table V). The deaminase would appear, therefore, to possessthe same capacity for allosteric regulation after its synthesis in vitro as when induced after T2 phage infection of E. coli. REFERENCES 1. MALEY, G. F., GUARINO, D. U., AND MALEY, F. (1967) J. Biol. Chem. 242, 3517. 2. TRIMBLE, R. B., MALEY, G. F., AND MALEY, F. (1972) J. Viral. 9, 454. 3. LEDKRMAN, M., AND%JBAY,G. (1968)Biochent. Bioyhys. Res. Commun. 32, 710. 4. SALSER, W., GESTELAND, R.F., ANDBOLLE, A. (1967) Nature London 216, 588. 5. GOLII, L. M., AND SCHWEIGER, M. (1970) J. Biol.

Chem.

246,

2255.

6. Yourut, E. T., II (1970) J. Mol. Biol. 61, 591. 7. WILI~IELM, J. M., AND HASELKORN, R. (1971) Virology 43, 198. 8. SAKI.YAMA, S., AND BUCHANAN, J. M. (1971) PI-IX. Nat. Acad. Sci. USA 68, 1376. 9. BRODY, E.N., GOLD, L.M., ANDBLACK, L. W. (1971) J. Mol. Biol. 60, 389. 10. PARKS, J.S., GOTTESMAN, M., PERLMAN, R.L., AND PATSBN, I. (1971) J. Biol. Chem. 246, 2419. 11. LAY~OCK, D. G., AND HUNT, J. A. (1969) Nature London 221, 1118. 12. GRAZIADEI, W. D., III, .~ND LENGYEL, P. (1972) Biochem. Biophys. Res. Commun. 46, 1816. 13. MCDOWELL, M. J., AND JOKLIK, W. K. (1971) Virology 14. CAFFIER,

46, 98.

46, 724.

H., AND GREEN,

M. (1971) Virology

DEOSYCYTIDYLATE 15. SIEGERT,

R. N. H., Bausn, H., P. H. (1972) Proc. -vat. Acad. Sci. USA 69, 888. SCHWEIGER, M., AND GOLD, L. M. (1970) J. Biol. Chem. 246, 5022. I)EVRIES, J. K., AND ZUBAY, G. (1969) J. Bacterial. 97, 1419. TRIMBLE, R. B., MALEY, G. F., AND M.~LEY, F. (1972) Fed. Proc. 31,443. ADAMS, M. H. (1959) in Bacteriophages, p. 446. Interscience, New York. M. LEY, G. F., AND MALEY, F. (1964) J. Biol. Chem. 239, 1168. X~THEWS, C. K. (1967) J. Biol. Chem. 242, 4083. B.IKER, B. R. (1967) J. Med. Chem. 10, 912. LORENSON, M. Y., MALEY, G. F., AND MALEY, F. (1967) J. Biol. Chem. 242, 3332. WAHBA, A. J., AND FRIEDKIN, M. (1961) J. Biol. Chem. 236, PC 11-12. X~LEY, G. F., AND MILEY, F. (1968) J. Biol. Chem. 243, 4506. BOLLE, A., EPSTEIN, R. H., SALSEE, W., BND GEIDUSCHEK, E. P. (1968) J. Mol. Biol. 31, 325. THOMAS, C. A., JR., .ZND ABELSON, J. (1966) in Procedures in Nucleic Acid Research (Cantoni, G. L., and Davies, D. R., eds.), pp. 553-561, Harper and Row, New York. LOWRY, 0. H., ROSEBROUGH, N. J., F.\RR, .IND

16. 17.

18. 19.

20. 21. 22. 23. 24. 25. 20.

27.

28.

W.,

DEAMINASE

KONINGS,

HOFSCHNEIDER,

29. 30. 31. 32. 33. 34. 35.

36. 37. 38.

39.

40.

41.

SYNTHESIS

in

vitro

525

A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265. YEH, Y.C., DUBOVI, E. J., AND TESSMAN, I. (1969) Viirology 37, 615. YEH, Y.C., AND TESSMAN, I. (1972) Virology 47, 767. TRIMBLE, R. B., G1~LIVAN, J., AND MALEY, F. (1972) Proc. Nat. Acad. Sci. USA 69, 1659. M~LEY, G. F., GUARINO, D. U., AND MALEY, F. (1972) J. Biol. Chem. 247,931. K~LEY, F., AND MALEY, G. F. (1971) Arch. Biochem. Biophys. 144, 723. GREENE, R., AND KORN, D. (1967) J. Mol. Biol. 28, 435. BOSE, s. K., AND WARREN, R. J. (1967) Biothem. Biophys. Res. Commun. 26, 385. YOUNG, E. T., II, AND VAN Houws, G. (1970) J. Mol. Biol. 61, 605. OKAZAKI, R., AND KORNBERG, A. (1964) J. Biol. Chem. 239, 269. REVEL, M., AVIV, H., GRONER, Y., AND POLLACK, Y. (1970) Fed. Eur. Biochem. Sot. Lett. 9, 213. VERMEER, C., TALENS, J., BLOEMSMBJONEMAN, F., AND BOSCH, L. (1971) Fed. Eur. Biochem. Sot. Lett. 19, 201. GRUNBERG-MANAGO, M., RABINOWITZ, J. C., DONDON, J., LELONG, J. C., AND GROS, F. (1971) Fed. Eur. Biochem. Sot. Lett. 19,193. MITZUTANI, S., AND TEMIN, H. M. (1971) J. Viral. 8, 499.