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Studies on phage development
VIROLOGY
%,
396403
(1966)
Studies
on Phage
Development.
III. The Fate of T4 DNA and Protein
Synthesized
in the
Presence of 9-Aminoacridine’J M. M. PIECHOWSKI Department
of Genetics,
AND
University
MILLARD
of Wisconsin,
Accepted November
SUSMAN Madison,
Wisconsin
2.4, 1965
9-Aminoacridine (9AA) inhibits maturation of phage T4. Upon diluting infected bacteria from the dye into a medium containing an inhibitor of protein synthesis, one obtains a crop of mature phage made out of the material accumulated before the removal of 9AA. DNA is synthesized in the presence of 9AA and continues to replicate after the removal of 9AA and addition of puromycin. By marking the DNA with B-bromodeoxyuridine (BUDR) and studying the light sensitivity of the phage produced by cells exposed to SAA, BUDR and puromycin in various programs, one can show that the DNA made in the presence of 9AA is incorporated later into viable phage. This early DNA is dispersed through the DNA synthesized after the removal of the dye. The DNA can in the presence of puromycin pass from the vegetative into the condensed state and subsequently be coated with the protein material synthesized in the presence of the dye. INTRODUCTION
Phage precursor protein appears in an infected cell in advance of the beginning of maturation. It is possible to follow the formation of mature phage from the precursor protein in the absence of protein synthesis (Piechowski and Susman, 1966). Precursor protein that has been accumulated in the presence of 9-aminoacridine (9AA) (an inhibitor of T4 maturation) can be assembled into mature phage in the presence of puromycin (Susman et cd., 1965). This gives us a method to study exclusively the precursor protein that has been made while maturation was prevented by 1 Contribution No. 1995 from the Department of Genetics. 2 This research was aided partially by a grant from The Wisconsin Alumni Research Foundation, University of Wisconsin, and partially by grant AI-05855 from the National Institute of Allergy and Infectious Diseases, U.S. Public Health Service. 396
9AA. In this paper we deal with the following questions : 1. How much maturable precursor protein accumulates in acridine? 2. How is this quantity affected by 9AA concentration? To study the fate of the DNA made in the presence of 9AA we have used 5-bromodeoxyuridine (BUDR) as a label. The incorporation of this base analog into phage DNA sensitizes the phage to visible light (Stahl et al., 1961). The shapes of the survival curves of BUDR-labeled phage exposed to visible light yield information not only about the amount of BUDR-DNA incorporated into viable phage but also about its distribution. Thus we can ask: 3. Is precursor DNA made in the presence of 9AA intermixed with new phage DNA made after the removal of t’he dye? By combining puromycin treatment with BUDR-labeling we can ask: 4. Can the transition of the DNA from
‘I’4 DNA AND PROTEIN MADE IN ACRIDINE
397
RESULTS the vegetative state into the condensedstat,e in the head of the mature phage occur in the When bacteria infected with phage are absenceof protein synthesis? incubated in the presenceof 9-aminoacridine (9AA) no mature phage appears.Upon diluMATERIALS AND METHODS Strains. Escherichia coli B/5 was used as host in all experiments. The indicators were E. coli B/5 and E. coli S/6/5. Wild-type bacteriophage T4D was used in all experiments. Wild-type T6 was used as a carrier. ildedia and growth. The synthetic medium was used as described by Susman et al. (1965). Growth conditions are the same as described by Piechowski and Susman (1966) and all experiments are tM = 23 experiments. Illumination. The illumination of phage with visible light (VL) was carried out as described by Stahl et al. (1961) except that t’he distance from the lamp was 18 cm. The lamp (Champion, F40CW-Cool White, 40 watts, Preheat-Rapid Start) was bare and used wit,hout reflector. 0.1 ml samples were withdrawn at intervals into broth dilution tubes and kept in darkness. All experiments with 5-bromodeoxyuridine (BUDR) incorporation were performed in yellow light. The preparation of lysates for illumination was carried out as follows. The lysates were spun at high speed (12,000 rpm, rotor 40, Spinco Uhracentrifuge model L) with T6 phage as carrier. The pellets were resuspended in buffer and after illumination assayed by plating on E. coli S/6/5. Chemicals. Crystalline 5-methyl-nn-tryptophan was purchased from Sigma Chemical Company, 5-bromodeoxyuridine and thymidine from California Corporation for Biochemical Research. The sources of and difhculties with different batches of puromycin have been described in the preceding paper (Piechowski and Susman, 1966). 9-Aminoacridine hydrochloride (9AA) was obtained from Mann Research Laboratories, Inc. A solution of 9AA is stable over long periods of time. Experiments with 9AA, in contrast to proflavine and acriflavine, can be carried out under ordinary fluorescent lamps since the dye does not photosensitize the infected complexes. 5-Fluorodeoxyuridine was kindly provided by Dr. R. Duschinsky of the Hoffmann-La Roche Company.
tion of the bacteria into an acridine-free medium mature phage begins to appear after a predictable delay (Susman et al., 1965). Even if protein synthesis is inhibited in the post-SAA incubation period, mature phage is assembled from the materials accumulated in the presence of the acridine. In Uchida’s (1958b) experiments on the assembly of mature T2 after removal of acriflavine chloramphenicol entirely prevented formation of mature phage. It is almost certain that chloramphenicol lysed the cells, which were yet more fragile because of the exposure to acriflavine. However, under our conditions 5-methyltryptophan or puromycin do allow phage maturation. We shall refer to the number of mature phage made under such conditions as the number of phage equivalents of maturable precursor per cell at the time of addition of the protein inhibitor (Piechowski and Susman, 1966).
Maturation of Phage Material Accumulated in the Presenceoj9AA. The number of phage equivalents of maturable precursor made in the presence of 9AA depends on the concentration of 9AA (Fig. 1). At higher concentrations (above 2.5 pg/ml), the amount of viable phage produced is lower than the input phage, which in the experiment shown in Fig. 1 was 8 particles per bacterium. The values obtained here for the number of phage equivalents of maturable precursor are overestimates since tiMT was used t,o inhibit protein synthesis in this experiment. 5MT, unlike puromycin, does not instantaneously inhibit protein synthesis, but requires l-2 minutes (Piechowski and Susman, 1966). The time of appearance of maturable precursor is independent of 9AA concentration and is about the same as in the control. The results are similar if transfer from 9AA is made into a medium cont,aining puromycin (Fig. 2): the first maturable precursor begins to appear at the 19th minute, which is not substantially
PIECHOWSKI AND SUSMAN 60-
I 50 MINUTES
AFTER
INFECTION
FIG. 1. Formation
of maturable precursor at different concentrations of 9AA as measured by transfer into 5-methyltryptophan. A culture containing 2.7 X lo9 bacteria/ml was infected with T4D at a multiplicity of 8. After 4 minutes of adsorption and 4 minutes of serum treat,ment the cultures were dilut,ed 104-fold and incLtbated at, 30”. Vigorous aeration was maintained from t,he beginning of infection. 9-Aminoacridine at concentrations indicated was present from the moment of infection. Each curve represents data obtained from a separate adsorption tube. At 5-minute irltervals, beginning at 20 minlltes after infection, samples were diluted lO@fold from 9AA into medium containing 20 pg of 5-methyltryptophan per milliliter. Thirty minutes after transfer the cultures were chloroformed and assayed. Each point represents the final yield of phage obtained in the presence of 5MT after dilrltion from SAA at the time on the abscissa. different from an untreated control (Piechowski and Susman, 1966). In these experiments the amount’ of intracellular phage at the moment of dilution from 9AA never exceeded 0.02 phage per infected cell.
The decline in phage yield after prolonged exposure to acridine is a common feature of these experiments. Incorporation of DNA Made in the Presence of QAA into Viable Phage
Uchida (195Sa) has shown for T2 that the DNA made in the presence of acriflavine is
FIG. 2. Formation of maturable precursor in 9AA as measured by transfer into puromycin. A culture containing 1.7 X 10” bacteria/ml was infected with T4D at a multiplicity of 12. After 4 minutes of adsorption and 4 minutes of serum treatment the culture was diluted lo4 and incubated at 30”. Vigorous aeration was maintained throughout. 9.Aminoacridine at a final concentration 1.5 pg/ml was added at the moment of infection. At 5-minute intervals beginning at 20 minIltes after infection samples were diluted 100.fold from SAA into medium containing 200 pg of puromycin per milliliter. Thirty minutes after transfer the cultures were chloroformed and assayed. Each point represents the final yield of phage obtained in the presence of pllromycin after dilution from 9AA at the time on the abscissa. Each point, therefore, refers to the material ready to become mature phage 11por1 dilution from 9AA into puromycin.
incorporated into viable phage after the dye is removed. The same is true for T4 DNA made in the presence of 9AA. Susman et al. (1965) demonskated reversion of a T4rII mutant by 5bromodeoxyuridine in t,he presence of 9AA. This indicat’es that the base analog is incorporated into precursor DNA in the presence of 9AA. This mutagenesis experiment does not reveal the diskibution of the DNA made in the presence of 9AA among the phages obt’ained after the removal of 9AA. In order to study the problem of DNA distribution, BUDR was used to label DNA. Incorporation of BUDR into DNA increases the sensitivity of the phage to visible light up t’o several hundred fold, depending on the degree of substituGon of t’hymidine by BUDR (Stahl et al., 1961). The phages containing DNA synthesized in the presence of BUDR were exposed to visible light (VL). We can expect the following possibilities
399
T4 DNA AND PROTEIN MADE IN ACRIDINE 4
2 b-h -e
IO
20
30
111THYMIDINE
50
0 CONTROL
K=
CONTROL
40
a DOSE (HOURS1
?
-o-
60 CONTROL
FIG. 3. Visible light inactivation of phage grown ill the presence of 9AA and BUDR and allowed to grow and mature after removal of both compouuds. The design of the experiment is given below the survival curves. A culture containing 1.9 X 109 bacteria/ml was infected with T4D at a mrrltiplicity of 6.5. 9.Aminoacridine at a concentration of 0.8 pg/ml and BUDR at a concent,ration of 200 pg/ml were present from the beginning of infection. After 4 minutes of adsorption and 4 minutes of serum treatment the culture Ts-as diluted lO@fold from the adsorption tube and maintained under vigorous aeration at 30”. At the times indicated, a N&fold dilution into medium containing per milliliter 100 pg of thymidine was made to remove 9AA and BUDR. The cultures u-ere chloroformed 30 minutes after transfer. The lysat,es were spnn at high speed with T6 as carrier. The pellets were taken up in buffer and exposed to visible light from a fluorescent lamp fixture at a distance of 18 cm. Samples were withdrawn at intervals and assayed on E. coli S/G/5. The control lysate (not exposed to BUDR at any time) was prepared in the same way.
-2\
\
t
m CONTROL: ALL BUDR
I
IO'
7.0'
3bl
4a
5b'
8' CONTROL
FIG. 4. Visible light inactivation of phage grown in the presence of 9AA and allowed to grow and mature in the presence of BUDR after removal of the dye. The design of the experiment is given below the phage survival curves. Adsorption and growth as in Fig. 3, but without BUDR. At times indicated, a lOGfold dilution into medium containing 206 rg/ml of BGDR was made. Preparation of lysates and illllmination as in Fig. 3. The all-BUDR control was prepared in t,he same way, but BUDR was present, throughout the entire period of phage growth.
replicate and recombine with the vegetative DNA formed after the removal of 9AA, then the progeny phages should all be light) sensitive. The design of the experiments is repreIf no BUDR goes into the DNA made in t#he sented in Figs. 3 and 4. Phage-infect.ed cells presence of 9AA, or if all DNA made in the were incubated for 20, 30, and 40 minutes in presence of 9AA is nonviable, then we do not medium with 9AA and BUDR, after which expect any light-sensitive phage progeny to they were diluted loo-fold into medium conappear aft’er the removal of 9AA. If, on the t,aining excess t)hymidine. We assume, as did other hand, the DNA made in t’he presence Pratt et al. (1961), t,hat thymidine rapidly of 9AA and BUDR matures separately from displaces BUDR from the intracellular pool. Chloroform was added 30 minutes after t,he new DNA synthesized after the removal of 9AA, then we should obtain two sub- transfer. The lysates were prepared for illumination as described under Methods. populations of phage progeny: one uniformly The light inactivation curves in Fig. 3 sensitive to light and one entirely resistant. If precursor DNA formed in the presence of show that the longer the time spent in 9AA acridine is not in a discrete pool but can and BUDR the more sensitive are the phages
400
PIECHOWSKI ,
1
\,\
=\pCONTROL:
AND
SUSMAN
113 LWSE(HOURS)
ALL BUDR
FIG. 5. Visible light inactivation of phage grown in the presence of SAA and BUDR and matured in the presence of puromycin after removal of bot,h compounds. The design of the experiment is the same as in Fig. 3 except that, besides thymidine, puromycin was added at 200 rg/ml. A culture containing 2.5 X 109 bacteria/ml was infected with T4D at a multiplicity of 4. 9AA (0.8 pg/ml) and BUDR (200 rg/ml) were present from the moment of infection. At the times indicated, samples were diluted lO@fold into medium containing thymidine and puromycin. Preparation of lysates and controls and conditions of illumination are described in the legend to Fig. 3.
produced after dilution from 9AA. From the inactivation curves we can estimate the minimum fraction of the progeny phage having incorporated BUDR. The fraction in the 20-minute population is at least 60%; in the 30- and 40-minute populations, it is at least 90 %. Figure 4 gives the VL-inactivation curves in an experiment where BUDR was present in the post-SAA period but not before. Phages coming out of cells that spent the longest time in 9AA had the least chance to incorporate BUDR. From the inactivation curves here we can estimate the maximum fraction of progeny phage containing no BUDR. For the 20-minute curve this fraction is less than 1 per cent, for the 30-minute curve less than 5%, and for the 40-minute curve less than 20%. These experiments suggest that almost all the DNA that enters mature phage after the removal of 9AA is DNA that was vegetat,ive at the time when the acridine was removed.
FIG. 6. Visible light inactivation of phage grown in the presence of 9AA and matured in the presence of puromycin and BUDR. Same culture and adsorption conditions as in the legend to Fig. 5 except that BUDR was omitted from the adsorption tube. The design of the experiment is the same as in Fig. 4 except that dilutions from 9AA were made into medium containing, besides BUDR, 200 pg of puromycin per milliliter. Preparation of lysates, controls, and conditions of illumination as in the legend to Fig. 3.
The Passage of Vegetative DNA into Mature Phage in the Absence of Protein Synthesis. The previous two experiments can be performed with the modification that in the post-SAA period puromycin is present so that the mature phage is coated only with the protein made prior to transfer. Under these conditions the maturation of DNA made after dilution from 9A.A into puromytin will be either limited or entirely excluded if it is true that the DNA cannot condense without concurrent protein synthesis (Kellenberger et al., 1959). Figures 5 and 6 present the results of two such experiments. One can see that except for the better enrichment of BUDR-containing DNA the pattern obtained in Fig. 5 is the same as in Fig. 3. When the infection proceeds in the presence of 9A.A only and the dilution from 9A.A is made into puromycin plus BUDR (Fig. S), one still obtains phages that are sensitive to visible light. This sensitivity could only result from incorporation of BUDR in the presence of puromycin. Thus, the DNA not only continues to replicate after dilution from 9AA, but is wrapped in coat protein that accumulated in the presence of 9AA, and the whole maturation process comes t#o completion in the absence of protein synthesis. Since the
T4 DNA
AND
PROTEIN
5MT + FUDR
0
E
1 IO
20 MINUTES
30 AFTER
40 INFECTION
FIG. 7. Formation of maturable precursor in SAA as measured by transfer into 5-methyltryptophan and 5-fluorodeoxyuridine. A culture containing 2.4 X 109 bacteria/ml was infected with T4D at a multiplicity of 4.5. After 4 minutes of adsorption and 4 minutes of serum treatment the cultures were diluted W-fold and incubat,ed at 30”. Vigorous aeration was maintained from the beginning of infection. 9.Aminoacridine at 1.0 pg/ml was present from the moment of infection. At 5-minute intervals beginning at 15 minutes after infection samples were diluted lOO-fold from 9AA into medium containing per milliliter either 20 pg of 5MT plus 50 pg of FUDR or 5MT alone (control). Thirty minutes after transfer the cultures were chloroformed and assayed. Each point represents the final yield of phage obtained in the presence of 5MT, or 5MT plus FUDR, after dilution from 9AA at the time on the abscissa.
inactivation curves do not contain a fraction as resistant as the unsubstituted control phage, one has to conclude that t,he DNA made in the presence of 9AA becomes dispersed among the DNA made after dilution from 9AA into puromycin. The only exception appears at, t’he 40-minute exposure to 9AA, but even here the resistant fraction is not as resistant as the control. The DNA made after dilution from 9AA into puromytin is vegetative and continues t,o replicate in t’he absence of protein synthesis. The ability of this DNA to condense and enter mature phage is in contrast to the observations of Kellenberger et al. (1959) on the
MADE
IN ACRIDINE
401
giant pool of DNA obt,ained in chloramphenicol. The experiment shown in Fig. 7 suggests that DNA synthesis is not involved in the final stages of phage assembly. Cells incubated in the presence of 9AA were diluted at intervals into medium containing 5MT plus FUDR. This treatment inhibits both DNA (Cohen et al., 1958; Kozinski and Kozinski, 1963) and the protein synthesis. The figure shows the number of phage particles t’hat appear after removal of 9AA. These phages must be assembled from precursor DnTA and precursor protein t.hat accumulated in the presence of the dye. In the control in which the dilution was made into 5MT alone the phage yield is higher. This suggests that in these SAA-t’reated cells the pool of precursor DNA t’hat can form viable phage is smaller than t#hepool of precursor protein. DISCUSSION
1. How much maturable precursor protein accumulates in acridine? We have described experiments showing that in a cell infected with T4 in the presence of 9AA there is an accumulation of all the protein necessary to make a complete phage. The amount of phage made after the removal of acridine is a minimum estimate of the amount of protein precursor accumulated in the presence of the dye. The higher t.he dye concent’ration the lower is the number of phage that appear after the removal of acridine. We do not, know how the treatment with 9AA limits the yield of infective phage. The 9AA might limit the amount of structural protein produced; it might divert some of the precursor into synthesis of noninfective phage; or it might not directly affect the protein precursor at all. The maximum value of 50 phage equivalents of maturable precursor per cell achieved in 1.0 pg per ml of 9AA (as measured by transfer into 5111T) comes near the value of 68 obtained in t’he control. The rate of accumulation of maturable precursor, however, is only half t#he rate in the control [compare with Fig. 5 in the preceding paper (Piechowski and Susman, 1966)]. It should be kept in mind that t’he values in 5MT are exaggerated.
402
PIECHOWSKl
AND
SUSMAN
2. Is precursor DNA made in the presence on messenger and ribosomal RNA in E. coli of 9AA vegetative or inert? The VL-inactiva(Soffer and Gros, 1964) suggests a possible explanation for the reduced yield of phage tion curves of the phage lysates prepared after prolonged acridine treatment. Howafter exposure to 9AA and BUDR strongly indicate that the viable DNA made in the ever, the concentrations of proflavine used in the experiments of Soffer and Gros (1964) presence of 9AA is vegetative. The fairly uniform light sensitivity of these phages surpass several hundredfold the concentrasuggests that the precursor DNA made in the tion necessary to suppress maturation of T4 (Couse, Piechowski and Susman, unpubpresence of 9AA occupies the same recombinlished) . ing pool as the DNA made after the removal 4. DNA condensation and protein syntheof the acridine. After prolonged 9AA treatsis. The condensation of DNA into a comments (e.g., 40 minutes) there appears a fraction that reaches a stage beyond further in- pact head structure does not appear to require protein synthesis. corporation of nucleosides after the removal of 9AA. We do not know whether this fracACKNOWLEDGMENTS tion represents a DNA that condensed in the presence of 9AA or that simply failed to The authors are deeply indebted to Drs. R. I. replicate and recombine after the removal DeMars, W. Szybalski, and H. Temin as well as to of the dye. Dr. and Mrs. H. K. Kubifiski for stimulating The decline of viable phage synthesis as criticism and helpful suggestions. well as the decline of the synthesis of matuREFERENCES rable precursor (or, more precisely, conversion of the precursor material into viable COHEN, S. S., FLAKS, J. G., BARNER, H. D., LOEB, M. R., and LICHTENSTEIN, J. (1958). The mode phage) after prolonged treatments with 9AA of action of 5-fluorouracil and its derivatives. can be interpreted to mean that there is an Proc. Natl. Acad. Sci. U.S. 44, 1604--1012. accumulation of nonviable phage material. Kellenberger et al. (1959) observed in t.hin KELLENBERGER, E., SECHAUD, J., and RYTER, A. (1959). Electron microscopical studies of phage sections of T2-infected cells treated with multiplication. IV. The establishment of the proflavine a great number of “badly filled” DNA pool of vegetative phage and the matura(with DN,4) phages. These authors also tion of phage particles. Vi’irology 8, 478498. cited the observations of Brenner and Horne KOZINSKI, A. W., and KOZINSKI, P. B. (1963). that abnormal phage heads made in the Fragmentary transfer of Paz-labeled parental presence of proflavine are unstable and their DNA to progeny phage. II. The average size of the transferred parental fragment. Two-cycle DNA leaks out slowly. Any fraction of nontransfer. Repair of the polynucleotide chain viable condensates accumulated in the presafter fragmentation. Virology 20, 213-229. ence of 9AA is not subject to detection in our PIECHOWSKI, M. M., and SUSMAN, M. (1966). experiments. Studies on phage development. II. The matura3. Timing of protein synthesis in the prestion of T4 phage in the presence of puromycin. ence of 9AA. It is important to note that Virology 28, 386-395. whatever t’he effect of acridine on DNA syn- PRATT, D., STENT, G. S., and HARRIMAN, P. D. thesis, phage condensation and phage syn(1961). Stabilization of s2P decay and onset of thesizing capacity, all the protein necessary DNA replication of T4 bacteriophage. J. Mol. Do make a mature phage begins to appear at Biol. 3, 409424. SOFFER, R. L., and GROS, F. (1964). Effects of the same time in acridine as in the control. dinitrophenol and proflavine on information The effect, t’hen, that 9AA exerts on the transfer mechanisms in Escherichia coli; a study assembly of the phage material into infecin vivo and in vitro. Biochim. Biophys. Acta 87, tive units and on the timing of maturation 423439. is independent of the synthesis of phage STAHL, F. W., CRASEMANN, J. M., OKUN, L., Fox, proteins or DNA. The inhibitory mechaE., and LAIRD, C. (1961). Radiation-sensitivity nism remains t,o be elucidated. The recent of bacteriophage containing B-bromodeoxyurireport on the degrading effect of proflavine dine. Virology 13, 98-104.
T4 DNAAND PROTEINMADEIN ACRIDINE &JSMAN, M., PIECHORSKI, M. M., and RITCHIE, D. A. (1965). Studies on phage development. I. An acridine-sensitive clock. Virology 26, 163174. UCHIDA, H. (1958a). Studies in the synthesis of bacterial viruses. I. On the accumulation of phage precursor nucleic acid when the matura-
tion step is inhibited Exptl.
by acriflavine.
403 Japan.
J.
Med. 28, 59-66.
UCHIDA, H. (195813).Studies in the synthesis of bacterial viruses. II. On the phage precursor protein components synthesized during the presence of acriflavine. Japan,. .T. 7hvtl. Med. 28, 67-72.
%,
396403
(1966)
Studies
on Phage
Development.
III. The Fate of T4 DNA and Protein
Synthesized
in the
Presence of 9-Aminoacridine’J M. M. PIECHOWSKI Department
of Genetics,
AND
University
MILLARD
of Wisconsin,
Accepted November
SUSMAN Madison,
Wisconsin
2.4, 1965
9-Aminoacridine (9AA) inhibits maturation of phage T4. Upon diluting infected bacteria from the dye into a medium containing an inhibitor of protein synthesis, one obtains a crop of mature phage made out of the material accumulated before the removal of 9AA. DNA is synthesized in the presence of 9AA and continues to replicate after the removal of 9AA and addition of puromycin. By marking the DNA with B-bromodeoxyuridine (BUDR) and studying the light sensitivity of the phage produced by cells exposed to SAA, BUDR and puromycin in various programs, one can show that the DNA made in the presence of 9AA is incorporated later into viable phage. This early DNA is dispersed through the DNA synthesized after the removal of the dye. The DNA can in the presence of puromycin pass from the vegetative into the condensed state and subsequently be coated with the protein material synthesized in the presence of the dye. INTRODUCTION
Phage precursor protein appears in an infected cell in advance of the beginning of maturation. It is possible to follow the formation of mature phage from the precursor protein in the absence of protein synthesis (Piechowski and Susman, 1966). Precursor protein that has been accumulated in the presence of 9-aminoacridine (9AA) (an inhibitor of T4 maturation) can be assembled into mature phage in the presence of puromycin (Susman et cd., 1965). This gives us a method to study exclusively the precursor protein that has been made while maturation was prevented by 1 Contribution No. 1995 from the Department of Genetics. 2 This research was aided partially by a grant from The Wisconsin Alumni Research Foundation, University of Wisconsin, and partially by grant AI-05855 from the National Institute of Allergy and Infectious Diseases, U.S. Public Health Service. 396
9AA. In this paper we deal with the following questions : 1. How much maturable precursor protein accumulates in acridine? 2. How is this quantity affected by 9AA concentration? To study the fate of the DNA made in the presence of 9AA we have used 5-bromodeoxyuridine (BUDR) as a label. The incorporation of this base analog into phage DNA sensitizes the phage to visible light (Stahl et al., 1961). The shapes of the survival curves of BUDR-labeled phage exposed to visible light yield information not only about the amount of BUDR-DNA incorporated into viable phage but also about its distribution. Thus we can ask: 3. Is precursor DNA made in the presence of 9AA intermixed with new phage DNA made after the removal of t’he dye? By combining puromycin treatment with BUDR-labeling we can ask: 4. Can the transition of the DNA from
‘I’4 DNA AND PROTEIN MADE IN ACRIDINE
397
RESULTS the vegetative state into the condensedstat,e in the head of the mature phage occur in the When bacteria infected with phage are absenceof protein synthesis? incubated in the presenceof 9-aminoacridine (9AA) no mature phage appears.Upon diluMATERIALS AND METHODS Strains. Escherichia coli B/5 was used as host in all experiments. The indicators were E. coli B/5 and E. coli S/6/5. Wild-type bacteriophage T4D was used in all experiments. Wild-type T6 was used as a carrier. ildedia and growth. The synthetic medium was used as described by Susman et al. (1965). Growth conditions are the same as described by Piechowski and Susman (1966) and all experiments are tM = 23 experiments. Illumination. The illumination of phage with visible light (VL) was carried out as described by Stahl et al. (1961) except that t’he distance from the lamp was 18 cm. The lamp (Champion, F40CW-Cool White, 40 watts, Preheat-Rapid Start) was bare and used wit,hout reflector. 0.1 ml samples were withdrawn at intervals into broth dilution tubes and kept in darkness. All experiments with 5-bromodeoxyuridine (BUDR) incorporation were performed in yellow light. The preparation of lysates for illumination was carried out as follows. The lysates were spun at high speed (12,000 rpm, rotor 40, Spinco Uhracentrifuge model L) with T6 phage as carrier. The pellets were resuspended in buffer and after illumination assayed by plating on E. coli S/6/5. Chemicals. Crystalline 5-methyl-nn-tryptophan was purchased from Sigma Chemical Company, 5-bromodeoxyuridine and thymidine from California Corporation for Biochemical Research. The sources of and difhculties with different batches of puromycin have been described in the preceding paper (Piechowski and Susman, 1966). 9-Aminoacridine hydrochloride (9AA) was obtained from Mann Research Laboratories, Inc. A solution of 9AA is stable over long periods of time. Experiments with 9AA, in contrast to proflavine and acriflavine, can be carried out under ordinary fluorescent lamps since the dye does not photosensitize the infected complexes. 5-Fluorodeoxyuridine was kindly provided by Dr. R. Duschinsky of the Hoffmann-La Roche Company.
tion of the bacteria into an acridine-free medium mature phage begins to appear after a predictable delay (Susman et al., 1965). Even if protein synthesis is inhibited in the post-SAA incubation period, mature phage is assembled from the materials accumulated in the presence of the acridine. In Uchida’s (1958b) experiments on the assembly of mature T2 after removal of acriflavine chloramphenicol entirely prevented formation of mature phage. It is almost certain that chloramphenicol lysed the cells, which were yet more fragile because of the exposure to acriflavine. However, under our conditions 5-methyltryptophan or puromycin do allow phage maturation. We shall refer to the number of mature phage made under such conditions as the number of phage equivalents of maturable precursor per cell at the time of addition of the protein inhibitor (Piechowski and Susman, 1966).
Maturation of Phage Material Accumulated in the Presenceoj9AA. The number of phage equivalents of maturable precursor made in the presence of 9AA depends on the concentration of 9AA (Fig. 1). At higher concentrations (above 2.5 pg/ml), the amount of viable phage produced is lower than the input phage, which in the experiment shown in Fig. 1 was 8 particles per bacterium. The values obtained here for the number of phage equivalents of maturable precursor are overestimates since tiMT was used t,o inhibit protein synthesis in this experiment. 5MT, unlike puromycin, does not instantaneously inhibit protein synthesis, but requires l-2 minutes (Piechowski and Susman, 1966). The time of appearance of maturable precursor is independent of 9AA concentration and is about the same as in the control. The results are similar if transfer from 9AA is made into a medium cont,aining puromycin (Fig. 2): the first maturable precursor begins to appear at the 19th minute, which is not substantially
PIECHOWSKI AND SUSMAN 60-
I 50 MINUTES
AFTER
INFECTION
FIG. 1. Formation
of maturable precursor at different concentrations of 9AA as measured by transfer into 5-methyltryptophan. A culture containing 2.7 X lo9 bacteria/ml was infected with T4D at a multiplicity of 8. After 4 minutes of adsorption and 4 minutes of serum treat,ment the cultures were dilut,ed 104-fold and incLtbated at, 30”. Vigorous aeration was maintained from t,he beginning of infection. 9-Aminoacridine at concentrations indicated was present from the moment of infection. Each curve represents data obtained from a separate adsorption tube. At 5-minute irltervals, beginning at 20 minlltes after infection, samples were diluted lO@fold from 9AA into medium containing 20 pg of 5-methyltryptophan per milliliter. Thirty minutes after transfer the cultures were chloroformed and assayed. Each point represents the final yield of phage obtained in the presence of 5MT after dilrltion from SAA at the time on the abscissa. different from an untreated control (Piechowski and Susman, 1966). In these experiments the amount’ of intracellular phage at the moment of dilution from 9AA never exceeded 0.02 phage per infected cell.
The decline in phage yield after prolonged exposure to acridine is a common feature of these experiments. Incorporation of DNA Made in the Presence of QAA into Viable Phage
Uchida (195Sa) has shown for T2 that the DNA made in the presence of acriflavine is
FIG. 2. Formation of maturable precursor in 9AA as measured by transfer into puromycin. A culture containing 1.7 X 10” bacteria/ml was infected with T4D at a multiplicity of 12. After 4 minutes of adsorption and 4 minutes of serum treatment the culture was diluted lo4 and incubated at 30”. Vigorous aeration was maintained throughout. 9.Aminoacridine at a final concentration 1.5 pg/ml was added at the moment of infection. At 5-minute intervals beginning at 20 minIltes after infection samples were diluted 100.fold from SAA into medium containing 200 pg of puromycin per milliliter. Thirty minutes after transfer the cultures were chloroformed and assayed. Each point represents the final yield of phage obtained in the presence of pllromycin after dilution from 9AA at the time on the abscissa. Each point, therefore, refers to the material ready to become mature phage 11por1 dilution from 9AA into puromycin.
incorporated into viable phage after the dye is removed. The same is true for T4 DNA made in the presence of 9AA. Susman et al. (1965) demonskated reversion of a T4rII mutant by 5bromodeoxyuridine in t,he presence of 9AA. This indicat’es that the base analog is incorporated into precursor DNA in the presence of 9AA. This mutagenesis experiment does not reveal the diskibution of the DNA made in the presence of 9AA among the phages obt’ained after the removal of 9AA. In order to study the problem of DNA distribution, BUDR was used to label DNA. Incorporation of BUDR into DNA increases the sensitivity of the phage to visible light up t’o several hundred fold, depending on the degree of substituGon of t’hymidine by BUDR (Stahl et al., 1961). The phages containing DNA synthesized in the presence of BUDR were exposed to visible light (VL). We can expect the following possibilities
399
T4 DNA AND PROTEIN MADE IN ACRIDINE 4
2 b-h -e
IO
20
30
111THYMIDINE
50
0 CONTROL
K=
CONTROL
40
a DOSE (HOURS1
?
-o-
60 CONTROL
FIG. 3. Visible light inactivation of phage grown ill the presence of 9AA and BUDR and allowed to grow and mature after removal of both compouuds. The design of the experiment is given below the survival curves. A culture containing 1.9 X 109 bacteria/ml was infected with T4D at a mrrltiplicity of 6.5. 9.Aminoacridine at a concentration of 0.8 pg/ml and BUDR at a concent,ration of 200 pg/ml were present from the beginning of infection. After 4 minutes of adsorption and 4 minutes of serum treatment the culture Ts-as diluted lO@fold from the adsorption tube and maintained under vigorous aeration at 30”. At the times indicated, a N&fold dilution into medium containing per milliliter 100 pg of thymidine was made to remove 9AA and BUDR. The cultures u-ere chloroformed 30 minutes after transfer. The lysat,es were spnn at high speed with T6 as carrier. The pellets were taken up in buffer and exposed to visible light from a fluorescent lamp fixture at a distance of 18 cm. Samples were withdrawn at intervals and assayed on E. coli S/G/5. The control lysate (not exposed to BUDR at any time) was prepared in the same way.
-2\
\
t
m CONTROL: ALL BUDR
I
IO'
7.0'
3bl
4a
5b'
8' CONTROL
FIG. 4. Visible light inactivation of phage grown in the presence of 9AA and allowed to grow and mature in the presence of BUDR after removal of the dye. The design of the experiment is given below the phage survival curves. Adsorption and growth as in Fig. 3, but without BUDR. At times indicated, a lOGfold dilution into medium containing 206 rg/ml of BGDR was made. Preparation of lysates and illllmination as in Fig. 3. The all-BUDR control was prepared in t,he same way, but BUDR was present, throughout the entire period of phage growth.
replicate and recombine with the vegetative DNA formed after the removal of 9AA, then the progeny phages should all be light) sensitive. The design of the experiments is repreIf no BUDR goes into the DNA made in t#he sented in Figs. 3 and 4. Phage-infect.ed cells presence of 9AA, or if all DNA made in the were incubated for 20, 30, and 40 minutes in presence of 9AA is nonviable, then we do not medium with 9AA and BUDR, after which expect any light-sensitive phage progeny to they were diluted loo-fold into medium conappear aft’er the removal of 9AA. If, on the t,aining excess t)hymidine. We assume, as did other hand, the DNA made in t’he presence Pratt et al. (1961), t,hat thymidine rapidly of 9AA and BUDR matures separately from displaces BUDR from the intracellular pool. Chloroform was added 30 minutes after t,he new DNA synthesized after the removal of 9AA, then we should obtain two sub- transfer. The lysates were prepared for illumination as described under Methods. populations of phage progeny: one uniformly The light inactivation curves in Fig. 3 sensitive to light and one entirely resistant. If precursor DNA formed in the presence of show that the longer the time spent in 9AA acridine is not in a discrete pool but can and BUDR the more sensitive are the phages
400
PIECHOWSKI ,
1
\,\
=\pCONTROL:
AND
SUSMAN
113 LWSE(HOURS)
ALL BUDR
FIG. 5. Visible light inactivation of phage grown in the presence of SAA and BUDR and matured in the presence of puromycin after removal of bot,h compounds. The design of the experiment is the same as in Fig. 3 except that, besides thymidine, puromycin was added at 200 rg/ml. A culture containing 2.5 X 109 bacteria/ml was infected with T4D at a multiplicity of 4. 9AA (0.8 pg/ml) and BUDR (200 rg/ml) were present from the moment of infection. At the times indicated, samples were diluted lO@fold into medium containing thymidine and puromycin. Preparation of lysates and controls and conditions of illumination are described in the legend to Fig. 3.
produced after dilution from 9AA. From the inactivation curves we can estimate the minimum fraction of the progeny phage having incorporated BUDR. The fraction in the 20-minute population is at least 60%; in the 30- and 40-minute populations, it is at least 90 %. Figure 4 gives the VL-inactivation curves in an experiment where BUDR was present in the post-SAA period but not before. Phages coming out of cells that spent the longest time in 9AA had the least chance to incorporate BUDR. From the inactivation curves here we can estimate the maximum fraction of progeny phage containing no BUDR. For the 20-minute curve this fraction is less than 1 per cent, for the 30-minute curve less than 5%, and for the 40-minute curve less than 20%. These experiments suggest that almost all the DNA that enters mature phage after the removal of 9AA is DNA that was vegetat,ive at the time when the acridine was removed.
FIG. 6. Visible light inactivation of phage grown in the presence of 9AA and matured in the presence of puromycin and BUDR. Same culture and adsorption conditions as in the legend to Fig. 5 except that BUDR was omitted from the adsorption tube. The design of the experiment is the same as in Fig. 4 except that dilutions from 9AA were made into medium containing, besides BUDR, 200 pg of puromycin per milliliter. Preparation of lysates, controls, and conditions of illumination as in the legend to Fig. 3.
The Passage of Vegetative DNA into Mature Phage in the Absence of Protein Synthesis. The previous two experiments can be performed with the modification that in the post-SAA period puromycin is present so that the mature phage is coated only with the protein made prior to transfer. Under these conditions the maturation of DNA made after dilution from 9A.A into puromytin will be either limited or entirely excluded if it is true that the DNA cannot condense without concurrent protein synthesis (Kellenberger et al., 1959). Figures 5 and 6 present the results of two such experiments. One can see that except for the better enrichment of BUDR-containing DNA the pattern obtained in Fig. 5 is the same as in Fig. 3. When the infection proceeds in the presence of 9A.A only and the dilution from 9A.A is made into puromycin plus BUDR (Fig. S), one still obtains phages that are sensitive to visible light. This sensitivity could only result from incorporation of BUDR in the presence of puromycin. Thus, the DNA not only continues to replicate after dilution from 9AA, but is wrapped in coat protein that accumulated in the presence of 9AA, and the whole maturation process comes t#o completion in the absence of protein synthesis. Since the
T4 DNA
AND
PROTEIN
5MT + FUDR
0
E
1 IO
20 MINUTES
30 AFTER
40 INFECTION
FIG. 7. Formation of maturable precursor in SAA as measured by transfer into 5-methyltryptophan and 5-fluorodeoxyuridine. A culture containing 2.4 X 109 bacteria/ml was infected with T4D at a multiplicity of 4.5. After 4 minutes of adsorption and 4 minutes of serum treatment the cultures were diluted W-fold and incubat,ed at 30”. Vigorous aeration was maintained from the beginning of infection. 9.Aminoacridine at 1.0 pg/ml was present from the moment of infection. At 5-minute intervals beginning at 15 minutes after infection samples were diluted lOO-fold from 9AA into medium containing per milliliter either 20 pg of 5MT plus 50 pg of FUDR or 5MT alone (control). Thirty minutes after transfer the cultures were chloroformed and assayed. Each point represents the final yield of phage obtained in the presence of 5MT, or 5MT plus FUDR, after dilution from 9AA at the time on the abscissa.
inactivation curves do not contain a fraction as resistant as the unsubstituted control phage, one has to conclude that t,he DNA made in the presence of 9AA becomes dispersed among the DNA made after dilution from 9AA into puromycin. The only exception appears at, t’he 40-minute exposure to 9AA, but even here the resistant fraction is not as resistant as the control. The DNA made after dilution from 9AA into puromytin is vegetative and continues t,o replicate in t’he absence of protein synthesis. The ability of this DNA to condense and enter mature phage is in contrast to the observations of Kellenberger et al. (1959) on the
MADE
IN ACRIDINE
401
giant pool of DNA obt,ained in chloramphenicol. The experiment shown in Fig. 7 suggests that DNA synthesis is not involved in the final stages of phage assembly. Cells incubated in the presence of 9AA were diluted at intervals into medium containing 5MT plus FUDR. This treatment inhibits both DNA (Cohen et al., 1958; Kozinski and Kozinski, 1963) and the protein synthesis. The figure shows the number of phage particles t’hat appear after removal of 9AA. These phages must be assembled from precursor DnTA and precursor protein t.hat accumulated in the presence of the dye. In the control in which the dilution was made into 5MT alone the phage yield is higher. This suggests that in these SAA-t’reated cells the pool of precursor DNA t’hat can form viable phage is smaller than t#hepool of precursor protein. DISCUSSION
1. How much maturable precursor protein accumulates in acridine? We have described experiments showing that in a cell infected with T4 in the presence of 9AA there is an accumulation of all the protein necessary to make a complete phage. The amount of phage made after the removal of acridine is a minimum estimate of the amount of protein precursor accumulated in the presence of the dye. The higher t.he dye concent’ration the lower is the number of phage that appear after the removal of acridine. We do not, know how the treatment with 9AA limits the yield of infective phage. The 9AA might limit the amount of structural protein produced; it might divert some of the precursor into synthesis of noninfective phage; or it might not directly affect the protein precursor at all. The maximum value of 50 phage equivalents of maturable precursor per cell achieved in 1.0 pg per ml of 9AA (as measured by transfer into 5111T) comes near the value of 68 obtained in t’he control. The rate of accumulation of maturable precursor, however, is only half t#he rate in the control [compare with Fig. 5 in the preceding paper (Piechowski and Susman, 1966)]. It should be kept in mind that t’he values in 5MT are exaggerated.
402
PIECHOWSKl
AND
SUSMAN
2. Is precursor DNA made in the presence on messenger and ribosomal RNA in E. coli of 9AA vegetative or inert? The VL-inactiva(Soffer and Gros, 1964) suggests a possible explanation for the reduced yield of phage tion curves of the phage lysates prepared after prolonged acridine treatment. Howafter exposure to 9AA and BUDR strongly indicate that the viable DNA made in the ever, the concentrations of proflavine used in the experiments of Soffer and Gros (1964) presence of 9AA is vegetative. The fairly uniform light sensitivity of these phages surpass several hundredfold the concentrasuggests that the precursor DNA made in the tion necessary to suppress maturation of T4 (Couse, Piechowski and Susman, unpubpresence of 9AA occupies the same recombinlished) . ing pool as the DNA made after the removal 4. DNA condensation and protein syntheof the acridine. After prolonged 9AA treatsis. The condensation of DNA into a comments (e.g., 40 minutes) there appears a fraction that reaches a stage beyond further in- pact head structure does not appear to require protein synthesis. corporation of nucleosides after the removal of 9AA. We do not know whether this fracACKNOWLEDGMENTS tion represents a DNA that condensed in the presence of 9AA or that simply failed to The authors are deeply indebted to Drs. R. I. replicate and recombine after the removal DeMars, W. Szybalski, and H. Temin as well as to of the dye. Dr. and Mrs. H. K. Kubifiski for stimulating The decline of viable phage synthesis as criticism and helpful suggestions. well as the decline of the synthesis of matuREFERENCES rable precursor (or, more precisely, conversion of the precursor material into viable COHEN, S. S., FLAKS, J. G., BARNER, H. D., LOEB, M. R., and LICHTENSTEIN, J. (1958). The mode phage) after prolonged treatments with 9AA of action of 5-fluorouracil and its derivatives. can be interpreted to mean that there is an Proc. Natl. Acad. Sci. U.S. 44, 1604--1012. accumulation of nonviable phage material. Kellenberger et al. (1959) observed in t.hin KELLENBERGER, E., SECHAUD, J., and RYTER, A. (1959). Electron microscopical studies of phage sections of T2-infected cells treated with multiplication. IV. The establishment of the proflavine a great number of “badly filled” DNA pool of vegetative phage and the matura(with DN,4) phages. These authors also tion of phage particles. Vi’irology 8, 478498. cited the observations of Brenner and Horne KOZINSKI, A. W., and KOZINSKI, P. B. (1963). that abnormal phage heads made in the Fragmentary transfer of Paz-labeled parental presence of proflavine are unstable and their DNA to progeny phage. II. The average size of the transferred parental fragment. Two-cycle DNA leaks out slowly. Any fraction of nontransfer. Repair of the polynucleotide chain viable condensates accumulated in the presafter fragmentation. Virology 20, 213-229. ence of 9AA is not subject to detection in our PIECHOWSKI, M. M., and SUSMAN, M. (1966). experiments. Studies on phage development. II. The matura3. Timing of protein synthesis in the prestion of T4 phage in the presence of puromycin. ence of 9AA. It is important to note that Virology 28, 386-395. whatever t’he effect of acridine on DNA syn- PRATT, D., STENT, G. S., and HARRIMAN, P. D. thesis, phage condensation and phage syn(1961). Stabilization of s2P decay and onset of thesizing capacity, all the protein necessary DNA replication of T4 bacteriophage. J. Mol. Do make a mature phage begins to appear at Biol. 3, 409424. SOFFER, R. L., and GROS, F. (1964). Effects of the same time in acridine as in the control. dinitrophenol and proflavine on information The effect, t’hen, that 9AA exerts on the transfer mechanisms in Escherichia coli; a study assembly of the phage material into infecin vivo and in vitro. Biochim. Biophys. Acta 87, tive units and on the timing of maturation 423439. is independent of the synthesis of phage STAHL, F. W., CRASEMANN, J. M., OKUN, L., Fox, proteins or DNA. The inhibitory mechaE., and LAIRD, C. (1961). Radiation-sensitivity nism remains t,o be elucidated. The recent of bacteriophage containing B-bromodeoxyurireport on the degrading effect of proflavine dine. Virology 13, 98-104.
T4 DNAAND PROTEINMADEIN ACRIDINE &JSMAN, M., PIECHORSKI, M. M., and RITCHIE, D. A. (1965). Studies on phage development. I. An acridine-sensitive clock. Virology 26, 163174. UCHIDA, H. (1958a). Studies in the synthesis of bacterial viruses. I. On the accumulation of phage precursor nucleic acid when the matura-
tion step is inhibited Exptl.
by acriflavine.
403 Japan.
J.
Med. 28, 59-66.
UCHIDA, H. (195813).Studies in the synthesis of bacterial viruses. II. On the phage precursor protein components synthesized during the presence of acriflavine. Japan,. .T. 7hvtl. Med. 28, 67-72.