Fate of parental ∅X174-DNA upon infection of starved thymine-requiring host cells

VIROLOGY

44, 168-187 (1971)

Fate of Parental

4X174-DNA

upon

Thymine-Requiring BERTOLD Molecular

Biology

Institute

Infection

Host

FRANCKE

AND

of Starved

Cells

DAN S. RAY

and Department of Zoology, University Los Angeles, California 90024

of California,

Accepted December 8, 1970 Escherichia coli HF 4704, a thymine-requiring host for +X174, and a derivative, defective in the replication of @X replicative form, were starved in buffer and infected under various conditions with 4Xam3 containing labeled DNA. If the infection was carried out in buffer without an energy source the parental DNA was recovered as int,aet single-stranded circles. If starved cells were infected in growth-medium a small percentage of the parental label was found in replicative form DNA whereas the majority was degraded. The noninfectious degradation-product was shown to contain parental DNA of varying sizes short.er than unit-length linear viral strands. The proportion of parental DNA being degraded was independent of the multiplicity of infection and the presence or absence of thymine in the medium. Washing of starved, infected cells with borate-EDTA liberated most of the parental label in particles that sedimented slower than intact phages but faster than intact DNA. They were resistant to sarcosyl but the DNA became freely sedimenting after treatment with sodium dodecyl sulfate and pronase. This DNA showed a similar distribution of degraded material as that recovered from the total lysate of infected cells, except that it contained no replicative form. All replicative form DNA synthesized under these conditions was found remaining with the cells after elution of the viral particles. These observations show that both penetration of the viral strand and synthesis of the complementary strand are impaired in starved cells and suggest, that active DNA-synthesis is required for productive infection-possibly replicating the infecting DNA out of the viral coat into the host cell. INTRODUCTION

The small bacteriophage +X174 is a spherical virus containing a single-stranded circular DNA of molecular weight 1.7 X 10”. The replication of this DNA in its host cell (23. coli C) has been studied intensely by several groups of investigators (review article : Sinsheimer, 1968)) leading to a three-step model of replication. The infecting viral DNA is first converted into a double stranded replicative form (RF)’ by host cell enzymes. RF can exist in two 1 Abbreviations: RF, replicative form; EDTA, ethylenediamine tetraacetate; sodium SDS, dodecyl sulfate; m.o.i., multiplicity of infection; PFU, plaque-forming units; cpm, counts per minute; 3HTdR, tritiated thymidine.

distinct types, as RFI, a supercoiled structure with both strands covalently closed, or as RFII, containing one or more discontinuities in either strand. The second and third steps, replication of parental RF and synthesis of progeny single-stranded DNA, both require the functioning of viral genes. Attempts to study the first step in this model in detail have met with several technical difficulties, the main problem being that the formation of the parental RF appears to be very rapid and no intermediates can be found in an unsynchronized infection. The model system of starved cells, first developed as a synchonization technique by Denhardt and Sinsheimer (1965), has been 168

ISFECTIOK

OF STARVED

used by Newbold and Sinsheimer (1969a,b, 1970) to study the first interactions of the infecting virus and it,s host,. These authors established that virus particles infecting st,arved cells at 37” in the absence of an energy source underwent, an eclipse step, that could be distinguished from an adsorption step taking place at’ lower temperatures. Earlier results by Iinippers et al. (1969) had suggested that the infectsion of starved, thymine-requiring cells in the absence of thymine and the presence of energy resulted in the penetration of the viral DNA without formation of RF. We were interested in the properties of the infecting viral DNA under the experimental conditions described above, especially whether after eclipse of the virus particle and/or penetration of the viral DNA into starved cells a product,ive infection could still be obtained. The results present’ed in this communication using starved, thymine-requiring cells, show that in the presence of an energy source most of the infecting viral DSA is degraded to some extent and the amount of functional RF is greatly reduced, even if the infection is carried out in the presence of thymine. Although this system has been very useful for a detailed understanding of the changes of t-he virus particle upon infection, the events on the level of DNA-synthesis appear to be abortive. Based on our result,s, especially the comparison of viral DNA still in particles containing parental viral coat protein with the DKA that appears to have penetrated, we suggest that active DNA synthesis is required for a product’ive infection-leaving the possibility open that 4x174 DiYA is replicated out of the viral coat’ in the course of a normal infection. XATERIALS

AND METHOD8

Phage and bacterial strains. 4Xam3, a lysisdefective amber mutant of 4X174, was used in all experiments. E. coli HF4704 her- is a t’hymine-requiring host for 4X174, which is nonpermissive for amber mutants. Hf4714 (susX) was used as a permissive host for plating +Xam3. Spheroplasts were prepared from I<12 W6. These &rains were kindly provided by Dr.

CELLS WITIT &Ylil

Ifi!)

Virginia Merriam. H 502, an Endonuclease I-deficient derivative of HF4704, was obtained from Dr. Robert L. Sinsheimer, aud the rep3 derivative of HF4704, one of the bacterial mutants unable to replicate 4X RF, was supplied by Dr. T>avid T. Denhardt. Biological assays. Cell densit,ies during gro&h in liquid medium were recorded in a Bausch and Lomb spect’rometer at, 440 nm in a l-inch tube against the same medium as a blank. Colony formers were plated by spreading the appropriate dih,&ons in starvation buffer (see below) on plates \\-ith bott)om agar. Viable phage were assayed by the usual soft agar overlayer I echnique from dilutions in 0.05 M borate. Spheroplasts for infectious DNA assays were prcpared as described by Hutchison and Sinsheimer (1966). The DNA was mixed with the spheroplast-preparation, and aft,er a 2-hour incubation the spheroplast,s were lysed and assayed for progeny phage. Media and chemicals. Full TPA medium contained per liter: 0.5 g NaCl; 8.0 g KCl; 1.1 g NH&l; 12.1 g tris base, 0.8 g sodium pyruvate, 1 ml 0.16 ]I!! N&O4 and 0.5 Y;# casamino acids (Difcoj; pH 7.4. After autoclaving, the following sterile solutions were added: 1 ml 20%’ JIgClz.(iHzO; 0.1 ml 1 rll CaC1,.2Hz0; 20 ml 10% glucose; 75 ml 0.1 JI KHzPO,; and 10 ml thymine (0.2 midmU. TPA-T contained no thymine. TPA with low phosphate contained only 2.5 X 1O-4 :If KH,PO, . TPA with low sulfate con1ained no NazS04 and only 0.1% technical casamino acids. St’arvation buffer contained per liter: 5 g KCl; 1 g SaCI; 1.2 g Tris base; 0.1 g MgSO,; 0.001 M CaCl,; and after autoclaving, 75 ml 0.1 M KH2POG (pH S.1). The supplement to full TPA medium, lo be added to an equal volume of starvation buffer, contained per liter: 5.5 g T
coniained addit ionul 0.006

170

FRANCKE

AND RAY

Tris-EDTA contained 0.01 M Tris and to keep as many viable phages as possible. The average 32P-~X am3 preparation con0.001 M EDTA, pH 8.0. tained a total of ca. 1 X 1013 PFU, ca. DNase I (pancreatic), RNase (pancreatic, 6 X lo7 cpm at the time of preparation, type Ia), lysozyme (grade I) were purchased 85 % infectious particles (calculated from from Sigma, and pronase (B grade) from the 32P:31P ratio in the medium), and 5-7 % Calbiochem. not in phage particles (calcuRadiochemicals and counting of radioac- radioactivity tiuity. 3HTdR CH-methylthymidine, 16.4 lated from the sucrose gradient profile). 3H-labeled +Xam3: The procedure was Ci/mmole, 0.5 mCi/ml), 32P-phosphate (carrier free, 15 mCi/ml), and 35S-sulfate the same as described for 32P, except that TPA + T was used, and instead of 32P a (carrier free, 10 mCi/ml) were purchased from Schwarz BioResearch. Samples were total of 0.75 mCi 3HTdR was added in three generally assayed for radioactivity by spot- portions at 1 min, 30 min, and 60 min after infection. An average 3H-$Xam3-preparating a measured volume onto a Whatman tion contained a total of ca. 2 X 1013PFU, No 3 cellulose filter disk, drying, and countca. 1 X lo7 cpm, 80% infectious particles, ing in a Beckman liquid scintillation counter and l-1.5% radioactivity not in phage in 5 ml toluene containing 13.6 g 2,5-diparticles. The DNA extracted from both, phenyloxazole per gallon. Acid-precipitable 32P- and 3H-labeled phages, consisted of radioactivity was counted after precipitating the sample with 5% trichloroacetic acid in mainly circular and 4-6 % linear molecules on sedimentation through alkaline sucrose the presence of 200 pg of calf thymus DNA gradients (see below). as a carrier. The precipitate was collected Double-labeled +XamS CH-DNA, 35S-proon a Schleicher and Schuell glass filter disk No. 24, washed two times with ea. 10 ml tein): The procedure was the same as 2% trichloroacetic acid, and counted as described for 3H-labeled phages, except that described above. Data are presented as TPA with low sulfate was used and that the cpm per volume measured and corrected for cells were sedimented by centrifugation and resuspended in fresh medium prior to inoverlap where necessary. Radioactively labeled phages. 32P-labeled fection. 35S (1 mCi) was added 5 min after infection. The preparation used for the +XamS: HF4704 was grown in 25 ml TPA with low phosphate to an OD440nmof 0.7. experiment described in Fig. 7 contained a After infection with $Xam3 (moi = 5) total of 8.8 X 10” PFU, 6.0 X lo6 3H-cpm, 4.4 X lo6 35S-cpm, and a maximum of 15 mCi 32P were added and the culture aerated for 2 hr at 37”. The infected cells 8.3 % 35S-label not in phage particles. For infection the phages were diluted were collected by centrifugation, washed twice with borate, resuspended in 2 ml directly from the sucrose gradient fractions borate-EDTA and lysed with lysozyme and into the cell culture. At the highest mul3 cycles of freeze-thawing. An excess of tiplicities used (moi = loo), this introduced 10-b M EDTA and 10m2M NaCl into the Mg2++ and 20 pg of DNase and RNase, each, were added, and incubated for 10 culture, which is unlikely to affect the infection process. min at 37”. After a low-speed centrifugation (5 min at 15,000 rpm) the superGrowth, starvation, and injection of cells. natant was layered onto a 56 ml 5% to 20% sucrose gradient in 1 M NaCl, TrisHF4704 or its rep3- derivative were diluted EDTA, and centrifuged in a SW 25.2 rotor 20-fold from an overnight culture (grown for 3.5 hr at 25,000 rpm and 5”. Fractions without aeration in medium with a reduced were collected through a needle in the bot- glucose content) into TPA + T and aerated tom of the tube and the position of the at 37°C. The ODMon, was followed and at phages was identified by their infectivity 0.4 (ea. 2 X lo8 cells/ml) the culture was and radioactivity. The peak fractions were centrifuged at room temperature in a Sorvall pooled and stored at 4”. No dialysis or any centrifuge until 15,000 rpm was reached further purification was performed in order and decelerated with breaking. All low speed

centrifugations were carried out this way unless stat,ed otherwise. The cells were washed three times with half the culture volume of starvation buffer by centrifugat,ion followed by decanting of the supernatant. After t,he last wash the cells were resuspended in half the culture volume of starvation buffer and aerated at 37” for 3..5 hr. During the first half hour of starvation t’he OD440nmdecreased by lo-15 70, reflecting the reduction in size of the cells, whereas t,he number of colony formers remained constant. If after the starvation period the supplement to full medium with thymine was added, both, the ODJ40nmand the number of colony- formers increased until st,ationary phase was reached. If the supplement was added without thymine, the number of colony formers remained constant, wereas the ODIJOnm increased for about 43 min and then leveled off. No loss of colony-forming abilit,y was observed in medium without thymine for 2 hr after starvation. The starvation procedure described here was followed for all experiments described. It should be noted, t,hat longer starvation periods (up to 6 hr) or starvation at lower cell densities (1 X 108/ml) have been performed and had no effect on the results. For infection, the following was added to the cells in starvation buffer simultaneously, depending on the type of experiment to be performed: (1) phage and one volume of starvation buffer, (2) phage and one volume of supplement to full medium without, or (3) wit,h 4 pg/ml of t,hymine. Aeration was discontinued for the first 5 min after infection and then resumed. If the total DXA of the infected complexes was to be examined, the infection was terminat,ed by diluting the culture 1: 1 into cold 0.1 31 SaCl-Tris-EDTA containing 0.01 M sodium c>-anide and 0.01 JI sodium azide, followed by washing once with t,he same solut,ion. After resuspension in borahe-EDTA the cells were lysed by lysozyme (100 pg/ml, 10 min, 37”). Chemical

elutiov

of infected

complexes.

Differentiation of det,ached phage particles from eluted phage part,icles and Dn’A re-

maining with the cells was att,ained b> following the technique of Xcwboltl and Sinsheimer (1969b). After the desired t,imtB of infection the culture was centrifuged in the cold. The supernatant cont,aining thtt detached particles \vas saved and t.hc pellet. eluted three times with borate-EDTA h\, resuspending the cells, vortexing for 1.5 se{ and centrifugation for 2 min at’ 17,000 rpm. The three supernatant fractions containing the eluted particles were combined, :antl the cells resuspended separately in borattkEDTA. All procedures were carried out :I I 4°C. Extraction of I),VA. l’hage and detached or eluted particles were each incubat,ed with 200 pg/ml of predigested pronase (30 mitl at 37” in Tris-EDTA) in the presence of 1 % SDS for 2 hr. The DNA was t,hen oxtracted twice wit,11 one volume of TrisEDTA-saturated phenol. Each phenol 1~1~:~~ was reextracted with 0.5 volume of TrisEDTA. The combined aqueous phases were precipitated with 2 volumes of ethanol in the presence of 0.2 ill sodium acetate for at least 4 hr at -20”. The precipitak was collected by centrifugation (30 min, 15,000 vm, - 5”), the supernatant decanted nnd the remaining ethanol removed by suction. Lysates from infected cells were incubated svith SDS and pronase as described above. After 2 hr >$ volume of 5 M KaCl was added, the sample chilled t.o 4” and cenkifuged for 5 min at, 15,000 rpm at 0”. Recovery of parental viral DNA in the supernatant. was always greater than 95 7;. The supernatant, fraction was concentrat.ed by ethanol prrcipitation directly, or after phenol-dcprot.einiz:ltion, as described above. Centrifugation Tdmiyues. All cent rifugations mere carried out in a Spinco I,?-65 preparative ultracentrifuge. Large high-salt sucrose gradients (separation of viral DNA forms) : Gradient R of 5 ‘.; to 20% sucrose in 1 :II NaCl-Tris-EDTA of a total volume of 34 ml were overlayeretl with 1 to 2 ml of sample and centrifuged n,t 24,000 rpm for 16 hr at 5” in an SW 27 rotor. Ca. 45 fractions were collected from the top of the tube by pumping 70% sucrose in from the bottom. Large low-salt sucrose gradient,s (purification of detached ant1 eclipsed part iclrsj :

172

FRANCKE

The procedure was the same, except that the sucrose contained no NaCl and the running time was reduced to 3.5 hr. Small high-salt sucrose gradients: Gradients of 5 % to 20% sucrose in 1 M NaClTris-EDTA of a total volume of 3.5 ml were overlayered with 0.2 ml of sample and centrifuged at 55,000 rpm for 3.5 hr at 5” in an SW 56 rotor. Ca. 42 fractions were collected from the bottom of the tube by pumping oil in from the top. Alkaline sucrose gradients (separation of circular and linear viral DNA): Gradients of 5 % to 20 % sucrose in 0.25 M NaOH and 0.005 M EDTA of a total volume of 3.5 ml were overlayered with 0.2 ml of sample in Tris-EDTA and centrifuged for 6 hr at 55,000 rpm at 5” in an SW 56 rotor. Fractions were collected as described for small high-salt sucrose gradients. Short alkaline sucrose gradients (separation of RF1 from other viral DNA): The procedure was the same, except that the running time was reduced to 90 min. Neutral CsCl density gradients: A bottom layer of 3.5 g CsCI, 1.9 ml Tris-EDTA and 0.1 ml of a 1% blue dextran solution was overlayered with 1.85 g CsCl dissolved in 2 ml Tris-EDTA containing the DNA sample. Centrifugation was for 27 hr at 40,000 rpm at 20” in a 65 angle head rotor. Fractions were collected from the bottom of the tube by pumping oil in from the top. Of a total of ca. 480 drops the first 100 drops, containing the blue dextran band, were collected as one fraction and the remainder of the gradient was collected in lo-drop fractions. RESULTS

Injection of Unstarved Cells The studies to be presented deal with the fate of the infecting 4X174 DNA in prestarved host cells. In order not to complicate the interpretation of the results, experiments were generally carried out under conditions not allowing the replication of the parental RF. The repa- mutant of HF4704 was used for most of the experiments reported here. Its properties have been described in detail by Denhardt et al. (1967). It is unable to replicate 4X RF, yet the

AND RAY

formation of the parental RF appears to be unaffected by the mutation. This is confirmed in the experiment shown in Fig. 1. The DNA extracted from HF4704 (Fig. la) and reps- (Fig. lb), each infected with 100 32P-4Xam3 per cell, was separated by sucrose gradient cent’rifugation. During the 30 min infection period both cultures had been labelled with thymidine-3H. RF replication in the HF4704 culture had been suppressed by t,he addition of 150 pg chloramphenicol per milliliter. The figure shows that practically all of the 32P labeled DNA was converted into RF II (the smaller, slower sedimenting peak) or RF I. That the faster sedimenting peak indeed consisted of RF I was established by analyzing the material from this peak in short alkaline sucrose gradient sedimentation, where 96 % (4704) and 89% (reps-) sedimented rapidly as a supercoil. The small amount of parental label sedimenting faster than RF I in Fig. 1 was usually seen in this type of experiment but was not investigated further. The total recovery of parental label in Fig. la and b was 85%, and the total amount of newly synthesized DNA (3H) recovered from the gradients did not exceed one single-stranded phage DNA equivalent per infecting viral DNA molecule. We therefore conclude that in both systems using unstarved cells and a multiplicity of 100 phages per cell, practically all viral DNA was converted into RF and no replication of the parental RF took place. These results with unstarved cells are of importance to show that the observations to be reported below were indeed caused by the starvation of the cells prior to infection and not by the use of the reps- mutation or high levels of chloramphenicol. Injection of Starved Cells in the Absence of Nutrient Medium Figure 2 shows an experiment in which starved reps- cells were infected in starvation buffer with 32Plabeled phage (m.o.i. = 100) for 20 min. The infected complexes were lysed, treated with SDS and pronase as described in Materials and Methods and the lysate was centrifuged through a large high-salt sucrose gradient (Fig. 2a). The

INFECTION

OF STARVED

CELLS

FRACTION

NUMBER

WITH

$5174

FIG. 1. Infection of unstarved cells in medium with thymine. A 20-ml culture each, of HF4704 and rep;-, grown to 2 X lo* cells/ml in TPA + T, was infected with 32P-+Xam3 for 20 min. At 20 min before infection, 150 rg of chloramphenicol/ml had been added t,o the IIF4704-culture. Both cult,ures were labeled with 0.75 mCi 3HTdR from the time of infect.ion. After a 1:l dilution into cold 0.1 M NaClTris-EDTA, containing 0.01 M sodium cyanide and 0.01 M sodium azide, the cells were washed in the same solution and lysed in 1 ml borate with EDTA-lysozyme. The lysate was treated with 100 rg/ml of predigested pronase in the presence of 1% SDS (2 hr, 37”C), layered onto a 34 ml gradient of 5?;, to 20y0 sucrose in 1 M NaCl-Tris-EDTA and centrifuged at 24,000 rpm for 16 hr at 5°C. Fractions were collected, 100 pl of each fraction spotted onto a cellulose paper disk and counted for radioactivity. Sedimentation in this and all other figures was from right to left. (a) HF4704 cells with 150 pg chloramphenicol/ml; (b) repa- cells: l ---0, 32P-parental label; O-----O, “H-label afler infection.

recovery of the input parental label from the gradient was 71%. The marked forward trailing of t’he peak was frequently seen in centrifugations of this type. The fractions indicated by the bracket in Fig. 2a were pooled, concentrated by ethanol precipitation and analyzed by sedimentation through a small high-salt sucrose gradient and an alkaline sucrose gradient with an added marker DNA ext,racted from 3H-

labeled phage. In both cases the DNA obtained from the infected complexes cosedimented with the marker. The slightly re-duced sedimentation rate in neutral sucrose (Fig. 2b) could possibly be due to a difference in the secondary structure of the DNA from infected complexes as compared to the viral marker DNA (Forsheit and Ray, 1970). RF II DNA would have sedimented much slower. The percentage of linear DNA

174

FRANCKE

FRACTION

AND RAY

NUMBER

FIG. 2. Infection of starved cells in starvation buffer. rep;- cells, grown in 20 ml TPA f T to 2 X 108/ml, were washed 3 times in 10 ml starvation buffer and aerated for 3.5 hr in 10 ml starvation buffer at 34”. At 20 min after infection with 32P-labeled +Xam3 (m.o.i. = lOO), the lysate was prepared and centrifuged through a sucrose gradient as described in legend to Fig. 1. The fractions indicated by the bracket were pooled and concentrated by ethanol precipitation. A 3H-labeled viral DNA marker was added; one half of the material was analyzed by small sucrose gradient, and the other half by alkaline sucrose gradient centrifugations. Both types of gradients, 5% to 20% sucrose in a total volume of 3.5 ml, were centrifuged at 55,000 rpm, the neutral gradient for 3.5 hr and the alkaline gradient for 6 hr. Five-drop fractions were collected directly onto cellulose paper disks and counted. (a) Large, high salt sucrose gradient; (b) fractions 10-28 of a total of 44 fractions collected from the small high-salt sucrose gradient; (c) fractions 12-30 of a total of 42 fractions collected from the alkaline sucrose gradient. The fractions not shown in (b) and (c) contained no radioactivity. O-0, 32Pparental label; O-----O, 3H-labeled viral DNA marker.

from the infected complexes (the slowersedimenting component in the alkaline sucrose gradient, Fig. 2~) was the same as in the phage preparation used in this experiment. Thus, after infection of starved cells in the absence of nutrient medium, about 70% of the infecting viral DNA could be recovered from the infected complexes as single-stranded, mostly circular DNA. The

same results were obtained with starved HF4704 cells in the presence or absence of 150 rg chloramphenicol per milliliter. Infection of Starved Cells in Nutrient Medium (a) DNA from infected complexes. rep3cells, starved for 3.5 hr, were infected with 100 3H-labeled 4Xam3 per cell in medium without thymine for 15 min. Figure 3a shows the radioactivity profile and the infectivity

of the parental D-1’A after sucrose gradient centrifugation. The infected complexes had been lysed as described for the experiment reported in Fig. 2. The recovery of input parental label from this experiment was 32 %t. This low recovery was due to more viral particles detaching from the infected complexes in nut,rient, medium than in starvation buffer, as will be shown later. Of the two major peaks the faster one, about 30% of the radioactivity, represented singlestranded, mostly circular DNA, as will be shown below; it, also contained most of the infectivit,y. The larger peak, although sedimenting roughly at the position where RF1 would be expected, was not infectious. Since RF can have a considerably lower infectivity

than single st’rands (Jaenisch et al., 1906), the material from this peak Teasexamined by short alkaline sucrose gradient sedimentation (not shown here) and found to contain lessthan 3 ‘2 rapidly sedimenting supercoiled DSA. The infectivity of these peak fractions was also t,est,edafter heat clennturation and no increase of infectivity \Y:LS seen, arguing that this peak did not, coni airi intact viral D?;A. Alkaline sucrose gradient sedimentation of the combined fractions containing the parental DNA from Fig. 3:~ showed t,hat, except for some circular 1)X-\ the majority of the parentsal DKA UX~ smaller Ohan unit-lengt’h linear viral 11X.1 (Fig. 3b). The position of unit-length lincb:rrs is indicated in the gradient by i hc sm:t~ll~

2

I-

: (b)

I-

FRACTION

FIG. 3. Infection

NUMBER

of starved cells in medium without, thymine. rep,cells, grown and st,arved as described in legend to Fig. 2, were infected in 10 ml of starvation buffer with 3H-labeled +Xam3 (m.o.i. = 100). At t,he time of infection 10 ml of supplement to full medium were added. At 20 min after infection, t,he lysate was prepared and centrifuged through a large high-salt sucrose gradient as described in legend to Fig. 1. Fractions were assayed for radioactivity and infectivity on spheroplasts. Fractions 11 to 33 were pooled, concentrated by ethanol precipitation and resedimented through an alkaline sucrose gradient as described in legend to Fig. 2. Three-drop fractions were collected from this gradient,. (a) Large high-salt sucrose gradient; (b) alkaline sucrose gradient, a---@, 3H parental label; O-----O, 32P viral DNA marker; X--X, infectivity.

176

FRANCKE

AND

RAY

i 8 s h X

E 8 a w

# IO

20 FRACTION

30

40

NUMBER

FIG. 4. Infection of starved cells in medium with and without thymine. Two cultures of rep;- cells were starved and infected with 32P-labeled +Xam3 (m.o.i. = lOO), and the lysates were sedimented through large high-salt sucrose gradients as described in the legend to Fig. 3. Infection was in both cases for 20 min. To one culture no thymine and to the other 2pg of thymine/ml and 0.375 mCi of 3HTdR were added. (a) infection in medium with thymine and 3HTdR; (b) infection in medium without thymine; a----0, 3zP parental label; O-----O, 3H label after infection.

peak of the added DNA marker extracted DNA, the majority of it was partially from 32P-labeledphage. (In similar gradients degraded, and a small fraction was conto be shown below, the position of circles verted into RF even in the absence of thyand linears of the viral DNA marker will mine, If the same type of experiment was caronly be indicated by arrows.) The shoulder on the slower sedimenting side of the major ried out in the presence of t,hymine, the peak in Fig. 3a contained some RF II with fate of the parental label was very similar. intact viral DNA, as indicated by the small Figure 4 shows a similar experiment to that peak of infectivity at that position. Sum- reported in Fig. 3a, with 32P-labeledphage. marizing these observations, in an infection In Fig. 4b infection was in medium without of starved cells in nutrient medium, only thymine, whereas in Fig. 4a the medium about 30% of the infecting viral DNA contained 2 pg/ml of thymine and 3H-labeled could be recovered from the infected com- thymidine. Short alkaline sucrose gradients plexes; while about 30% of this DNA sedi- of the material sedimenting at the position mented at the position of infectious viral of RF I (fractions 19-21) in Fig. 4a showed

INFECTION

OF

STARVED

that S5% of the 3H-labeled newly synthesized DNA, but only 7 % of the 32P parent,aI label sedimented rapidly as supercoils. Alkaline sucrose gradient sedimentat.ion of t’he total DNA pooled from this gradient (Fig. 4a) revealed the same profile for the

CELLS

WITH

6x174

177

parental DNA as shown in Fig. 3b. Thus, the presence or absence of thymine in the medium (also tested at 20 pg/ml) had no effect on t’he degradation of the infecting DNA or the amount of RF synthesized. To further invest,igate the nature of thr>

IO

IO

IO

FRACTION

NUMBER

DROP

NUMBER

FIG. 5. Analysis of four regions from Fig. 4b. Regions 1 to 4, as indicated in Fig. 4b, were separately pooled and concentrated by ethanol precipitation. 3H-labeled viral DNA was added to each fraction. Alkaline sucrose gradient sedimentation was as described in legend to Fig. 2. CsCl density gradient, centrifugation (using the two layer technique described in Materials and Methods) was for 27 hr at 40,000 rpm and 20”. Ten drop fractions were collected directly onto cellulose paper disks. (a, b, c, d) alkaline sucrose, and (e, f, g, h) CsCl density gradients of portions 1 (a, e), 2 (b, f), 3 (c, g), and 1: (d, h), from Fig. 4b. @---a, s2P parental label; O-----O, 3H labeled viral DNA marker. The positions of the marker DNA components are indicated by arrows (C = circular molecules, /, = full-length linear molecules). The CsCl gradients are shown from drop No. 100 to drop No. 300 (of a total of ca. -180 drops collected). The remainder of the gradients contained no radioactivity.

178

FRANCKE

degradation product, four different regions (indicated by brackets in Fig. 4b) of the parental DNA were separately pooled and examined in alkaline sucrose (Fig. 5a, b, c, d) and neutral CsCl density gradients (Fig. 5e, f, g, h). To both types of gradients a 3H-labeled DNA extracted from phage was added as marker. Region 4, the fastest sedimenting parental DNA consisted mainly of intact circles and contained some degraded DNA. Regions 3, 2, 1 consisted mainly of degraded DNA. The difference in the sedimentation rate of the degraded DNA as compared to the position of the full-length linears of the added marker increased from region 4 to 1, indicating that the degra.dation product was not uniform in length. Two peaks of parental DNA at the position of intact circles and full-length linears were seen in region 1 (Fig. 5a), representing the small amount of RF II made under these conditions. Whether the small peaks of circular molecules observed in regions 2 and 3 (Fig. 5b and c) were due to trailing of region 1 into these regions or arose from partially finished RF II molecules containing a still intact parental circle, could not be decided. The latter possibility was supported by the finding that regions 2 and 3 contained double-stranded material banding as shoulders on the light side in CsCl density gradients (Fig. 5f and g). This shoulder appeared as a separate peak in region 1 (Fig. 5e), whieh was known to contain intact RF II. It was further noticed that the denser single-strand peak from regions 1 to 3 always banded slightly on the light side of the intact single-strand marker (Fig. 5e, f, g), whereas the parental DNA from region 4 cobanded exactly with the marker. Whether this property of the degraded parental DNA was due to a partial doublestrandedness or was caused by the preferential degradation of a G-C-rich region of the infecting DNA, could not be decided and will be discussed later. A reason for the degradation of the infecting viral DNA might have been that too high phage to cell ratios were used for starved cells. To examine this possibility a series of infections was carried out with starved cells at multiplicities ranging from 0.1 to

AND

RAY

100. A summary of t’hese results is presented in Table 1. Both the degree of degradation and the relative amount of RF I synthesized were independent of the multiplicity of infection. HF4704 in the presence of chloramphenicol and reps- behaved similarly in this respect. The possibility that more functional RF II had been synthesized at low multiplicities could be excluded, since this should have increased the relative amounts of undegraded parental DNA seen in long alkaline sucrose gradient sedimentations. We would like to conclude from this result that the capacity of starved TABLE RF

1

SYNTHESIS AND DEGRADATION OF INFECTING +X174 DNA IN STARVED CELLS AT DIFFERENT MULTIPLICITIES OF INFECTION” yo of input

Cell type

M.o.i.

Recovered from infrfd

In RF1

parental

label

In degraded DNA

In circular DNA

16.2 16.9 15.0 19.1 16.2 20.0

5.3 3.9 4.15 4.0 3.75 4.1

16.2 18.8 18.4

2.75 3.2 3.05

plexes

rep3-

HF4704

100 20 1.75 30 20 1.8 10 22 1.6 3 20 1.65 1 22 1.55 0.3 20 1.5 (+150 pg chloramphenicol/ml) 100 19 1.8 10 22 2.1 1 21 1.9

a Starved cells were infected in medium with thymine for 30 min with 32P-+Xam3 at the m.o.i.‘s indicated. After pelleting, the infected complexes were resuspended in borate-EDTA and a sample was counted for radioactivity (column 3). The DNA was extracted with phenol after sodium dodecyl sulfate-pronase treatment and concentrated by ethanol precipitation. The amount of RF1 (column 4) was calculated as rapidly sedimenting material in short alkaline sucrose gradients (90 min at 55,QQO rpm). Degraded and circular DNA were measured after long alkaline sucrose gradient sedimentations (6 hr at 55,000 rpm) as sedimenting with the circular DNA peak or slower than the linear DNA peak of the added viral DNA marker, respectively. Full-length linear DNA could not be calculated, because no distinct peak was seen at this position.

INFECTION

OF

STARVED

cells to synthesize +X-complementary strands is generally impaired and that the primary reason for the degradation is not that there is a limited number of sites in starved cells. Even at multiplicities below one infecting phage per cell the relative amount of RF made was as low as at high multiplicities. The fact that some RF was synthesized at all even in the absence of thymine, could either be explained by a leakiness of t,he T- mutation in HF4704 and reps- or by taking into account that starvation in buffer in t.he absence of an energy source may not have depleted the intracellular pool of thymidine nucleotides. The mechanism that is impaired in st,arved cells leading to the partial breakdown of the viral DNA is unknown. But it is conceivable that the degradation is a result of attempted DNA synthesis. Table 2 summarizes the results obtained from studies of the time course of the degradation and of RF synthesis in starved reps- cells after infection in medium with thymine. While the formation of the small amount of RF was very rapid, the breakdown of the remaining viral DNA appeared to be a much slower process. One reason why the presence of thymine does not prevent the degradation could be that extraneous thymine-added after starvaTABLE

2

TIME COURSE OF RF SYNTHESIS AND DEORADATI~X OF INFECTING $X174 DNA IN STARVED CELLS" $JO of input MinUteS after infection 2 7 20 30

parental

label

- Recovered from infected complexes 52 26 20 22

In RF1

In degraded DNA

In unitlength linear DNA

0.1 1.1 1.05 1.2

4.2 8.75 12.2 16.4

3.5 2.45 -

In circular DNA

33.2 8.4 2.4 1.9

5 Starved repa- cells were infected in medium with thymine with 32P +Xam3 (m.o.i. = 100) and samples were withdrawn at the times indicated (column 1). The procedures and calculations were as described in footnot,e to Table 1.

CELLS

WITH

17!)

&X174 TABLE

3

INFECTION OF ST.&WED CELLS WITH $X171 .\T DIFFEI~ENT TIMES AFTER TRANSFER INTO MEDIUM WITH THYMINED Minutes Unst,arved

after cells 0 10 30

transfer -...-.

rO RF1

synthesized

.-.~-~~ 100 27.2 81.5 91.0

___--.-~~ ..__ n Starved reps- cells were transferred into medium with thymine and infected immediately, 10 min, and 30 min after the transfer with 32P +XamS (m.o.i. = 100) for 30 min. The l)xA from the infected complexes was extracted by sodium dodecyl sulfate-pronase and phenol and analyzed by short alkaline sucrose sedimentation. The amount of rapidly sedimenting DNA (ItFI) was calculated as percent of that synthesized in 11r1starved cells under the same conditions.

tion-is not. readily utilized for viral DSA synthesis once the infection process has begun under unfavorable conditions immediately after starvation. If starved cells were transferred into thvmille-colltnirliIlg medium prior to infection rather than at. the time of infection, they fast rrgnined their ability to synt.hesize normal amounts of RF (Table 3). A preincubation of starved cells in medium without thymine up to 30 min, on the other hand, did not change the fat’e of the infecting DSA as compared to an infection immediately after starvation. b. Spontaneous detach,ment and d~emical elution of pal-tides containiny vital Il:YA. In the experiments reported so far, the fate of the infecting DNA was examined after extraction of the tot.al DNA from infected complexes, i.e., t,hat portion of the viral DNA that was not removed from the cells by simple washing in 0.1 M SaQ TrisEDTA. Newbold and Sinsheimer (1YSSb) have reported that two types of viral DNAcontaining particles could be obtained after infection of starved cells in starvation buffer. One type, called spontaneously detached particles, remained in the supernatant, aft,er pelleting of the infected complexes, and the part#icles, could be other type, “eclipsed” chemically eluted by several washings of the infected complexes with borat.r-EDT-4. Using the spume procedure the results ~howrt

180

FRANCKE

AND

TABLE RECOVERY

OF 32P-LABELED

+X174 MATERIAL

Cell type rep3HF4704 H502

DNA AFTER

4

AS SPONTANEOUSLY DETACHED AND INFECTION OF STARVED CELLS~

After infection in Starvation Medium Medium Medium Medium

RAY

buffer without thymine with thymine with thymine with thymine

CHEMICALLY

ELUTED

70 of input parental label Spontaneously detached

Chemically eluted

38.3 61.5 72.0 58.4 (32.7) 56.3

50.5 19.25 22.5 29.0 (26.2) 25.5

Q Cells of the type indicated (column 1) were starved in 2.5 ml of starvation buffer at a density of 4 x 108 cells/ml for 3.5 hr. At the time of infection (50 EzP+Xam3/cell), 2.5 ml of starvation buffer, or the supplement to full medium with or without thymine was added (column2). After 30 min the cultures were centrifuged in the cold, and the supernatant, containing the detached material, was saved (column 3). The pellet was eluted 3 times with borate-EDTA (1 X 1 ml, 2 X 2 ml) and the eluates were combined (column 4); 200 pl of each fraction were spotted onto a glass filter disk and counted for radioactivity. Acid-precipitable radioactivity (determined as described in Materials and Methods) is presented in parentheses for the experiment with HF4704 cells.

in Table 4 were obtained. In this experiment the number of borate-EDTA washings was limited to three cycles. Up to seven or more cycles removed more than 90 % of the input label from the cells (not included in Table 4). The amount of detached material after an infection in medium was relatively greater than in starvation buffer, which account’s for the difference in parental label recovered from infected complexes under these different conditions, observed above (Figs. 2 and 3). The sedimentation behavior of the material eluted from cells infected in medium was similar to that reported for “eclipsed” particles by Newbold and Sinsheimer (196913). It contained a small amount of acid-soluble material, whereas the majority of the radioactivity sedimented as a broad peak intermediately between intact phage and free viral DNA (Fig. 6). A similar experiment was performed using double-labeled 4Xam3 (“-DNA and 35S-protein). Figure 7a and d show sucrosegradient sedimentations of detached and eluted particles after infection of starved HF4704 cells in medium with thymine. The detached particles sedimented as a sharp band at the rate of the slow portion of the eluted particles. Figure 7b, c, e, and f show the same particles after treatment with sarcosyl (b and e) or SDS-pronase (c and f). SDSpronase liberated the viral DNA from both

IO FRACTION

20 NUMBER

FIG. 6. Sucrose gradient sedimentation of particles eluted from prestarved cells infected in medium with and without thymine. Prestarved repa- cells, infected for 30 min with 3H-labeled +Xam3 (m.o.i. = 100) were pelleted and eluted 3 times with 0.5 ml borate-EDTA as described in legend to Table 4. The three eluates were combined and mixed with azP-labeled intact phage and free DNA at a ratio of 3: 1; 0.2 ml was sedimented in a small high-salt sucrose gradient as described in legend to Fig. 2 except that the running time was reduced to 50 min at 55,ooO rpm. (a) Eluted particles after infection of starved cells in medium with and (b) without thymine. O-0, 3H parental label; O-----O, 32P labeled phage and viral DNA markers.

IKFECTION

OF

STARVED

types of particles; but, whereas the coat protein of t,he detached particles appeared to be resistant to t.his treatment, that of t,he eluted particles was degraded. The small amount of residual 35S-label left at the original posit’ion was probably due to incomplete removal of detached particles by simple pelleting of the infected cells. The difference between the prot.ein components of the two types of particles is not’ understood. It can also be seen from Fig. 7 (b and e) that sarcosyl had no effect on the detached particles, whereas eluted particles sedimented more uniformly and at the rate of detached particles aft.er this treat.ment. This observation indicated that eluted particles contained an additional component causing their faster and heterogeneous sedimentation behavior. It is assumed that the additional component was of cellular origin.

41

(a)

^

CELLS

WITII

+X174

IS1

Similar results were obtained when eluted particles from an infection in starvation buffer were studied. The importance of these results for the fate of infecting 4X-DNA in starved cells in nutrient, medium, is that ca. 60% detached spontaneously, that the majority of the attached DNA could be eluted in borateEDTA and was still associated with paren.tal viral protein. Also at lower multiplicities (t,ested at) m.o.i. = 1) the percenbages of detached and eluted parental label w-cre similar (not included in Table 4). c. Viral DXA e&acted from detached a& eluted particles. Since t,he detachment and elution of particles as described in t,he preceding paragraph provides a means of locating the infecting DNA, it was of interest to see which of the different forms of viral DKA described in the first paragraph of the

(b)

FRACTION

NUMBER

FIG. 7. Double-labeled (DNA-3H, 35S-protein) detached and eluted particles from starved cells infected in medium with thymine. Prestarved HF4704 cells were infected in medium wit.h thymine with 3HJ5S-labeled &Xam3 (m.o.i. = 50) for 30 min. Detached particles were obtained from the supernatant after pelleting of the infected complexes and eluted particles prepared as described in legend to Table 4. Both types of particles were either kept in the cold, treated for 10 min with 0.5% sarcosyl at 37”, or digested with pronase (100 rg/ml, 2 hr, 37”) in the presence of 1% SDS. Small high-salt sucrose gradients were overlayered with 0.2 ml of each preparation and centrifuged for 70 min at 55,000 rpm and 5°C. (a, b, c) detached part.icles, (d, e, f) eluted particles; (a, d) no treatment, (b, e) sarcosyl treatment, (c, f) SDS-pronase treat.ment,. @---a, 3H parental DNA label; O-----O, 3% parental protein label.

182

FRANCKE

FRACTION

NUMBER

AND

RAY

DROP

NUMBER

8. DNA from detached and eluted particles and DNA remaining with the cells after an infection of starved cells in medium with thymine. Starved rep3- cells were infected with z2P labeled +Xam3 (m.o.i. = 100) for 30 min in medium with thymine. Three fractions containing the detached particles, the eluted particles and the DNA remaining with the cells were obtained as described in legend to Table 4. After SDS-pronase treatment the DNA of the three fractions was extracted with phenol and concen3H-labeled viral DNA was added as a marker and all three DNA-prepartrated by ethanol precipitation. ations analyzed by neutral and alkaline sucrose and by CsCl density gradient centrifugations. (a, b, c) DNA from detached, and (d, e, f) eluted particles, and (g, h, i) remaining with the cells, in small highsalt (a, d, g), and alkaline sucrose gradients (b, e, h), and (c, f, i), and in CsCl density gradients. l ---0, 32P parental label; 0 -----0, 3H-labeled viral and, in (f) only, RF-DNA. The position of the viral DNA marker is indicated by arrows as SS (= single strands) in neutral sucrose as an C (= circular DNA), and L (full-length linear DNA) in alkaline sucrose. FIG.

section was contained in which type of particle. Figure 8 shows neutral sucrose gradient-, alkaline sucrose gradient- and neutral CsCl density gradient centrifugations of the DNA from detached and eluted particles and of the DNA remaining with the cells. For this experiment starved reps- cells were infected with 32P-labeled phage in medium with thymine. To all gradients a marker of 3H-labeled viral DNA was added. The gradient shown in Fig. 8f also contained and 3H-RF II marker. The detached particles contained some still circular molecules, a definite peak of unit-length linears, and degraded material with a different size dis-

tribution than had been seen in the DNA from infected complexes. These particles were the only source of unit-length linear molecules. The eluted particles showed a distribution in their DNA similar to that seen in the infected complexes, i.e., some still circular DNA and a majority of linear material shorter than unit-length. The CsCl density gradient profiles indicated that the DNA remaining with the cells was enriched for double-stranded material, which was the only source from which a definite peak of RF could be obtained. Whether the somewhat lighter density of the DNA from eluted particles compared to the viral DNA

ISFECTIOS

OF

STARVED

marker KM caused by some degree of double-strandedness was invest’igated in two different ways. Figure 9 shows two different attempts I o incorporate thymidine-3H label into the DNA from eluted part,icles. In Fig. !)a the cells had been prelabeled with 3HTdR, starved and infected in the absence of thymine. In Fig. 9b unlabeled cells were starved and infect ccl in the presence of thymine and “HTdlZ. III both cases no peak of 3H label

20-

CELLS

WIT11

4S174

ISIS

was found to sediment with the eluted particles. In the case of the prelabeled cells :I, slow sedimenting DSA was also eluted from the cells, indicatjing that the elution procedure did not liberate only phage particles from the cells. The nadure of this DNA was not further investigated. Another approach involved CC1 banding of the DSA from eluied particles before and after lieat denaturation. If the shift to the light densit!

(b)

I I P I

10 -

:

5~~~olL&&Lk 20 FRACTION

NUMBER

FIG. 9. Eluted particles from prelabeled starved host cells infected in medium without thymine, and unlabeled, starved host cells infected in medium containing tritiated thymidine. Two 20-ml cultures of HF4704 in TPA + T were grown to 2 X lo* cells/ml. One culture was labeled with a total of 1.4 mCi of 3HTdR added in 0.2 mCi portions at 30-min intervals. Both cultures were washed and starved in 10 ml of starvatiou buffer for 3.5 hr. Infection with 32P-labeled +Xam3 was for 30 min (m.o.i. = 100) in medium wit,hout thymine in case of t,he prelabeled culture and in medium with 2 *g/ml of thymine and 0.75 mCi 3HTdR for the other culture. After pelleting of the infected complexes, they were eluted three times with borate-EDTA (1 X 1 ml and 2 X 0.5 ml). The separately combined supernatant fractions from each culture were layered onto 34 ml 5y0 to 20% sucrose gradients (in Tris-EDTA) and centrifuged for 3.5 hr at 24,090 rpm. (a) starved, 3HTdR-prelabeled cells, infected in medium without thymine; (b) starved, unlabeled cells, infected in medium with thymine and 3HTdR. @---a, 32P parental viral label; 0 -----0, 311 prelabel (a) or 3H label after infection (b).

184

FRANCKE

AND RAY

? (a)

I

lb)

FRACTION

NUMBER

DROP

NUMBER

FIG. 10. Comparison of the total DNA from infected complexes and the DNA extracted from eluted particles after infection of starved cells in medium with thymine. A culture of starved rep3 cells in starvation buffer was split into two halves. Each half was infected separately for 30 min in medium with thymine, one with aH- and the other with 32P-labeled +Xam3 (m.o.i. = 100 in both cases). Both cultures were pelleted and resuspended in 1 ml borate-EDTA. The 3H culture was lysed with lysozyme, and the a2P culture was eluted 3 times with borate-EDTA. The eluates were combined with the 3H lysate and the DNA extracted with phenol after SDS-pronase treatment. After ethanol precipitation the DNA was analyzed by neutral sucrose and alkaline sucrose gradients and by CsCl density gradient centrifugation. (a) Small high-salt sucrose gradient; (b) alkaline sucrose gradient; (c) CsCl density gradient (drop No. 170 to 440, of a total of 440 drops collected). a----@, 3H parental label from the infected complexes; 0 -----0, 32P parental label from the eluted particles.

of this DNA had been caused by some degree of double-strandedness it should have cobanded exactly with the viral DNA marker after denaturation. Since it was found that the denaturation did not affect its banding properties we favor the interpretation that it had been due to a possibly specific degradation of the single-stranded DNA. A comparison of DNA from eluted particles with the total DNA from infected complexes is shown in Fig. 10. In this experiment two parallel infections with 3Hand 32P-labeled phages were carried out. Eluted particles prepared from the 32Pculture

were

mixed

with

the lysate

of infected

complexes of the 3H culture and the DNA extracted together. All characteristics of double stranded DNA were only seen in the

3H profiles: (1) a separate peak of RF in CsCl density banding, (2) a slow sedimenting shoulder at the RF II position in neutral sucrose, and (3) a relatively greater amount of still intact DNA in alkaline sucrose. The proportionally lower recovery of 32Plabel in single-stranded and degraded DNA was due to the limitation to three borate-EDTA washings while preparing the detached particles. The DNA from detached and eluted particles after infection in starvation buffer was also examined as described in the experiment reported in Fig. 8. These gradients are not shown here but were included in Table 5, giving a summary of the forms of parental DNA found in det,ached and eluted particles and remaining with the cells after infection of starved cells under various con-

INFECTION TABLE

OF

STARVED

5

FATE OF PARENTAL +X174 DNA UPON INFECTION OF STARVED CELLS" Consists DNA

from

,IflLr DNl

Fulllength linear L DNA

of (%) Degraded DNA

RFDNA

CELLS

WITH

+X174

1 U.j

Endonuclease I-deficient derivative (IS HF4704) gave us essentially t’he same results (compare also Table 4), with the minor difference that’ aft’er a 30 min infection in medium with thymine the amount of surviving circular DNA was about t,wice that usually seen in HF4704 or reps--. DISCUSSION

Based on the finding that after infect’ion of unstarved cells even at high multiplicities of infection (Fig. 1) essentially all infecting 90 4 None None $X Dn’A was converted into RF, we conclude that the degradat,ion of the viral in medium with thymine DNA observed in prest,arved cells was due 7 40 53 None partito a change in the cells caused by the star(40%) vation. ?Jo degradation or RF formation particles 10 90 None was seen if infection was carried out in @O%‘o) starvation buffer (Fig. 2). Under this conCells after bor13* 54b 36 dition, as already described by Newbold ate-EDTA-eluand Sinsheimer (1969b), a small amount of tion (10%) virus particles detached spontaneously Acid-soluble material (30%) whereas the remainder had not penetrated and could almost completely be &ted in a Starved repscells were infected with 32P borate-EDTA as particles containing the +Xam3 (m.o.i. = 100) for 30 min either in starvaparental DNA still as single-stranded circlrs. tion buffer or in medium with thymine. Detached If starved cells were transferred into and eluted particles were prepared as described in nutrient medium and simultaneously infootnote to Table 4. The eluted cells were resusfected, with increasing t,ime after infection pended in borate-EDTA. The DNA was extacted more and more parental label detached from and analyzed by alkaline sucrose and CsCl density gradient centrifugation as shown in Fig. 8. The the cells (Table 2). After a 30 min infect.ion, amount of RF-DNA was calculated from CsCl these detached parkles contained, in :iddidensity gradients and circular, full-length linear, tion to some surviving circles, :L considerable and degraded DNA from alkaline sucrose gradiamount of unit-length linear molecules and ents. The percentages in parentheses indicate the some degraded DNA. JIost of thr remainamount of parental label found in each particular ing DKL4 could also be eluted from the fraction as an estimated average from several infected complexes under these conditions. similar experiments. Such eluted particles contained mostly dcl* These two values were calculated from a long graded DNA. The parental DKL4 rt~ctovcretl alkaline sucrose gradient in which RF1 is pelleted, from both types of particles was still singleand since RF11 appears both as RF-DNA in CsCl stranded and attached to pnrent,al viral coat, and as linear DNA in alkaline sucrose, the sum of these componentjs exceeds 100%. protein. That they indeed represented t\~o different types of particles was seen in tht: ditions. We would like to repeat here t’hat sedimentation behavior and different s;tubilthese results were essentially independent of it’y in detergents (%‘ig. 7). Srwbold an(l the multiplicity of infection, of the presence Sinsheimer (1969b) have sho\vn that or absence of thymine in the medium, and “eclipsed” particles obtained after elation of the type of cell used (HF4704 and reps-). of cells infected in starvation buffer have Additionally, the effect of various levels of varying port’ions of their Dn’A protruding chloramphenicol (30-150 pg/ml) was found from the particle, and suggested thk :LS:L to have no effect on the amount of degra- reason for their heterogeneous, faster s&ition. mentation rate, as compared to tktacht:tl :ln infection of starved H502 cells (an particles. We ha,ve rcportrcl here 11~11~ch Infection Detached cles Eluted (65%) Znjection Detached cles Eluted

in starvation parti(25%) particles

buffer 38

37

25

None

186

FRANCKE

particles both, after infection in starvation buffer or in medium, could be converted into the slower sedimenting type by sarcosyl treatment and that this conversion was not accompanied by loss of parental protein label. We therefore suggest that the eluted particles contained a host cell component of unidentified nature. RF synthesis in starved cells was minimal and the amount of RF found independent of the presence of thymine in the medium. Whatever RF had been made remained with the cells after the elution procedure and no double-stranded DNA could be demonstrated in either type of particle. Since in starved cells both, RF synthesis and penetration of the infecting DNA, appeared to be greatly reduced, we suggest that in normal infection of unstarved cells active viral DNA synthesis is required for penetration, possibly replicating the parental single strand out of its coat. The observations of Newbold and Sinsheimer (1969b), that UV-inactivated +X fails to penetrate, could then not only be explained by possible DNA-protein crosslinking but also by a DNA synthesis stop at the site of the UV damage. In light of this hypothesis, the degradation observed in starved cells might follow an initial attempt of synthesis. This interpretation was supported by two observations. The degraded DNA banded in CsCl density gradients slightly on the light side of intact viral DNA, indicating that possibly a specific G-C-rich region was degraded preferentially. Also, the result obtained with the Endonuclease-I-deficient mutant H502 suggested that the degradation was not caused by a relatively higher activity of this enzyme in starved cells. At this time, we have no direct explanation for the observed phenomena. The reduction of sites for the viral DNA replication in starved cells to a limited number per cell (Yarus and Sinsheimer, 1967), cannot be made responsible since the relative amounts of degradation and RF synthesis were independent of the phage to cell ratio even at low multiplicities (Table 1). One would rather have to envisage a mechanism under which any given infecting viral DNA has the same chance of being degraded or, at a low probability forming an RF. Uninfected,

AND

RAY

starved cells resume normal growth and their capability to replicate $X in medium with thymine (Table 3), while even after a long incubation of infected complexes in thymine-containing medium the yield of RF is not increased. Thus, the event leading to degradation probably occurs very shortly after the adsorption of the phage. Since the degradation product is acid soluble material (Table 4) and linear pieces of DNA, the first event could be a single endonucleolytic nick. Whether this nicking is specific and might also be required for the normal synthesis of the first RF cannot be decided. Short pulses with 3HTdR after infection of starved cells have given inconclusive results unpublished data). To so far (Francke, study this mechanism the system of starved cells was not suitable because of the predominance of the degradation process. ACKNOWLEDGMENT This research was supported by a USPHS International Postdoctoral Fellowship to B.F. (No. 5 F05 TWO 1536-02) and by research grants from the U.S. Public Health Service (AI 07717) and the National Science Foundation (GB 18074). We would like to thank Dr. Virginia Merriam, Dr. Arleen Forsheit, and Mr. Randy Schekman for cooperation and stimulating discussions. REFERENCES DENHARDT, D. T., and SINSHEIMER, R. L. (1965). The process of infection with bacteriophage #X174.111. Phage maturation and lysis after synchronized infection. J. Mol. Biol. 12, 641646. DENHARDT, D. T., DRESSLER, D. H., and HATRAWAY, A. (1967). The abortive replication of +X174 DNA in a recombination-deficient mutant of Escherichia coli. Proc. Nat. Acad. Sci. u. s. 57,813~820. FORSHEIT, A. B., and RAY, D. S. (1970). Conformations of the single-stranded DNA of bacteriophage M13. Proc. Nat. Acad. Sci. U. 8. 67, 1534-1541. HUTCHISON, C. A., and SINSHEIMER, R. L. (1966). The process of infection with bacteriophage +X174. X. Mutations in a +X Lysis Gene. J. Mol. Biol. 18, 429447. JAENISCH, R., HOFSCHNEIDER, Ph.H., and PREUSS, A. (1966). gber Infektiijse Substrukturen aus Escherichia Coli Bakteriophagen. VIII. On the Tertiary Structure and Biological Properties of +X174 Replicative Form. J. Mol. Biol. 21, 501-516.

ISFECTION

OF STARVED

It., SALIVAR, W. O., NEWBOLD, J. E., and SINSHEIMER, R. L. (1969). The process of infection with the bacteriophage +X174. XXVI. Transfer of the parental DNA of bacteriophage +X174 into progeny bacteriophage particles. J. Mol. Biol. 39, 64-654. NEWBOLD, J. E., and SINSHEIMER, R. L. (1969a). The process of infection with bacteriophage +X174. XXXI. Abortive infection at low temperatures. J. ,Ilol. Biol. 49, 2348. NEWBOLD, J. E., and SINSHEIMER, R. L. (1969b). The process of infection with bacteriophage +X174. XxX11. Early steps in the infection

KNIPPERS,

CELLS

WITII

4X17-l

1\;

process: Attachment, eclipse and DNA penttration. J. Mol. Biol. 49, 49-66. NEWBOLD, J. E., and SINSHEIMER, R. L. (1971)). The process of infection with bacteriophage +X174. XXIV. Kinetics of the attachment and eclipse steps of the infection. .7. 17irlrb. 5, 427431. SINSHEIMER, R. L. (1968). Bacteriophage +Xli4 and related viruses. Progr. Nucl. Acid Res. Mol. Biol. 8, 115169. YARUS, M. J., and SINSHEIMER, It. L. (196i). The process of infection with bacteriophage +X174. XIII. Evidence for an essential bacterial “site.” J. Viral. 1, 135-144.