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Replication process of single-stranded DNA of bacteriophage φX174
J. Dfol. Biol.
(1967) 29, 297-306
Replication V. Production
Process of Single-stranded DNA of Bacteriophage $X1741of Replicative Form in Escherichiu coli Cells irradiated by Ultraviolet Light
KE~I-CHI MATSUBARA, KAZUNORI SHIMADA .4x1) ‘SITASTATJKI TAK~GI Department oj’ Biochemistry Kyushu University School of Medicine Fukuoktr, Japan (Received 24 October 1966, and in rwised form 1
I?fa’?~ 1967)
The process of replicative form production w-as studied in ult,raviolct,-irradiatec~ bacteria infected with +X174, and the results wwc: compared wit,h the process in non-irradiated bacteria. It was observed that in irradiated bactwia., double-&anded parental repliwtivc form is produced as a result of conversion of the injcctcxcl phagc single-strantled DPI’A, 1~111, subsequent production of infectious progwy rcplicntive form tlocs not tako place. On the ot.hor hand, in non-irradi:~t~etl ~11s thrl injcctcd phage single-stranded DNA is converted to dc,llblc-atrantlr,n parental rcq)licativc f’orm. x\ hi& then replicates semiconservatively to accumulatc~ l>rog~ny rcplicat,iw form n~oleculcs (Denhardt &, Sinshoimor, 1965; Matsuhara, Shim&la & Takagi, 1965). Several lines of evidence suggest that t,he double-stranded pnrcnta.1 rc~pliwtivo form produced in irradiated cells is normal, but its sobscqnc~nt rrplicat~ion is blocked il:. ultraviolet-irradiated ~11s.
1. Introduction Upon infection of Escherichia coli mit,h bad-eriophage 4x174, parental single-stranded DNA is converted to double-stranded replicat,ive form (Sinshcimer, Sta,rman, Kagl~,r & (kthrie 1962) via de noro synthesis of a strand complcmcnta,ry to t,he phage L)?r;,J (Denhardt~ & Sinsheimer, 1965; Matsubara, Shimada Br.Talragi: 1967). In the normal infwtion process, the RF then replicates twmiconscrvativclp, leading to the sppea.rancc and accumulation of progeny RF molecules in the cell (Dcnhardt $ Sinshcimcr. 1965; Mat8subarn et al., 1967). Several different propcrtiw of the systems that carry out the conversion and replication procwscs of the rrplicative form are 1~now11: thth tn 0 (I) chloramphcnicol, an inhibitor of prol-ein synthesis. affects diffcwntly pro~ws.ws, na,mely, production of the primary RF: and its subsequent, replication (Tessman. 1966)s; (2) the latter process seems to be more asynchronous than the fonntr (Ma~,sl~bnra et nl., 1967); (3) some phage mutants have been obtninrd that wrrv out tlw former but not t,hc latter process (Tcssman, 1!Wi).
298
K.
MATSUBARA,
K.
SHIMADA
ASl)
Y.
'l'AKA(;l
It is the purpose of this paper to describe the effect of ultraviolct-irradiation ei bacteria on the processes related to $XRF production and replication. Evitk~ncc \z.ill be presented that in heavily ultraviolet-irradiated bacteria,, coilversion of par~~~t;ti phage DNA to double-stranded RF takes place, and the RF thus produced fails to replicate.
2. Materials and Methods The experimental
details are the 1967), except where noted. L medium is a Tris-salt-glucose acids. D medium is the same as L E. digest of 15N- and 2H-labelled
same as those presented
olsewhore
(Matsuba.ra
et al..
medium with the addition of thymine and Casamino medium, except that it has D,O, (15NH,),S0, and a
coli cells in place of HzO, (NH,),SOI and Casamino acids. Lm and Dm media are the L and D media with 200 pg of 5-methyltryptophan/ml. purified 4X174 Host bacteria are E. coli 15T- D3 and phages are 32P-, lsN-, 2H-labelled (32P.D.+X) or 15N-labelled purified +X174 (N.+X). 15N-labelled, and 15N-, 2H-labelled DNA are mom denso than the unlabelled DNA by 0.014 and 0.031 g cmm3, respectively. Bacteria were grown in either L or D medium to 3 x lO*/ml. and irradiated in a thin layer with a Mazda germicidal lamp or with a General Electric germicidal lamp at, a dose of 1.2 x lo4 ergs/mm2 at 0°C in the presence of O,Ol rvr-CaCl,. After irradiation, tho surto viving fraction of colony-forming bacteria is 0.5 to 1 x 10W7 and the residual capacity take up [i4C]thymidine in 30 min into cold trichloroacetic acid-precipitable material is 0.50,” of that of the non-irradiated bacteria. The subsequent experiments were carried out under dim light. The irradiated bacteria were infected with density-labelled phage at a multiplicity of about 0.7 and incubated in an appropriate medium containing met’hyltryptophan. (Methyltryptophan, an inhibitor of protein synthesis, was added to the irradiated bacteria in the process of $X infection so that the results obtained could bo compared directly with those for non-irradiated bacteria infected with #X (Matsubara et al., 1967). In the lat’ter system, tlic drug is necessary t*o prevent production of single-stranded DNA.) The nucleic acid was then extracted from the cells, mixed with a reference 3H-labcllotl E. coli DNA mixture which was used to calibrate the density. The sample was then centrifuged to equilibrium in a C&l density-gradient, and radioactivity and/or infectivity of RF on E. coli spheroplasts were assayed on separate portions. In the equilibrium centrifugation profiles, the patterns of the reference [3H]DNA’s are omitted for clarity. The expected positions of phage DNA and RF’s are indicatctl by lines accompanied by symbols: thus D, D.D, etc., D, N and L st’and for laN- and 2Hsubstituted, l”N-substituted and non-substituted phage DNA, respectively. Combinations of t’1v-o letters represents double-stranded RF constituted with single-strandctl DNA of the rcspoctivo density. Thus N.D represents a double-stranded DNB in which 15N 11:~s been substit’uted in one strand and laN and 2H in the other.
3. Results (a) The conversion
process
In those host cells which have had their own DNA synthesizing capacity dcst.royed by heavy doses of ultraviolet light prior to infection, +X174 phage are unable to replicate, and production of infectious single-stranded DNA does not take place. However, the early process of the growt’h cycle of +X begins normally. In Fig. l(a) and (b), non-irradiated and irradiat’rtl bacteria ncre infected with 32P-labclled D.+X and incubated for 15 minutes in Lm medium. The DNA was extracted from each culture and centrifuged to equilibrium in a CsCl drnsity-gratlient. It can be seen that in both irradiated and non-irradiated cells the radioactive phage singlcstranded DNA is converted to the same extent to double-stranded RF with the
$XRF
PRODUCTION
IN
U.V.
:!!,!I
CELLS
Density
Fraction no. FIG. 1. Fate aof parental 32P-labelled D.4X DSA. Density distribution diagrams of radioactive DNA. Bacteria grown in L medium were infected with 32P-labelled D.$X (multiplicity of infection =: 0.7) and the infected complex incubated in Lm medium for 16 min. (a) Non-irradiated cells; (b) cells irradiated with ultraviolet immediat,ely before infection; (c) cells irradiated with ultraviolet followed by incubation in L medium for 15 min prior to infection. After incubation, nucleic acids were extracted from the infect,ed cells, banded in C&l density-gradients and radioactivit,y essayed. For symbols, see Materials and Metho&. A line with an accompanying symbol “coli L.1,” represents the density at which fully light E. coli DNA forms a band. Some 32P radioactivity in this area in (a) results from breakdown of the phage DNA and is re-utilized for host, DNA synthesis. Another 32P banding around D is the parental D. single-stranded DNA which failed to become RF. For details, see Matsubara et nl. (1967).
density of D.L. Figure l(c) shows that irradiated bacteria retain the capacity for converting single-stranded DNA to RF even when an incubation period follows the irradiation (post-incubation). In another experiment, it was shown that the capacity is unimpaired for up to 25 minutes post-incubation. The rate of the conversion process was roughly compared for irradiated and nonirradiated cells. The two kinds of cells were infected with 32P-labelled D.$X, incubated for 7 and 15 minutes in Lm medium and the amounts of radioactive D.L RF as determined by banding in CsCl were compared. The results were 1400 and 1420 ct’slmin at 7 and 15 minutes in non-irradiated cells, and 1380 and 1500 cts/min at 7 and 15 minutes in irradiated cells. This proves that the rate of the conversion process in irradiated bacteria is normal and is completed by 7 minutes after the addition of phagc. (b) Properties
of the parental
replicative
form produced in irradiated
The a.bove experiment (Fig, 1) showed that D.+X infection in Lm medium. When irradiated
bacteria
D.L RF is produced as a result, of bacteria were infected with N.+X in
thcsizcd de ~~~07~1 in the process of convcrsioii barn et al.. 1967).
(lhilu~ndt
Density I 74 I
1.72 I
I 70 I
0 Fraction
no
FIG. 2. Production of infectious parental RF in irradiated bacteria. Density distribution diagrams of infectious DNA. Bacteria grown in L medium (a) or in D medium (b) were irradiated, infectjed with S.&X (multiplicity of infection = 0.3) and incubated in the respective medium with methyltr\-pt,ol)hal~. After incubation for 15 min, nucleic acids WXY? extracted from the infected cells, handed il) CkCl density-gradienk and t’he infectivity assayed. For symbols, see Materials and Methods. A peak around fract,ion 50 is the N. singlo-stranded DSA which failed to become RF.
In order to distinguish the RF which cont’ains parentSal phage DNA as one of the constituents from the RF synthesized de r~ouo after infection, we shall call the former parental RF and the latter progeny RF. From non-irradiated and irradiated cells infected with 32P-labelled D.Q and incubated in Lm medium as in Fig. 1, the radioactive parental RF with a density of D.L was prepared by C&l density-gradient centrifugation. The ratio of the number (ct’s/min) of plaque-forming units to spheroplasts in the preparation was 1.3 for BP produced in the non-irradiated cells and 1.2 for RF produced in the irradiated ~11s. Figure 3(a) and (b) shows that both RF’s exhibit the same sedimentation patkerns if
,#,XRF
PRODUCTIOS
IN
U.V.
::o I
C!EII,LS
The t\r-o components i II are centrifuged through neutral sucrose gradients. sample may correspond t’o t#he components I and 11 dcscribrXtl hy ot h1,1,i1.t1rk1.1.:. (Kl!rton & Sinshc~imt~r, 1965; Jaenisch, HofschrGl(~r & I%VIWS, l!Wi).
they radr
I
I
1
I
I
/ /
(cl
Cd)
--i
PPt
jl
A w
IO
20
30 Top Fraction
n
no.
Frc. 3. Sucrose sedimentation diagrams of ratlioactive parental RF preparations tinrn ultr ,violet -irradiated and non-irradiated cells. Sedimentation was through neutral sucrose gradients of purified parental RF’s ~woducorl ill non-irradiated cells (a) and in irradiated cells (b). 32P-labelled D. 4X was added to bacteria grows in L medium, incubated in Lm medium for 15 min as described in the legend to Fig. I(a) alld (I I) and nucIeic acids were extracted and banded in CsCl. Fractions of CsCl centrifugation volltainitlg 32P-labeIled parental RF with a density of D.L -we pooled and dialysed against, water in the ~1~1. The dialysed materials (0.2 ml.) were layered on fop of 4.8 mI. sucrose solutions (3 to l;j”;, lirww gradient) cont,aiuing O.Ob M-Tris (pH 7.6), 0.15 M-N&Cl, and centrifuged for 5 hr at 38,000 rev./mm using Hitachi ultracentrifuge RPS40 rotor. Sedimentation through alkali-salt sucrose gradient,s of nucleic acid preparatjons frown 11011. irradiated cells (c) and from irradiated cells (d). 32P-labellod +X wits added to bacteria grow, III L medium, incubated in Lm medium for 15 min, and nucleic acids were extract,ed and illrmediatcLl>donatured by t.he addition of NaOH to a concentr&ion of 0.4 N at 0°C. The mixt,urrs WC’T~~ incubat(vl at 25°C for 30 nlirl and 0.3.m1. portions were layvred on top of 4.6 ml. sucrose soluf.ions (5 to ZO”,, linear gradient) containing 0.1 N-NaOR, 0.9 M-K&X, 4 x 1O-3 X-E:DTA and rentrifuga(l for 3 hr
In another experiment, recorded in Fig. 3(c) and (d), radioactive parental RF’s produced in irradiated and non-irradiated cells were sedimented through alkali-eahDNA extracted from infectr~tl sucrose gradients. In this particular experiment, cells was used without further purification in a C&l density-gradient. The pr
302
K.
>IATSUBAKA,
K.
SHlMADr\
AND
Y. ‘L‘AliA(;
I
addition to some other material resulting from breakdown of injcctcd l)SA\ ii;):’ details, see Matsubara et al., 1967). Thcsc~ materials colnprisc t IN: slo\\--sc~(litnc’lit i IX component in 6hc alkali-sucrose sedimentation profil(h. 111 atltlition to SIIC:~ PA I\\ sedimenting materials, the presence of a fast-scdimentin g co~nptc~~t is c,vitltwt. ‘l’iiih is the cyclic double-helix RF molecule consisting of two Sc~Jarat(‘ly cont)inuotls l~>l> I nucleotidc strands (Powels & Jansz, 1964; Burton & Sinshtbimer, 1965; Jnctlisclt c:t aZ., 1966), and is clearly seen in nucleic acid preparations from both irradiated and IJO~).non-irradiated cells. Therefore, joining ends to produce a closed circular nucleotide from a strand complementary to the parental viral DNA occurs in irradiated as well as in non-irradiated bacteria. So far as we have observed, there is no evidence to suggest any abnormal propcrt,icbs of parental RF formed in irradiated bacteria. (c) The replication
process of replicative
form
The striking difference between non-irradiated and irradiated bacteria is seen in the production of infectious progeny RF. Irradiated and non-irradiated bacteria were infected with D.+X and incubated in Lm medium for 15 minutes (Fig. 4). Nucleic acids were extracted, banded in C&l density-gradients and RF’s were assayed by their infectivity. Here, again, the same amount of parental RF with a density of D.L is produced in both samples. However, production and accumulation of infectious progeny L.L RF, both of which strands
Density
I.74
172
Fraction
170
no.
FIQ. 4. Comparison of the production of RF in irradiated and non-irradiated bacteria. Density distribution diagrams of infectivity. (a) Bacteria were grown in Lm medium, irradiated, infected with D.$X (multiplicity of infection = 0.7) and incubated in Lm medium for 15 min. (b) Same as (a), except that the irradiation of cells was omitted. Nucleic acids were extracted from the infected cells, banded in CsCl densitygradients and the infectivity assayed. For symbols, see Materials and Methods.
c$XRF
PRODUCTION
IN
U.V.
CELLS
:KO
are synthesized de nova after infection, is completely missing in irradiated cells. On the other hand, in non-irradiated control cells, five times as much progeny L.L RF as parental D.L RF was accumulated. We have shown (Matsubara et al., 1967) that, in non-irradiated cells, the production of progeny RF is carried out as a result of semiconservative replication of the RF 1965). The natural outcome of these molecules (see also Denhardt & Sinsheimer, observations is to infer that in the irradiated bacteria, phage DNA is convert,ed to parental RF which then fails to replicate. To test for t,he possible replication of RF in irradiated bacteria, we adopted the technique which was used in the studies of the replication of RF in non-irradiat,ed bacteria (Ma,tsubara et al., 1967). The irradiat.ed bacteria were infected with D.4X and incubat’ed in Dm medium to allow accumulation of RF with a density of D.D. Ha,lf the infected complex was saved and t’he other half was bransferred t)o Ltn mrdium and incubated for another 10 minutes. Semiconservative replication should result, in t’he production of D.L RF in addition to the progeny RF’s with a density of L.L. Nucleic acids were prepared from both batches of cells, banded in CsCl densitygradients and RF’s were assayed by their infectivity. The results show that infectious l%F produced while the complex was in .Dm medium has the density of D.D (Fig. 5(a)): and remained as such even after incubation of the infected complex in Ltn medilun (Fig. 5(b)). In the control system, namely in D.+X infection of non-irradiated ~~4s. D.D RF was the sole RF component before the change of medium, but production of D.L RF and accumulation of L.L progeny R#F took place after incubat’inn in 1,111medium. In another experiment to confirm the above result, N.$X was used in place of D.$X for infection in Dm medium. Here, the RF produced as a result of conversion from parental single-stranded N. DNA should have a density of N.D, and progeny RF should have a density of D.D during incubation of the infected complex in Dm meclium. The result shows the production of N.D RF but not D.D RF in irradiated cc~lls (Fig. 5(c)). Upon further incubation of the infected complex in Lm medium for 20 minutes. the infectivity still remained at N.D RF, without appreciable change in density (Fig. 5(d) ). The components expected as a result of the semiconservative replication of N.D RF (N.L and D.L in this case) did not accumulate. Nor did any L.1, progeny RF appear. The amount of infectious N.D RF was the same before and aft,rr the incubation in Lm medium. On the other hand, in non-irradiated bact,eria, all the RF components with different densities expected from the semiconservative replication model have been found (Mat’subara et a,Z., 1967). These observations are all compatible with the idea that the replication of tlrcs primary RF produced as a result of conv:>rsion from phage single-stranded DNA is blocked in irradiat,ed bnct,eria.
4. Discussion The conversion of phage single-stranded DNA to double-stranded RF (parent)al RF’I occurs in t,hose bacteria which havo had their capacity to synthesize DN:2 desi royed by I~avy doses of ultraviolct light. It has been shown that in the irradiated bacteria, the P&and complementary to the phage single-stranded DNA is synthesized de ?(OVOand that several properties of the conversion reaction and its product, including the approximate rate of the conversion process, the specific infectivity and
I<.
MATSUBARA,
K.
SHIMADA
.-4x1)
I’.
‘l-i\
I; .\(:
1
Density
b Fraction
40
50
60
no.
FIG. 5. Absence of replication of parental RF and production of infectious progeny RF ill irradiat,ed bacteria. Densit’y distribution diagrams of infectious DNA. In (a) and (b), bacteria were grown in D medium, irradiated, infected with D.&K (multiplirit> of infection = 0.7) and incubated in Dm medium for 7 min. 4 half of tbc infected complex was saved (a) and the other half was incubated in Lm medium for another 10 min (b). In (c) and (d), bacteria were grown in D medium, irradiated, infected wit,h N.#X (multiplicity of infect,ion = 0.9) and incubated in Dm medium for 15 min. a half of the infectled cornl~lru was saved (c) and tlltx other half was incubated in Lm medium for another 20 min. Nucleic acids were extracted from the infected cells, banded in C&l density-gradients and the infectivity assayed. For symbols, see Materials and Methods. The peaks around fraction 20 in (a), (b) and around fraction 45 in (c), (d) are the phage single-stranded D. and N. DNA, respectively, which failed t’o become RF.
sedimentation properties of the parental RF’s, the production of twisted circular RF (and hence the react’ion to close ends of a DNA molecule), are all indistinguishable from that seen in non-irradiated bacteria. We can, therefore, conclude that ultraviolet,-irradiation prior to infection does not affect the conversion proccss, and that the parental RF DNA thus produced is normal. The capacity to carry out the conversion process remains unaffected during about 25 minutes of incubation following the irradiation. Hence, under the experimental condition in which the reaction does not require a, longer incubation period, it, is unlikely that the results are affected by general loss of progressive rcduetion in gent4 synthetic capacities of cells due to irradiation. Density studies have shown that after the parental RF is formed, t,hc events related to the multiplication of RF in irradiated cells arc abnormal; no infectious progeny RF is made under the various incubat,ion condit,ions test,ed. This implies tha,t in the irradiated bacteria, either the progeny RF’s arc not, produwcl, or if t’1lc.y are made they are non-infectious. We prefer the former vion- from tlw folloninp considerations. (1) Density studies have shown that, the products which appear as a result of semiconservative replication of parenta, RF in non-irradiated ~11s (Matsu-
cJXRF
PRODUCTION
IN
U.V.
CELLS
3ll.i
bara et al., 1967) fail to appear in irradiated bacteria. (2) The density of parental RF does not change upon incubation of the infected complex in a medium with different density. (3) The total amount of infectious parental RF produced in irradiated crlls is approximately the same as that of parental RF produced in non-irradiated bact,cri;i, and this infectivity remains constant during prolonged incubation of the infcctcrl complex. The simplest way of explaining all of these observations is to assume that. in irradiated bacteria the parental RF fails to replicat’c. Other ideas require more we have reservations, because in irradiated complicat~cd assumptions. However, cells we have not t,horoughly investigated the DNA precursor pool problem and this may a8ffect the conclusions drawn from density studies (1) a,nd (2), and bccausc t Ilr sort of quant,ita,tirc discussion (3) on the amount of RF molecules as based on thtkir alterat,ion of that I{ 1: biological Assam’ might be erroneous sinw a mcrc structural n~ol~~c~~leaffect,~ t,hc specific infectivity (Pcwels & Jansz. 196-C; Burton & Sinshc~irnc~r. I%;,). TXraviolct irradiation of the host bacteria prior to infection separates the WIIversion process from the subsequent production of t’he infectious progeny RF. :\lthough t,lw mechanism of ultraviolet damage to the system(s) that, participate in RF muhiplkltion is not known, our observations provide st’rong evidence tha,t such a system(b) must be provided by the host bacteria. This is because the irradiation is gircn prior to infection. and because the parental RF produced in irradiated bacteria seems to 1~: normal. OW can predict, based on this view, that it may bepossiblc to find bacteriill 1tlllt~tJltS in I:-hich t,he replication process of the RF is affected. Rcwrltly. son~.c r~,cc,ln~)illatiotI-drficient bacterial mutants that fail to ;~llo~~ normal protlllctiou of RF’ havcx IWCII isolated (Friedman $ Tessman. personal communication; lkrllli\rcit, Drc~sskr R- Ha,thanay, 1967). Stud& along t’his linrb may furthw c~lncichr~tc~t /I(’ ]‘rohlcnl.
On the other hand, there is evidence t,llat the multiplication of RF to prodnc~~ infc,ctious progeny RF depends upon the function of a phage gene. Tcssman (I!Mi) observed that, production of infect,ious progeny RF is blocked by high concentration of (.17loramplrrcnicol administered to the infected bacteria, and that production t101~s not seem to lake place in bacteria infected with a mutant phage 513 although tllc inj~&~d singl+st~randed DNA is converted to double-stranded RF. WC may spcculatr, thwcforr, that the multiplication of RF and production of infect’ious progeny RI;‘ rcquiw the co-op,cration of both an unimpaired phage-dircctcd system and a systcbm (q) provitlcd by i hr. host cell. ‘l’hc separalion of parental RF formation from the pro&&ion of infectious progcwy RF by making LISCof irradiated bacterial cells or mutant phage might bc usefIll :n cluklnting the functional roles of parental and progeny RF’s in the procws of plunge inffbrtion. In I his connection, it will be recalled that no single-stranded DNA syntlwis takw plaw in such irradiated bact,eria with or wkhout nwthyltryptophan. althorl~ll t hc~re sums 1,:) bc normal parental RF production. Bacteria infected with a mutant i)f ph;lgc 513 ~vhich fails to show RF mult iplicat,iou giw the same result (Tessma n. 19M), suggesting some correlat,ion between t,lre capacity to replicate th, parf~l~tnl JC1.’and tlrth ‘-II twqucnt~ production of single-strantlcd 1)N.I. \Vc, tl~anli 111,i\lat~t~hcw S. Mewlson for allowing onI. ,lf’ 11s (I<. ix.) to cllrry olrt iL prt t,hi.+ \ro~.lc ill his laboratory. ‘i’llis investigation was :liclcd by a grant from tllc: J~MIP (‘offin C’hilds 3lc:mori:tl Frmtl Mrctical Kcscarc~h.
or for
300
K.
MA4TSUISAllil,
I<. SHIRIADA
AKI)
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REFERENCES Burton, A. & Sinsheimer, R. L. (1965). J. MoZ. Biol. 14, 327. Denhardt, D. T., Dressier, D. H. & Hathaway (1967). Pro?. Nat. Amd. S-i.. Il'rrslt. 813. Denhardt, D. T. & Sinsheimer, R. 2. (1965). J. Mol. Biol. 12, 647. Jaenisch, R., Hofschneider, P. H. & Preuss, A. (1966). J. iMoZ. Biol. 21, 501. Matsubara, K., Shimada, K. & Takagi, Y. (1967). J. Biochem., Jqczn, in the press. Pow&, P. H. & Jansz, H. S. (1964). Biochim. biophyls. Acta, 91, 177. Shimada, K. (1965). Virus (Japan), 15, 29. Sinsheimer, R. L., Starman, B., Naglcr, C. & Guthrie, S. (1962). J. 4101. Biol. 4, 142. Tessman, E. (1966). J. Mol. BioZ. 17, 018.
57.
(1967) 29, 297-306
Replication V. Production
Process of Single-stranded DNA of Bacteriophage $X1741of Replicative Form in Escherichiu coli Cells irradiated by Ultraviolet Light
KE~I-CHI MATSUBARA, KAZUNORI SHIMADA .4x1) ‘SITASTATJKI TAK~GI Department oj’ Biochemistry Kyushu University School of Medicine Fukuoktr, Japan (Received 24 October 1966, and in rwised form 1
I?fa’?~ 1967)
The process of replicative form production w-as studied in ult,raviolct,-irradiatec~ bacteria infected with +X174, and the results wwc: compared wit,h the process in non-irradiated bacteria. It was observed that in irradiated bactwia., double-&anded parental repliwtivc form is produced as a result of conversion of the injcctcxcl phagc single-strantled DPI’A, 1~111, subsequent production of infectious progwy rcplicntive form tlocs not tako place. On the ot.hor hand, in non-irradi:~t~etl ~11s thrl injcctcd phage single-stranded DNA is converted to dc,llblc-atrantlr,n parental rcq)licativc f’orm. x\ hi& then replicates semiconservatively to accumulatc~ l>rog~ny rcplicat,iw form n~oleculcs (Denhardt &, Sinshoimor, 1965; Matsuhara, Shim&la & Takagi, 1965). Several lines of evidence suggest that t,he double-stranded pnrcnta.1 rc~pliwtivo form produced in irradiated cells is normal, but its sobscqnc~nt rrplicat~ion is blocked il:. ultraviolet-irradiated ~11s.
1. Introduction Upon infection of Escherichia coli mit,h bad-eriophage 4x174, parental single-stranded DNA is converted to double-stranded replicat,ive form (Sinshcimer, Sta,rman, Kagl~,r & (kthrie 1962) via de noro synthesis of a strand complcmcnta,ry to t,he phage L)?r;,J (Denhardt~ & Sinsheimer, 1965; Matsubara, Shimada Br.Talragi: 1967). In the normal infwtion process, the RF then replicates twmiconscrvativclp, leading to the sppea.rancc and accumulation of progeny RF molecules in the cell (Dcnhardt $ Sinshcimcr. 1965; Mat8subarn et al., 1967). Several different propcrtiw of the systems that carry out the conversion and replication procwscs of the rrplicative form are 1~now11: thth tn 0 (I) chloramphcnicol, an inhibitor of prol-ein synthesis. affects diffcwntly pro~ws.ws, na,mely, production of the primary RF: and its subsequent, replication (Tessman. 1966)s; (2) the latter process seems to be more asynchronous than the fonntr (Ma~,sl~bnra et nl., 1967); (3) some phage mutants have been obtninrd that wrrv out tlw former but not t,hc latter process (Tcssman, 1!Wi).
298
K.
MATSUBARA,
K.
SHIMADA
ASl)
Y.
'l'AKA(;l
It is the purpose of this paper to describe the effect of ultraviolct-irradiation ei bacteria on the processes related to $XRF production and replication. Evitk~ncc \z.ill be presented that in heavily ultraviolet-irradiated bacteria,, coilversion of par~~~t;ti phage DNA to double-stranded RF takes place, and the RF thus produced fails to replicate.
2. Materials and Methods The experimental
details are the 1967), except where noted. L medium is a Tris-salt-glucose acids. D medium is the same as L E. digest of 15N- and 2H-labelled
same as those presented
olsewhore
(Matsuba.ra
et al..
medium with the addition of thymine and Casamino medium, except that it has D,O, (15NH,),S0, and a
coli cells in place of HzO, (NH,),SOI and Casamino acids. Lm and Dm media are the L and D media with 200 pg of 5-methyltryptophan/ml. purified 4X174 Host bacteria are E. coli 15T- D3 and phages are 32P-, lsN-, 2H-labelled (32P.D.+X) or 15N-labelled purified +X174 (N.+X). 15N-labelled, and 15N-, 2H-labelled DNA are mom denso than the unlabelled DNA by 0.014 and 0.031 g cmm3, respectively. Bacteria were grown in either L or D medium to 3 x lO*/ml. and irradiated in a thin layer with a Mazda germicidal lamp or with a General Electric germicidal lamp at, a dose of 1.2 x lo4 ergs/mm2 at 0°C in the presence of O,Ol rvr-CaCl,. After irradiation, tho surto viving fraction of colony-forming bacteria is 0.5 to 1 x 10W7 and the residual capacity take up [i4C]thymidine in 30 min into cold trichloroacetic acid-precipitable material is 0.50,” of that of the non-irradiated bacteria. The subsequent experiments were carried out under dim light. The irradiated bacteria were infected with density-labelled phage at a multiplicity of about 0.7 and incubated in an appropriate medium containing met’hyltryptophan. (Methyltryptophan, an inhibitor of protein synthesis, was added to the irradiated bacteria in the process of $X infection so that the results obtained could bo compared directly with those for non-irradiated bacteria infected with #X (Matsubara et al., 1967). In the lat’ter system, tlic drug is necessary t*o prevent production of single-stranded DNA.) The nucleic acid was then extracted from the cells, mixed with a reference 3H-labcllotl E. coli DNA mixture which was used to calibrate the density. The sample was then centrifuged to equilibrium in a C&l density-gradient, and radioactivity and/or infectivity of RF on E. coli spheroplasts were assayed on separate portions. In the equilibrium centrifugation profiles, the patterns of the reference [3H]DNA’s are omitted for clarity. The expected positions of phage DNA and RF’s are indicatctl by lines accompanied by symbols: thus D, D.D, etc., D, N and L st’and for laN- and 2Hsubstituted, l”N-substituted and non-substituted phage DNA, respectively. Combinations of t’1v-o letters represents double-stranded RF constituted with single-strandctl DNA of the rcspoctivo density. Thus N.D represents a double-stranded DNB in which 15N 11:~s been substit’uted in one strand and laN and 2H in the other.
3. Results (a) The conversion
process
In those host cells which have had their own DNA synthesizing capacity dcst.royed by heavy doses of ultraviolet light prior to infection, +X174 phage are unable to replicate, and production of infectious single-stranded DNA does not take place. However, the early process of the growt’h cycle of +X begins normally. In Fig. l(a) and (b), non-irradiated and irradiat’rtl bacteria ncre infected with 32P-labclled D.+X and incubated for 15 minutes in Lm medium. The DNA was extracted from each culture and centrifuged to equilibrium in a CsCl drnsity-gratlient. It can be seen that in both irradiated and non-irradiated cells the radioactive phage singlcstranded DNA is converted to the same extent to double-stranded RF with the
$XRF
PRODUCTION
IN
U.V.
:!!,!I
CELLS
Density
Fraction no. FIG. 1. Fate aof parental 32P-labelled D.4X DSA. Density distribution diagrams of radioactive DNA. Bacteria grown in L medium were infected with 32P-labelled D.$X (multiplicity of infection =: 0.7) and the infected complex incubated in Lm medium for 16 min. (a) Non-irradiated cells; (b) cells irradiated with ultraviolet immediat,ely before infection; (c) cells irradiated with ultraviolet followed by incubation in L medium for 15 min prior to infection. After incubation, nucleic acids were extracted from the infect,ed cells, banded in C&l density-gradients and radioactivit,y essayed. For symbols, see Materials and Metho&. A line with an accompanying symbol “coli L.1,” represents the density at which fully light E. coli DNA forms a band. Some 32P radioactivity in this area in (a) results from breakdown of the phage DNA and is re-utilized for host, DNA synthesis. Another 32P banding around D is the parental D. single-stranded DNA which failed to become RF. For details, see Matsubara et nl. (1967).
density of D.L. Figure l(c) shows that irradiated bacteria retain the capacity for converting single-stranded DNA to RF even when an incubation period follows the irradiation (post-incubation). In another experiment, it was shown that the capacity is unimpaired for up to 25 minutes post-incubation. The rate of the conversion process was roughly compared for irradiated and nonirradiated cells. The two kinds of cells were infected with 32P-labelled D.$X, incubated for 7 and 15 minutes in Lm medium and the amounts of radioactive D.L RF as determined by banding in CsCl were compared. The results were 1400 and 1420 ct’slmin at 7 and 15 minutes in non-irradiated cells, and 1380 and 1500 cts/min at 7 and 15 minutes in irradiated cells. This proves that the rate of the conversion process in irradiated bacteria is normal and is completed by 7 minutes after the addition of phagc. (b) Properties
of the parental
replicative
form produced in irradiated
The a.bove experiment (Fig, 1) showed that D.+X infection in Lm medium. When irradiated
bacteria
D.L RF is produced as a result, of bacteria were infected with N.+X in
thcsizcd de ~~~07~1 in the process of convcrsioii barn et al.. 1967).
(lhilu~ndt
Density I 74 I
1.72 I
I 70 I
0 Fraction
no
FIG. 2. Production of infectious parental RF in irradiated bacteria. Density distribution diagrams of infectious DNA. Bacteria grown in L medium (a) or in D medium (b) were irradiated, infectjed with S.&X (multiplicity of infection = 0.3) and incubated in the respective medium with methyltr\-pt,ol)hal~. After incubation for 15 min, nucleic acids WXY? extracted from the infected cells, handed il) CkCl density-gradienk and t’he infectivity assayed. For symbols, see Materials and Methods. A peak around fract,ion 50 is the N. singlo-stranded DSA which failed to become RF.
In order to distinguish the RF which cont’ains parentSal phage DNA as one of the constituents from the RF synthesized de r~ouo after infection, we shall call the former parental RF and the latter progeny RF. From non-irradiated and irradiated cells infected with 32P-labelled D.Q and incubated in Lm medium as in Fig. 1, the radioactive parental RF with a density of D.L was prepared by C&l density-gradient centrifugation. The ratio of the number (ct’s/min) of plaque-forming units to spheroplasts in the preparation was 1.3 for BP produced in the non-irradiated cells and 1.2 for RF produced in the irradiated ~11s. Figure 3(a) and (b) shows that both RF’s exhibit the same sedimentation patkerns if
,#,XRF
PRODUCTIOS
IN
U.V.
::o I
C!EII,LS
The t\r-o components i II are centrifuged through neutral sucrose gradients. sample may correspond t’o t#he components I and 11 dcscribrXtl hy ot h1,1,i1.t1rk1.1.:. (Kl!rton & Sinshc~imt~r, 1965; Jaenisch, HofschrGl(~r & I%VIWS, l!Wi).
they radr
I
I
1
I
I
/ /
(cl
Cd)
--i
PPt
jl
A w
IO
20
30 Top Fraction
n
no.
Frc. 3. Sucrose sedimentation diagrams of ratlioactive parental RF preparations tinrn ultr ,violet -irradiated and non-irradiated cells. Sedimentation was through neutral sucrose gradients of purified parental RF’s ~woducorl ill non-irradiated cells (a) and in irradiated cells (b). 32P-labelled D. 4X was added to bacteria grows in L medium, incubated in Lm medium for 15 min as described in the legend to Fig. I(a) alld (I I) and nucIeic acids were extracted and banded in CsCl. Fractions of CsCl centrifugation volltainitlg 32P-labeIled parental RF with a density of D.L -we pooled and dialysed against, water in the ~1~1. The dialysed materials (0.2 ml.) were layered on fop of 4.8 mI. sucrose solutions (3 to l;j”;, lirww gradient) cont,aiuing O.Ob M-Tris (pH 7.6), 0.15 M-N&Cl, and centrifuged for 5 hr at 38,000 rev./mm using Hitachi ultracentrifuge RPS40 rotor. Sedimentation through alkali-salt sucrose gradient,s of nucleic acid preparatjons frown 11011. irradiated cells (c) and from irradiated cells (d). 32P-labellod +X wits added to bacteria grow, III L medium, incubated in Lm medium for 15 min, and nucleic acids were extract,ed and illrmediatcLl>donatured by t.he addition of NaOH to a concentr&ion of 0.4 N at 0°C. The mixt,urrs WC’T~~ incubat(vl at 25°C for 30 nlirl and 0.3.m1. portions were layvred on top of 4.6 ml. sucrose soluf.ions (5 to ZO”,, linear gradient) containing 0.1 N-NaOR, 0.9 M-K&X, 4 x 1O-3 X-E:DTA and rentrifuga(l for 3 hr
In another experiment, recorded in Fig. 3(c) and (d), radioactive parental RF’s produced in irradiated and non-irradiated cells were sedimented through alkali-eahDNA extracted from infectr~tl sucrose gradients. In this particular experiment, cells was used without further purification in a C&l density-gradient. The pr
302
K.
>IATSUBAKA,
K.
SHlMADr\
AND
Y. ‘L‘AliA(;
I
addition to some other material resulting from breakdown of injcctcd l)SA\ ii;):’ details, see Matsubara et al., 1967). Thcsc~ materials colnprisc t IN: slo\\--sc~(litnc’lit i IX component in 6hc alkali-sucrose sedimentation profil(h. 111 atltlition to SIIC:~ PA I\\ sedimenting materials, the presence of a fast-scdimentin g co~nptc~~t is c,vitltwt. ‘l’iiih is the cyclic double-helix RF molecule consisting of two Sc~Jarat(‘ly cont)inuotls l~>l> I nucleotidc strands (Powels & Jansz, 1964; Burton & Sinshtbimer, 1965; Jnctlisclt c:t aZ., 1966), and is clearly seen in nucleic acid preparations from both irradiated and IJO~).non-irradiated cells. Therefore, joining ends to produce a closed circular nucleotide from a strand complementary to the parental viral DNA occurs in irradiated as well as in non-irradiated bacteria. So far as we have observed, there is no evidence to suggest any abnormal propcrt,icbs of parental RF formed in irradiated bacteria. (c) The replication
process of replicative
form
The striking difference between non-irradiated and irradiated bacteria is seen in the production of infectious progeny RF. Irradiated and non-irradiated bacteria were infected with D.+X and incubated in Lm medium for 15 minutes (Fig. 4). Nucleic acids were extracted, banded in C&l density-gradients and RF’s were assayed by their infectivity. Here, again, the same amount of parental RF with a density of D.L is produced in both samples. However, production and accumulation of infectious progeny L.L RF, both of which strands
Density
I.74
172
Fraction
170
no.
FIQ. 4. Comparison of the production of RF in irradiated and non-irradiated bacteria. Density distribution diagrams of infectivity. (a) Bacteria were grown in Lm medium, irradiated, infected with D.$X (multiplicity of infection = 0.7) and incubated in Lm medium for 15 min. (b) Same as (a), except that the irradiation of cells was omitted. Nucleic acids were extracted from the infected cells, banded in CsCl densitygradients and the infectivity assayed. For symbols, see Materials and Methods.
c$XRF
PRODUCTION
IN
U.V.
CELLS
:KO
are synthesized de nova after infection, is completely missing in irradiated cells. On the other hand, in non-irradiated control cells, five times as much progeny L.L RF as parental D.L RF was accumulated. We have shown (Matsubara et al., 1967) that, in non-irradiated cells, the production of progeny RF is carried out as a result of semiconservative replication of the RF 1965). The natural outcome of these molecules (see also Denhardt & Sinsheimer, observations is to infer that in the irradiated bacteria, phage DNA is convert,ed to parental RF which then fails to replicate. To test for t,he possible replication of RF in irradiated bacteria, we adopted the technique which was used in the studies of the replication of RF in non-irradiat,ed bacteria (Ma,tsubara et al., 1967). The irradiat.ed bacteria were infected with D.4X and incubat’ed in Dm medium to allow accumulation of RF with a density of D.D. Ha,lf the infected complex was saved and t’he other half was bransferred t)o Ltn mrdium and incubated for another 10 minutes. Semiconservative replication should result, in t’he production of D.L RF in addition to the progeny RF’s with a density of L.L. Nucleic acids were prepared from both batches of cells, banded in CsCl densitygradients and RF’s were assayed by their infectivity. The results show that infectious l%F produced while the complex was in .Dm medium has the density of D.D (Fig. 5(a)): and remained as such even after incubation of the infected complex in Ltn medilun (Fig. 5(b)). In the control system, namely in D.+X infection of non-irradiated ~~4s. D.D RF was the sole RF component before the change of medium, but production of D.L RF and accumulation of L.L progeny R#F took place after incubat’inn in 1,111medium. In another experiment to confirm the above result, N.$X was used in place of D.$X for infection in Dm medium. Here, the RF produced as a result of conversion from parental single-stranded N. DNA should have a density of N.D, and progeny RF should have a density of D.D during incubation of the infected complex in Dm meclium. The result shows the production of N.D RF but not D.D RF in irradiated cc~lls (Fig. 5(c)). Upon further incubation of the infected complex in Lm medium for 20 minutes. the infectivity still remained at N.D RF, without appreciable change in density (Fig. 5(d) ). The components expected as a result of the semiconservative replication of N.D RF (N.L and D.L in this case) did not accumulate. Nor did any L.1, progeny RF appear. The amount of infectious N.D RF was the same before and aft,rr the incubation in Lm medium. On the other hand, in non-irradiated bact,eria, all the RF components with different densities expected from the semiconservative replication model have been found (Mat’subara et a,Z., 1967). These observations are all compatible with the idea that the replication of tlrcs primary RF produced as a result of conv:>rsion from phage single-stranded DNA is blocked in irradiat,ed bnct,eria.
4. Discussion The conversion of phage single-stranded DNA to double-stranded RF (parent)al RF’I occurs in t,hose bacteria which havo had their capacity to synthesize DN:2 desi royed by I~avy doses of ultraviolct light. It has been shown that in the irradiated bacteria, the P&and complementary to the phage single-stranded DNA is synthesized de ?(OVOand that several properties of the conversion reaction and its product, including the approximate rate of the conversion process, the specific infectivity and
I<.
MATSUBARA,
K.
SHIMADA
.-4x1)
I’.
‘l-i\
I; .\(:
1
Density
b Fraction
40
50
60
no.
FIG. 5. Absence of replication of parental RF and production of infectious progeny RF ill irradiat,ed bacteria. Densit’y distribution diagrams of infectious DNA. In (a) and (b), bacteria were grown in D medium, irradiated, infected with D.&K (multiplirit> of infection = 0.7) and incubated in Dm medium for 7 min. 4 half of tbc infected complex was saved (a) and the other half was incubated in Lm medium for another 10 min (b). In (c) and (d), bacteria were grown in D medium, irradiated, infected wit,h N.#X (multiplicity of infect,ion = 0.9) and incubated in Dm medium for 15 min. a half of the infectled cornl~lru was saved (c) and tlltx other half was incubated in Lm medium for another 20 min. Nucleic acids were extracted from the infected cells, banded in C&l density-gradients and the infectivity assayed. For symbols, see Materials and Methods. The peaks around fraction 20 in (a), (b) and around fraction 45 in (c), (d) are the phage single-stranded D. and N. DNA, respectively, which failed t’o become RF.
sedimentation properties of the parental RF’s, the production of twisted circular RF (and hence the react’ion to close ends of a DNA molecule), are all indistinguishable from that seen in non-irradiated bacteria. We can, therefore, conclude that ultraviolet,-irradiation prior to infection does not affect the conversion proccss, and that the parental RF DNA thus produced is normal. The capacity to carry out the conversion process remains unaffected during about 25 minutes of incubation following the irradiation. Hence, under the experimental condition in which the reaction does not require a, longer incubation period, it, is unlikely that the results are affected by general loss of progressive rcduetion in gent4 synthetic capacities of cells due to irradiation. Density studies have shown that after the parental RF is formed, t,hc events related to the multiplication of RF in irradiated cells arc abnormal; no infectious progeny RF is made under the various incubat,ion condit,ions test,ed. This implies tha,t in the irradiated bacteria, either the progeny RF’s arc not, produwcl, or if t’1lc.y are made they are non-infectious. We prefer the former vion- from tlw folloninp considerations. (1) Density studies have shown that, the products which appear as a result of semiconservative replication of parenta, RF in non-irradiated ~11s (Matsu-
cJXRF
PRODUCTION
IN
U.V.
CELLS
3ll.i
bara et al., 1967) fail to appear in irradiated bacteria. (2) The density of parental RF does not change upon incubation of the infected complex in a medium with different density. (3) The total amount of infectious parental RF produced in irradiated crlls is approximately the same as that of parental RF produced in non-irradiated bact,cri;i, and this infectivity remains constant during prolonged incubation of the infcctcrl complex. The simplest way of explaining all of these observations is to assume that. in irradiated bacteria the parental RF fails to replicat’c. Other ideas require more we have reservations, because in irradiated complicat~cd assumptions. However, cells we have not t,horoughly investigated the DNA precursor pool problem and this may a8ffect the conclusions drawn from density studies (1) a,nd (2), and bccausc t Ilr sort of quant,ita,tirc discussion (3) on the amount of RF molecules as based on thtkir alterat,ion of that I{ 1: biological Assam’ might be erroneous sinw a mcrc structural n~ol~~c~~leaffect,~ t,hc specific infectivity (Pcwels & Jansz. 196-C; Burton & Sinshc~irnc~r. I%;,). TXraviolct irradiation of the host bacteria prior to infection separates the WIIversion process from the subsequent production of t’he infectious progeny RF. :\lthough t,lw mechanism of ultraviolet damage to the system(s) that, participate in RF muhiplkltion is not known, our observations provide st’rong evidence tha,t such a system(b) must be provided by the host bacteria. This is because the irradiation is gircn prior to infection. and because the parental RF produced in irradiated bacteria seems to 1~: normal. OW can predict, based on this view, that it may bepossiblc to find bacteriill 1tlllt~tJltS in I:-hich t,he replication process of the RF is affected. Rcwrltly. son~.c r~,cc,ln~)illatiotI-drficient bacterial mutants that fail to ;~llo~~ normal protlllctiou of RF’ havcx IWCII isolated (Friedman $ Tessman. personal communication; lkrllli\rcit, Drc~sskr R- Ha,thanay, 1967). Stud& along t’his linrb may furthw c~lncichr~tc~t /I(’ ]‘rohlcnl.
On the other hand, there is evidence t,llat the multiplication of RF to prodnc~~ infc,ctious progeny RF depends upon the function of a phage gene. Tcssman (I!Mi) observed that, production of infect,ious progeny RF is blocked by high concentration of (.17loramplrrcnicol administered to the infected bacteria, and that production t101~s not seem to lake place in bacteria infected with a mutant phage 513 although tllc inj~&~d singl+st~randed DNA is converted to double-stranded RF. WC may spcculatr, thwcforr, that the multiplication of RF and production of infect’ious progeny RI;‘ rcquiw the co-op,cration of both an unimpaired phage-dircctcd system and a systcbm (q) provitlcd by i hr. host cell. ‘l’hc separalion of parental RF formation from the pro&&ion of infectious progcwy RF by making LISCof irradiated bacterial cells or mutant phage might bc usefIll :n cluklnting the functional roles of parental and progeny RF’s in the procws of plunge inffbrtion. In I his connection, it will be recalled that no single-stranded DNA syntlwis takw plaw in such irradiated bact,eria with or wkhout nwthyltryptophan. althorl~ll t hc~re sums 1,:) bc normal parental RF production. Bacteria infected with a mutant i)f ph;lgc 513 ~vhich fails to show RF mult iplicat,iou giw the same result (Tessma n. 19M), suggesting some correlat,ion between t,lre capacity to replicate th, parf~l~tnl JC1.’and tlrth ‘-II twqucnt~ production of single-strantlcd 1)N.I. \Vc, tl~anli 111,i\lat~t~hcw S. Mewlson for allowing onI. ,lf’ 11s (I<. ix.) to cllrry olrt iL prt t,hi.+ \ro~.lc ill his laboratory. ‘i’llis investigation was :liclcd by a grant from tllc: J~MIP (‘offin C’hilds 3lc:mori:tl Frmtl Mrctical Kcscarc~h.
or for
300
K.
MA4TSUISAllil,
I<. SHIRIADA
AKI)
Y.
‘l’:\K-\(:I.
REFERENCES Burton, A. & Sinsheimer, R. L. (1965). J. MoZ. Biol. 14, 327. Denhardt, D. T., Dressier, D. H. & Hathaway (1967). Pro?. Nat. Amd. S-i.. Il'rrslt. 813. Denhardt, D. T. & Sinsheimer, R. 2. (1965). J. Mol. Biol. 12, 647. Jaenisch, R., Hofschneider, P. H. & Preuss, A. (1966). J. iMoZ. Biol. 21, 501. Matsubara, K., Shimada, K. & Takagi, Y. (1967). J. Biochem., Jqczn, in the press. Pow&, P. H. & Jansz, H. S. (1964). Biochim. biophyls. Acta, 91, 177. Shimada, K. (1965). Virus (Japan), 15, 29. Sinsheimer, R. L., Starman, B., Naglcr, C. & Guthrie, S. (1962). J. 4101. Biol. 4, 142. Tessman, E. (1966). J. Mol. BioZ. 17, 018.
57.