J. Mol. Hiol. (1958)
121, 95-111
Mutant of Escherichiu cob which Blocks T7 Bacteriophage Assembly: Accumulation of Short T7 DNA YOSHIHIKO YAMADA,JAXET
~ILNVTZER AND DAI NAKAIM
Department of Biochemistry University of Pittsburgh School of N dirin~~ Pittsburgh, Pa 15261, C:.S.;1. (Received 23 September 1977, and in revised form 27 January
I97S)
A mut,ant strain which blocks bacteriophage T7 assembly, presumably at the from li’wherichiu cdi DlO F ~-. phage DNA packaging process, was isolat,rd This mutant, Y49, was selected for its ability to plate TT+ phage but’ not 7’7 gene 0.7 mutant phage (T7K-). The plating efficiency of Ti+ phagc on strain Y49 was o~rr SOa; of t’hat on the parental &rain but the yield of T7 + phagr WAS phagr, ‘L-49 cells synthr~sizc~tl less t~han loo’ ,O’ Upon infection with T7 + or T7IC T7-specific RNA and protein almost, normally but, thf3 synthrsis of ‘I?7 progqv by the DNA was slightly reduced, about 70 to 80:; of thr amount synthesized parental strain. In Y49 cells infected with T7 + or T7Kphage, T7 progeny DNA appeared first as concatemers larger in size than the mature T7 phage DNA but, it than the size of T7 phage later was converted to pieces about Xl:,, shorter DNA. When Y49 cells were infect,cd with T7 mutant phagrs known to accumulate concatemcric T7 progeny DNA in t,he normal host, cells, T7 progeny DNA also accumulat,ed as concatemcrs and was not clcavetl to smaller pieces. These rc,sults suggest t,hat, the cleavage of concatemr,ric T7 DPJA in ‘I”i+ or T7K-~ phapcbinfc?ct,ed Y49 cells, which generates short DNA species, appears to bc site-specific* and probably results from a blockage of the phapc DNA packaging process d\le to the host mutat)ion, implying that a host function is invol\-c,d in the 7’7 phagcs DNA packaging process.
1. Introduction Bact)eriophage T7 gene 0.7 codes for a protein kinase which phosphorylat,es many proteins of the host Escherichia coli including RNA polymerase (Rahmsdorf et ad.. 1973,1974; Zillig et al., 1975). Phosphorylation of the host E. coli RNA polymerasr has been implicated in the inactivation of t,he enzyme and thus the “host shut,-off” b~l !I’7 phage inf&ion (Rothman-Denes et aZ.. 1973; Pont,a et al.. 1974). However. recent studies in t,his laboratory revealed t,hat the inactivabion of E. coli RPI’A polymerase in T7-infected cells is mainly due to the binding to the enzyme of an inhibitor prot,ein. termed 1 protein, coded by T7 gene 2 although gene 0.7 product is also partly involved in the inactivation of the enzyme (Hcssclbach & Xakatla, 1975,1977a,b). T7 gene 0.7 has been considered as one of the non-essential genes of T7 (Studiw, can be propagated normally in t’he most 1973): T7 gene 0.7 mutant phage (T7K-) commonly used host’ E. coli B st)rain. Hirsch-Kauffman et al. (1975) found, however. that) T7K- phage gave only few, if any, progeny phagrs when used t,o infect E. coli 9.i 10 1978 ~2catltTTlicPress Inc. (London) Ltcl. c,o~n~~s3e/7s/l:'lrrt.i11 $02.00/O
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K12 st’rain gro\\~ in a minimal mrdium whcrvas thv \vild-t>,pc T’i ’ IJlag~~ u ah propagat,etl normally. Thcb mechanism and significance of this “hc~lpc~r funrt iolr ” (II’ T7 protein kinase (Hirsch-Kauffmann et ~1.. 1955) is not kno\+,n. We thought that an isolation of host mutants in which the prc~sonc~’ of ‘I’7 fm)tclill kinase is essential for the development’ of ‘I’7 phage \vould br useful for th(b vlucitiation of the fun&ion of T7 gene 0.7. Thus, we have isolated several mutant’ strains from E. coli K12 and B by select’ing mutagenized bacteria which can plat,e ‘1’7 + pha,gc but not T’iKphage. Suclier (1973) has also isolat’ed a mutant, BRS. from E. roli I3 which support’s the growbh of T7+ but’ not T7K- phage. While studying the synthesis of phage-specific macromolecules in the mutant host8 bacteria, we found t’hat the host mutant infect,ed wit’h eit,her T7 + or T7K phaytx blocks T7 phage assembly, presumably the phage DNA packaging process. In this paper, we describe an accumulation of Ti progeny DI\‘A molecules t,hat are shorter than the mature T7 phage DNA in T7-infected mutant host cells. The result’s suggest, that a host function is involved in the T7 phago packaging process. In support, of this notion, we describe the accumulation of T7 phage head precursor particles in T7infected mubant host cells in a forthcoming paper.
2. Materials and Methods L-[3H]Leucine (57.4 Ci/mmol), [3H]t’hymidine (5.7 Ci/mmol) and [14C]amino ncitls mixture were purchased from Now England Nuclear Corp. and [35S]methionine (200 t,o Non-ionic detergent NP40 was obtained from 500 Ci/mmol) from Amersham/Searlc. Schell Chemicals; DNase I (RNase-free) from Worthington Biochemicals; lysozymc (grade I) from Sigma Chemical Co.; Pronase (B grade) from Calbiochem; Sarkosyl (NL-97) from K and K Lab. Other chemicals \~ere obtained from common commer&d sources. (b) Culture T-broth contained tained 7 g Na,HP04,
media
10 g Bacto-tryptone and 5 g NaCl in 1 1 of watrr. M9 medium 3 g KH,PO,, 1 g NH,Cl and 0.065 g MgSO, in 1 1 water.
(c) Bacteriophages
al&
con-
bacteria
T7 phage stocks were from F. W. Studier (St,udier, 1973). T7+ (wild type), H3 (deletion in gene 0.7), A57 (point mutation in gene O.?‘), 3-29 (amber mutation in gene 3) and 9-17 (amber mutation in gene 9) were used in t,his experiment. T3 phagc \vas from M. Malamy’s collection. T4 and $80 phages were from our laboratory st,oek. E. coli DlO F- (met-, Bl-, RNusc I-) was our laborat,ory strain and derived from DlO F+ (Yamada et al., 1974). E. coli 011’ was from F. 1%‘. Stutliclr and used for the growth of T7 amber mutant’ phages (Studier, 1973). A mutant strain Y49 was isolated from lj:. roli DlO F- by selcct,ing t,he abilit,y to plat,c* T7 + but not, T7K(H3) phagc after nitrosoguanidinc mlltagenesis. Similarly, sevcxral mutant st,rains were isolated from E. coli B by &tjrosogljanidine troatmcnt,.
(d)
T 7 phage infection
E. coli DlO or Y49 cells were grown in MY-glucose medium pg L-methionine/ml and 2.5 pg vitamin Bl/ml to a cell density of infected with T7 phage at a multiplicity of infection of 10. For [35S]methionine, the concentration of methionine in M9 medium When indiaated, E. coli cultures were irradiated with ultraviolet
supplemented with 50 3 x 108/ml at, 30°C and labeling of protein with was reduced to 5 pg/ml. light for 11 min and
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UItraviolrt.-irradiated cells were infected with T7 phage at a multiplicity of infection 01’ 10 at 30°C. Various times after infection 0.1.ml portions of the culture, w-err withtlraun ;Intl incuhatcd with 5 PCi [35S]methionine for 4 min at 30°C’. Non-radioactive mcthionincb \vas a(id~i to a tinal concentrat,ion of 2 mg/nrl, and thp incbubation ww continued for an additional 1.5 min and terminated by quickly chilling the culture in an ice, bath. (‘~~11s \vr’ro pell~~tctl by cent,rifugation and suspended in 0.05 ml of SDS? bllff’er (0.05 M-Tris .Hf ‘I (pH 6.X), l”, SDS, 0.142 M-mercaptoethanoi, lOqo glycerol and O.OOloi; brornophclnol I)lr~). Sampltss \\-cre heated to 100°C for 1 min Ijefore r!ectrophoresin. Preparation of aerylamitlr/SDS slab gels and the conditions of rlrct.rophoresis mere described prcAviollsl> (Yamada di Nakatla, 1976). Slab gels were dried and autoradiogra,phrd in contact with a Kodak SB-54 X-ray film (Maizel, 1971). (t’)
Analysis
of 1’7 progeny
DNA
by sucrose
gradient
cerrtrifuyation
‘1’7 phago-infect,ed cells were labeled wit,h 20 ,Ci [3H]t,hymidinc/ml from 9 to 10.5 min af%cr infection (a 1.5min pulse labeling) and t,hrn chased with 1 mg non-radioactivcx thymidine/ml. At vxrious time intervals 0.2-ml samples woe withdram& and mixed wit.11 .HCl buffer (pH 8.0) cont,aining 0.02 X-E:DTA, 0.02 M-KC% 0.2 ml of icca-cold 0.2 M-‘h-is anti 0.01 ,w-iotloacrtat,c. After adding 32P-labeled purified ‘l’7 phages as a srdimmtat ion ~narkrr, ~11s \vc’r(’ lysetl by the met,hod of Encluist & Skalka (1973). Lysozymc> \vas ;uld~t to t,hnt (‘(‘llt,rifilgnt,iorl. Phenol c,xt,raction of DNA from th(t lys:\tr: (iicl not c*hangt’ thr srdimc>nt ation l~rofilc of the DNA. Dialyzed lysatcs. 0.2 ml each, were laycrtld on 4.6 ml of linear 5?,, to 20”,, nclrlt ral su(*ros< gradients in 0.01 M-Tris .HCl buffer (pH 7.9) cont)aining 0.001 M-EDT-~ and 0.9 M-KnCl, anti t,ht> gradients were centrifuged in an SW 50.1 rotor at 38,000 revs/min for 2 h. For alkaline s~~crose gradient centrifugatioll, DNA samples in dialyzed lysatcs ww d(Xllat,rlrcd t)y mixing with an equal volume of 1 M-N&H and thrn layered on 4.6 ml of 3”,, to 20”,, stlrros3c gradients cont,aining 0.3 M-N&H, 0.7 M-NaC‘l and 0.001 M-EDT.4. In all ncxutral and alkaline gradients 0.4 ml of TO’]’0 sucrose was prescxnt, at the bottom of the cc~ntrif’upe t u hrs as a cushion. After centrifllgat’ion, gradirnt fractions UWY collrct~(l on \Vhatman no. 3 filter pa.per discs. Filter pap, IO-.” IO - 2
burnt size.
Y49 cells grow in both rich medium (T broth) and minimal medium (M9 medium) at about bhe same rate as parental DlO cells. Y49 strain appears to be normal in T7 phage adsorption and T7 DNA penet’ration into the cell. At a multiplicity of infection of 10, T7+ and T7Kphage killed 98 to 99% of bot’h DlO and Y49 cells in five minutes. When Y49 cells were infected with T7 + or T7K- pha,ge at a multiplicity of infection of about 10e3 and lysed with chloroform five minutes after infection, about 98”/; of the phages lost infectivit,y; a similar result was obtained with the DlO strain. Both T7+ and T7Kphage caused lysis of Y49 cells at about 28 minutes after infection at 3O”C, again similar to the lysis of parental strain DlO. Our preliminary experiments indicate t,hat, Y49 strain possesses an activity of protein kinase which is equivalent to that of parental strain DlO. Both strains were grown in t’he presence of [32P]orthophosphate, and hydrolysates of total cellular proteins were analyzed to assay the content of phosphoserine residues. No significant difference was found in phosphoserine content between Y49 cells and DlO cells.
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‘I(:. 1. Gel electrophoretic profiles of proteins synthesized in u.v.-irradiated Dl0 and Y-l!) 1~11s rted with T7+ phago. u.v.-irradiated DlO and Y49 cells were infected with T7+ pbagc as *ribed in Materials and Methods. At indicated times after infection, 0.1 -ml portions of the. ,11re were labeled with [35S]methionine for 4 min, then chased with non-radioactive mc-thionilw I.5 min at 30°C. Labeled cells were processed and subjected to electrophorwis in a I:i”,, ;lamido/O~l “/A SDS slab gel at 30 m.4 as &scribed in Matwials and Mrthotls. The slab g~nthrsizc~cI were almost t#he same as t)hat in DIO cells (Fig. 1 (h) to (n)). Upon infection uit’h T7K- phage. both u.v.-irradiated DlO and Y49 cells synt,hesized T7-specific protjeins similarly as upon infect,ion w-it11 1’7 + phagt~. Tllus, it appears
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FIG. 2. (:(>I elwtrophoretic profiles of proteins synthesizrrl in u.v.-irratliatccl Y49 c~c~llsinfwtrtl Y49 ~1311s~VCW~infc~rtetl with phrtg” ((f) to (j)). u v .- irmtliatrtl wit,h T7 + phapc ((a) to (c)) or T7KTT + or T7K (Aj7, amber mutation in gcuc: 0.7) phapr: and labcle~l with 1“%]methionint~ for 1 min at indicatzd times after infkrtion and chasctl for 1.5 min. Labrlrd I)rott*ins \VPI‘P analyzrd t>>’ 10% acrylitmid~/O. I O,& SDS slab gel eloctrophorssia as described in the legerrcl for Pig. 1, u.v.-irradiatrd Y49 cplls infertctl with T7 + phage ((a) ix) (c)). (a) Iabr~ling I to 5 min aftvr infection; (h) 5 to 9 min; (c) 9 to 14 min; (d) 13 to 17 min: (e) 17 to 2 t min. u.v.-irradiated Y49 cells infwtcd with T7Kphagr ((f) to (j)). (f) IA:ttxSling I to 6 min after infection; (g) 5 to 9 min: (h) 9 t)cl 13 min; (i) I3 to 17 min: (j) 17 to 21 min.
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that Y49 cells can synthesize T7-specific prot’eins normally lvhen infected with either T7+ or T7K- phage, as shown in Figure 2. As expected from these results, T7-specific RNA synthesis in Y49 cells is also normal. Comparison of RNA synthesis bet’ween T7+ phage-infected DlO and Y49 wlls showed no significant difference in t,he tota’ amount of radioactive uridine incorporation into RNA nor in the amount, of radioact’ive RNA hybridizable to T7 DNA. Similarlp, no difference was observed in the Ti-specific RNA-synthesizing aLlit>bet’\\een TTK-- -infected DlO and Y49 cells. Therefore. T7 phage genes all appear t,o be well expressed in Y49 cells even though the production of progeny phage is extremely poor in T7K--infectjed Y49 cells. \Vr assume that’ the mutation in Y49 strain is notI blocking the expression of '1'7 gwt~~ atIc1 t’he prodtrction of direct gene products.
(c) ,!!ynthesis of T7 I)L$‘~ in! j-d!) ds with T 7+ or T’iK- p,hcuy
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Rates of DNA synthesis in T7+ or T7K phagr-infected DlO and Y49 cells were measured by pulse-labeling with [3H]t,hynlidinr for one mituue at various times after infect~ion. As shown in Figure 3(a), the rate of DKA synthesis in Tit-infect’ed DlO cells rapidly increased at about eight minutes after infection and reached a maximum tenfold increase about 19 minutes after infection. In 7’7 +-infected Y49 cells, init,ial rate of DNA synthesis was verv similar to t.hat observed with D10 cells. However, the increase in the rate of DNA synthesis in Y49 cells terminated sooner than in I)10 cells. These pulse-labeled DNA samples taken after ten minmes of infection \vore sl~owa to bc exclusively T7-specific h,v hybridization with Ti DEA. Total amornn of’ T7 DN_A svnthesizcd in Y49 cells was about SO’);, of that in DlO cells.
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V‘rc:. 3. ltate of DNA synthesis in DlO and Y49 cells infwtt~tl with T7+ phugt: (a) or ‘1’7K phagr (b). Cells were grown to 3 > lO*/ml in MS-glurow medium at 30’C and infected with T7 + phage (a) or T7Kphage (b) at a multiplicity of infection of 10. At indic>ated times after infection @d-ml portions of the culture were withdrawn and pulse-labeled with PO pCi [3H]thymidinc: for I rnin at 30°C. The incorporation of [3H]thymidine was terminated by the addition of cold SO,, trivhloroacctiv acid and acid-precipitable radioactivity rr~t~ainc(! on a glass fiber filttar was tlvtwmined. (a) -0-0-, T7+ phage-infected DlO cells; -3-C’-. TT+-infected Y49 cells. (b) -e-e--, T7K(A5i) phage-infected DlO cells; -5 _- “,-, T7K--infected Y49 ~11s.
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7. DNA ligase activity in wll-free extracts from DlO and Y49 cells. Souiratrd crll-free extracts prepared from DlO and Y49 cells were assayed for DNA ligase activity as described in Materials and Methods. The assay measured acid-precipitable radioactivity due to the formation of covalently closed circular molecules from 5.8 nmol (4600 cts/min per nmol) of linear 3H-labeletl [d(A-T)], oligomers in 0.1 ml reaction mixture after 30 min at 37°C. -a--@--, Cell-free extract from DlO cells; -O-a-, cell-free extract from Y49 cells. FIG.
However, since the u.v.-sensitivity of T49 cells is the same as that of DlO cells and T7K- phage can grow well on a polA- mutant strain of E. coli, Y49 st,rain is not considered as a polAmutant. DNA ligasc activity in Y49 cells was examined in comparison with that in DlO cells. Cell-free extracts were prepared from I)10 and Y49 cells and assayed for DNA ligase activity. The result shown in Figure 7 indicat)es t)hat, the DNA ligase activities in both cell-free extracts are about the same. (c) Accumulation of concatemeric T7 DNA ill YPY cells infected u*ith other T7 mutant phages Two classes of T7 mutant phages are known to be defective in processing T7 DNA. Mutant phages in t,he first class have mutations in either gene 3 (endonuclease), gene 18 (maturation protein), or gene 19 (maturation protein). In host cells infected wit)h one of these phages, concatemeric T7 DNA remains even late in infection (Hausmann & LaRue, 1969; Striitling et al., 1973; Miller et al., 197G). The second class of phage mutant,s has mutations in either gene 8 (head protein), gene 9 (head assembiy
HOST
MUTATION
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T7 PHAGE
ASSEMBLY
107
protein, or scaffolding protein), or gene 10 (major head protein). This class of mutant, phages fails to produce phage head precursor structures (prohead) (Studier, 1972) and concatemeric T7 DNA accumulates (Hausmann & LaRue, 1969). Tn order to examine whether the generation of short T7 DNA in T7 phage-infected Y49 cells is due to a defect in cleaving the concatemeric DNB t,o proper genonw size DNA, Y49 cells were infected with T7 mutant phages known to accumulate concat,emeric T7 DNA in normal host. Pulse-labeling DNA wit.h [ 3H~lthvmidinc~ followt~cl by a chase was carried out similarly to that performed with ?i + and T7K--infectetl cells. Labeled T7 DNA in the lysates from DlO and Y49 cells infect,ed \vith th(w mutant phages was analyzed by sucrose gradient centrifugation. Figure 8 shows sedimentation profiles of T7 DKA in DlO and Y49 cells infectoti
6
2
Froctlon
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FIN. 8. Sedimentation patterns of pulse-labeled DNA from T7 3- phage-infected DlO and Y49 cells. The conditions for T7 3- (3-29, amber mutation in gene 5) phage infection, pulse-labeling with L3H]thymidine followed by chase, and sedimentation of DNA samples in neutral HIWIWW gradients were the same as described in the legend to Fig. 4. (a) and (b). DNA from T7 3- phage-infected DIO cells. (a) Pulse-labeled I.5 min; (b) pula~laheled and chased for 9.5 min. (c) and (d). DNA from T7 3- phage-infc&cd 1149 cells. (c) l’ula~-labeled for 1.5 min: (cl) 1~11lne. labeled and chased for 9.5 min. -m-e-, 3H-labeled DNA; - -c)-CJ--, 32P-labeltd marker 7’7 phage DNA.
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with T7 3- phage (3-29, amber mutation in gcallr?SX rvn;;bin(~(l ;tf’tcv appeared as concatemers (Fig. 8(s)) ant1 most of concatc~riwrir chase (Fig. 8(b)). A kmall portion of ttlfa concakmcric DXA molccwlf~s \\‘as coll\~cdf~ti t,o smaller pieces but’ the cleavage appears to be non-specific. and no spc&kl sixc vtass of DNA was generated aft,cr a long chase (Fig. 8(b)). A similar result, \vas obtainrd xlith the DNA from T7 3- -inf&ecl Y49 cells as sho\vn in Figurv X(c) and ((1). i\gaill. most of t,he pulse-labeled concatemeric T7 DKA (Fig. 8(c)) remained after chase nntl only a small port,ion of concatemers n.as clraved randomly to gencratc smaller lkcc~s after ;L long chase (Fig. S(d)). Figure 9 shows sedimentation profiles of T7 D1L’A in D10 and Y49 cells infected with T7 9- phage (9-l 7, amber mutation in gene !,), represenhing bhe second class of T7 mutant phages. The results shown in Figure 9 arc similar to those obt~ainetl with
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FIG. 9. Sedimentation p&terns of pulse-labeled DNA from T7 9- phage-infected D10 and Y49 cells. The conditions for T7 9- (9-l 7, amber mutation in gens 9) phage infection, pulse-labeling with [3H]thymidine followed by chase, and sedimentation of DNA samples in neutral SU~~OSC gradients wcrc the same as described in the legend to Fig. 4. (a), (b) and (c). DNA from T7 9- phage-infected DlO cells. (a) Pulse-labeled for 1.3 min; (b) pulse-labeled and chased for 3 min; (c) pulse-labeled and chased for 9.5 min. (d), (e) and (f). DNA from T7 Y- phage-infected Y49 cells. (d) Pulse-labeled for I.5 mill; (e) pulse-labeled and chased for 3 min; (f) pulse-labeled and chased for 9.5 min. “ZP-labeled marker T7 phagc D N.4. --e--e-, 3H-labeled DNA; --c)-Cl--,
HOST
MUTATION
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ASSEMBLY
109
T7 3- infect,ion shown in Figure 8. In both DlO and Y49 cells, T7 9- infection caused an accumulation of concatcmeric T7 DNA even late in infection. Only a small portion of the concatemer DNA was randomly cleaved and converted to smaller size species after a long chase (Fig. 9(c) and (f)). The results presented here showed that t,he mutation in E-49 strain has no e&ct on the defect, in the cleavage of concatemeric T7 DNA resulting from either ‘I’S 3- or ‘1’7 !)- mutation. Therefore, it’ appears t,hat the T7-specific expression of tjhe host) mutation inY49 strain, resulting in a generation of homopeneonsshort DNS molecules. requires the function of T7 genes 3 and 9.
4. Discussion A new mutant, strain, Y49, which causes an abnormal processing of ‘I’7 phat#t> progeny DNA, was isolated from E. coli K12. Y49 cells synthesize T7-specific RKAand prot,eins normally when infected with T7 + or T7K- phage: t#hc amount of T7 DX.‘X synthesized in Y49 cells is only slightly less than t’hat in parental, normal host Dl.0 cells. Sucroxc gradient centrifugal analysis revealed that, TS + or T7Kphage-infcctecl Y49 cells produce concatemeric T7 progeny DNA molecules as parental DlO cells do. but labe in infection these concatemers are converted to a class of DNA molecules with a size about 30% shorter than the T7 genome. Kcplicat ion of T7 DNA was shown to involve concatemeric intermediat,e molecult\s. thrc~~~to four times longer than the size of the T7 genome. before these concatemrtrs are cleaved t,o the genome size and packaged int,o phage particles (Kelly & Thomas. 1969: Schlcgtll & Thomas, 1972). Therefore, it leas thought’ t’hat the formation of concatemeric TT progeny DNA requires host enzymes such as DNA polymerasr 1 and DNA ligase in addition to T7 gene &coded T7 DNA polymerasc (IfTatson, 1972). Since concatrmeric T7 DNA was formed in the mutant Y49 cells. t,he mut,at.ion in Y4!) strain does not seem to be involved in the concat.cmer format,ion. In fact., WV havct shown t,hat Y49 stra,in possesses a normal Irvel of DSA ligase activit!r. In addition. normal u.v.-sensit,ivity of Y49 stmin suggests that DNA polymerast~ I activit,y is not. affected by the Y49 mut,ation. It has been shown that, certain mut’atjions in T7 genes block t,he processing of ‘1’7 concat,emeric DNA to t)he genome-size DNA. Concatemeric T7 DNA remains mostl! uncleavcd even late in the infection in norma,l host cells infect’ed wit,h these mut’ant phagt,s. These mut,at,ions are eit,her in a, class of genes (genes 8. II and 70) which are involved in 7’7 phage head formation (Hausmann & LaRue. 1909: St)udicr, 1972: our unpublished results), or in a class of genes (gtbnes 3. 18 and I.!?) involved in ‘I’7 DNA processing (Hausmann $ LaRue, 1969: StrIitling rt al., 1972; Miller ct al., 1976). I &&on of the host with mutant phages of the latter class causes an accumulation of caoncatcmctr ‘I’7 DNA with a concomit’ant accumulation of phage head precursor particles, prohtlad (Studier, 1972). I\S prrsent.rd in this paper, Y49 cells infcct~ed with T7 3- or ‘I’7 Q- phagc accumulatt>d concatcmt+c T7 DNA molecules, and most of the concatrmers were not’ clt>avecl in a similar manner as in parental DlO cells. Thereforc, it appears t’hat, the abnormal processing of TT DNB in Y49 ~11s \\~hich generates short Dlt’A molecules from tlw concatemerie T7 DNA occurs after t,he formation of proheads, because the mere presence of concatemer DNA (in T7 9- phage infection) or concatemer DNA together
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with proheads (in T7 3 - phage infection) in Y49 cells does not result’ in t!hci clenvag~* nf concatemeric DNA to generat’e the “short DNA.” Our results suggtlst t,lrat 1111’ mutated gene in Y49 strain exerts it,s effect beyond t’ho formabion of both c*oncatc>meric DNA and proheads, probably in a furt)her progressed st,age of TS DNA packaging into phage head. This prompted us to investigate t)he head assembly proc(‘ss in T7 phage-infected Y49 cells in comparison to that in DlO cells, as will be describc,cI in a forthcoming paper. Finally, we would like tjo mention that T7 DNA has a unique, non-permut,otl$ redundant region with a length about l”/ of the T7 genome at both ends of t hc double-stranded molecule (Richie et al.. 1967). Replication and processing of ‘I’7 DNA, t,herefore, involve certain specific mechanisms by which the terminall) redundant sequence is recognized and preserved (Watson, 1972). The short T7 DNA molecules which accumulated in Y49 cells infected with T7+ or T7K- phage, with a size about 307; shorter than the T7 genome, must have lost either one or both of t,hc terminally redundant regions. Electron microscopic visualization of a heteroduplcx, formed between the short DNA from Y49 cells and T7 phage DNA, t,o determine t)he missing region(s) in bhe short DNA is in progress. We thank Dr F. W. Studier for his supply of T7 mutant phages and advice. We also thank Dr M. H. Malamy for T3 phage and Dr W. S. Kelley for poZA mutants of E. ~01%. This work was supported by a U.S. Public Health Service Research Grant GM21504 (to D.N.). REFERENCES
Center, M. S. (1973). J. Viral. 12, 847-854. Enquist, L. W. & Skalka, A. (1973). ,J. Mol. Biol. 75, 185-212. Freifelder, D. (1970). J. Mol. Biol. 54, 567-577. Hausmann, R. & LaRue, K. (1969).J. Viral. 3,278-281. Hesselbach, B. A. & Nakada, D. (1975). Nature (London), 258, 354-357. Hesselbach, B. A. & Nakada, D. (1977a). J. Viral. 24, 736-745. Hesselbach, B. A. & Nakada, D. (19773). J. Tlirol. 24, 746-760. Hirsch-Kauffmann, M., Herrlich, P., Pont)a, H. & Schweiger, M. (1975). Nature (London), 255, 508-510. Kelly, T. J. Jr & Thomas, C. A. Jr (1969). J. Mol. BioZ. 44, 459-475. Maizel, J. V. (1971). Methods Viral. 5, 179-246. Miller, R. C. Jr, Lee, M., Scraba, D. G. & Paetkau, V. (1976). J. ;yloZ. Biol. 101, 223-234. Modrich, P. & Lehman, J. R. (1970). J. BioZ. Chem. 245, 3626-3631. Ponta, H., Rahmsdorf, H. J., Pai, S. H., Hirsch-Kauffman, M., Herrlich, P. & Schweigcr. M. (1974). Mol. Gen. Genet. 134, 281.-297. Rahmsdorf, H. J., Herrlich, P., Pai, S. H., Schweiger, M. & Witt,mann. H. U. (1973). AfoZ. Gen. Genet. 127, 259-271. Rahmsdorf, H. J., Pai, S. H., Ponta, H., Herrlich, P., Roskoski, K. Jr, Slchweigrr, M. & Studier, F. W. (1974). Proc. Nat. Acad. Sci., U.S.A. 71, 586589. Ritchie, D. A., Thomas, C. A. Jr, MaeHattie, I,. A. & Wensink, P. C. (1967). J. AlfoZ. Biol. 23, 365-376. Rothman-Denes, L. B., Muthukrishnan, S., Haselkorn, R. & S:tudier, F. W. (1973). Press, Virus Research (Fox, C. F. & Robinson, W. S., eds), pp. 227-239, Academic New York. Schlegel, R. A. & Thomas, C. A. Jr (1972). J. MoZ. BioZ. 68, 319-345. Striitling. W., Krause, E. & Knippers, R. (1973). T’irology, 51, 109-119. Studier, F. W. (1972). Science, 176, 367-376. Studier, F. W. (1973). J. MoZ. BioZ. 79, 227-236. Watson, J. D. (1972). Nature Nezo Biol. 239, 197-201.
HOST Yama.da, Yamada, Zillig, W., H. &
MUTATION
AND
T7 PHAGE
ASSEMBLY
III
Y. & Nakada, D. (1976). J. Mol. Biol. 100, 35-45. Y., Whitaker, P. A. & Nakada, D. (1974). Nature (London), 248, 335-338. Fujiki, H., Blum, W., Janekoviit, D., Schweiger, M., Rahmsdorf, H. J., Ponta, Hirsch-Kauffman, M. (1975). Proc. Nut. Acad. Sci., C.S.A. 72, 2506-2510.
121, 95-111
Mutant of Escherichiu cob which Blocks T7 Bacteriophage Assembly: Accumulation of Short T7 DNA YOSHIHIKO YAMADA,JAXET
~ILNVTZER AND DAI NAKAIM
Department of Biochemistry University of Pittsburgh School of N dirin~~ Pittsburgh, Pa 15261, C:.S.;1. (Received 23 September 1977, and in revised form 27 January
I97S)
A mut,ant strain which blocks bacteriophage T7 assembly, presumably at the from li’wherichiu cdi DlO F ~-. phage DNA packaging process, was isolat,rd This mutant, Y49, was selected for its ability to plate TT+ phage but’ not 7’7 gene 0.7 mutant phage (T7K-). The plating efficiency of Ti+ phagc on strain Y49 was o~rr SOa; of t’hat on the parental &rain but the yield of T7 + phagr WAS phagr, ‘L-49 cells synthr~sizc~tl less t~han loo’ ,O’ Upon infection with T7 + or T7IC T7-specific RNA and protein almost, normally but, thf3 synthrsis of ‘I?7 progqv by the DNA was slightly reduced, about 70 to 80:; of thr amount synthesized parental strain. In Y49 cells infected with T7 + or T7Kphage, T7 progeny DNA appeared first as concatemers larger in size than the mature T7 phage DNA but, it than the size of T7 phage later was converted to pieces about Xl:,, shorter DNA. When Y49 cells were infect,cd with T7 mutant phagrs known to accumulate concatemcric T7 progeny DNA in t,he normal host, cells, T7 progeny DNA also accumulat,ed as concatemcrs and was not clcavetl to smaller pieces. These rc,sults suggest t,hat, the cleavage of concatemr,ric T7 DPJA in ‘I”i+ or T7K-~ phapcbinfc?ct,ed Y49 cells, which generates short DNA species, appears to bc site-specific* and probably results from a blockage of the phapc DNA packaging process d\le to the host mutat)ion, implying that a host function is invol\-c,d in the 7’7 phagcs DNA packaging process.
1. Introduction Bact)eriophage T7 gene 0.7 codes for a protein kinase which phosphorylat,es many proteins of the host Escherichia coli including RNA polymerase (Rahmsdorf et ad.. 1973,1974; Zillig et al., 1975). Phosphorylation of the host E. coli RNA polymerasr has been implicated in the inactivation of t,he enzyme and thus the “host shut,-off” b~l !I’7 phage inf&ion (Rothman-Denes et aZ.. 1973; Pont,a et al.. 1974). However. recent studies in t,his laboratory revealed t,hat the inactivabion of E. coli RPI’A polymerase in T7-infected cells is mainly due to the binding to the enzyme of an inhibitor prot,ein. termed 1 protein, coded by T7 gene 2 although gene 0.7 product is also partly involved in the inactivation of the enzyme (Hcssclbach & Xakatla, 1975,1977a,b). T7 gene 0.7 has been considered as one of the non-essential genes of T7 (Studiw, can be propagated normally in t’he most 1973): T7 gene 0.7 mutant phage (T7K-) commonly used host’ E. coli B st)rain. Hirsch-Kauffman et al. (1975) found, however. that) T7K- phage gave only few, if any, progeny phagrs when used t,o infect E. coli 9.i 10 1978 ~2catltTTlicPress Inc. (London) Ltcl. c,o~n~~s3e/7s/l:'lrrt.i11 $02.00/O
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K12 st’rain gro\\~ in a minimal mrdium whcrvas thv \vild-t>,pc T’i ’ IJlag~~ u ah propagat,etl normally. Thcb mechanism and significance of this “hc~lpc~r funrt iolr ” (II’ T7 protein kinase (Hirsch-Kauffmann et ~1.. 1955) is not kno\+,n. We thought that an isolation of host mutants in which the prc~sonc~’ of ‘I’7 fm)tclill kinase is essential for the development’ of ‘I’7 phage \vould br useful for th(b vlucitiation of the fun&ion of T7 gene 0.7. Thus, we have isolated several mutant’ strains from E. coli K12 and B by select’ing mutagenized bacteria which can plat,e ‘1’7 + pha,gc but not T’iKphage. Suclier (1973) has also isolat’ed a mutant, BRS. from E. roli I3 which support’s the growbh of T7+ but’ not T7K- phage. While studying the synthesis of phage-specific macromolecules in the mutant host8 bacteria, we found t’hat the host mutant infect,ed wit’h eit,her T7 + or T7K phaytx blocks T7 phage assembly, presumably the phage DNA packaging process. In this paper, we describe an accumulation of Ti progeny DI\‘A molecules t,hat are shorter than the mature T7 phage DNA in T7-infected mutant host cells. The result’s suggest, that a host function is involved in the T7 phago packaging process. In support, of this notion, we describe the accumulation of T7 phage head precursor particles in T7infected mubant host cells in a forthcoming paper.
2. Materials and Methods L-[3H]Leucine (57.4 Ci/mmol), [3H]t’hymidine (5.7 Ci/mmol) and [14C]amino ncitls mixture were purchased from Now England Nuclear Corp. and [35S]methionine (200 t,o Non-ionic detergent NP40 was obtained from 500 Ci/mmol) from Amersham/Searlc. Schell Chemicals; DNase I (RNase-free) from Worthington Biochemicals; lysozymc (grade I) from Sigma Chemical Co.; Pronase (B grade) from Calbiochem; Sarkosyl (NL-97) from K and K Lab. Other chemicals \~ere obtained from common commer&d sources. (b) Culture T-broth contained tained 7 g Na,HP04,
media
10 g Bacto-tryptone and 5 g NaCl in 1 1 of watrr. M9 medium 3 g KH,PO,, 1 g NH,Cl and 0.065 g MgSO, in 1 1 water.
(c) Bacteriophages
al&
con-
bacteria
T7 phage stocks were from F. W. Studier (St,udier, 1973). T7+ (wild type), H3 (deletion in gene 0.7), A57 (point mutation in gene O.?‘), 3-29 (amber mutation in gene 3) and 9-17 (amber mutation in gene 9) were used in t,his experiment. T3 phagc \vas from M. Malamy’s collection. T4 and $80 phages were from our laboratory st,oek. E. coli DlO F- (met-, Bl-, RNusc I-) was our laborat,ory strain and derived from DlO F+ (Yamada et al., 1974). E. coli 011’ was from F. 1%‘. Stutliclr and used for the growth of T7 amber mutant’ phages (Studier, 1973). A mutant strain Y49 was isolated from lj:. roli DlO F- by selcct,ing t,he abilit,y to plat,c* T7 + but not, T7K(H3) phagc after nitrosoguanidinc mlltagenesis. Similarly, sevcxral mutant st,rains were isolated from E. coli B by &tjrosogljanidine troatmcnt,.
(d)
T 7 phage infection
E. coli DlO or Y49 cells were grown in MY-glucose medium pg L-methionine/ml and 2.5 pg vitamin Bl/ml to a cell density of infected with T7 phage at a multiplicity of infection of 10. For [35S]methionine, the concentration of methionine in M9 medium When indiaated, E. coli cultures were irradiated with ultraviolet
supplemented with 50 3 x 108/ml at, 30°C and labeling of protein with was reduced to 5 pg/ml. light for 11 min and
HOST
MUTATION
AND
tjlrm inrubat~r~d for 15 min a,t 30°C before et az., 1974). (v)
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T’7 PHAGE
phapc
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ASSEMBLY as dcscrihed
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UItraviolrt.-irradiated cells were infected with T7 phage at a multiplicity of infection 01’ 10 at 30°C. Various times after infection 0.1.ml portions of the culture, w-err withtlraun ;Intl incuhatcd with 5 PCi [35S]methionine for 4 min at 30°C’. Non-radioactive mcthionincb \vas a(id~i to a tinal concentrat,ion of 2 mg/nrl, and thp incbubation ww continued for an additional 1.5 min and terminated by quickly chilling the culture in an ice, bath. (‘~~11s \vr’ro pell~~tctl by cent,rifugation and suspended in 0.05 ml of SDS? bllff’er (0.05 M-Tris .Hf ‘I (pH 6.X), l”, SDS, 0.142 M-mercaptoethanoi, lOqo glycerol and O.OOloi; brornophclnol I)lr~). Sampltss \\-cre heated to 100°C for 1 min Ijefore r!ectrophoresin. Preparation of aerylamitlr/SDS slab gels and the conditions of rlrct.rophoresis mere described prcAviollsl> (Yamada di Nakatla, 1976). Slab gels were dried and autoradiogra,phrd in contact with a Kodak SB-54 X-ray film (Maizel, 1971). (t’)
Analysis
of 1’7 progeny
DNA
by sucrose
gradient
cerrtrifuyation
‘1’7 phago-infect,ed cells were labeled wit,h 20 ,Ci [3H]t,hymidinc/ml from 9 to 10.5 min af%cr infection (a 1.5min pulse labeling) and t,hrn chased with 1 mg non-radioactivcx thymidine/ml. At vxrious time intervals 0.2-ml samples woe withdram& and mixed wit.11 .HCl buffer (pH 8.0) cont,aining 0.02 X-E:DTA, 0.02 M-KC% 0.2 ml of icca-cold 0.2 M-‘h-is anti 0.01 ,w-iotloacrtat,c. After adding 32P-labeled purified ‘l’7 phages as a srdimmtat ion ~narkrr, ~11s \vc’r(’ lysetl by the met,hod of Encluist & Skalka (1973). Lysozymc> \vas ;uld~t to t,hnt (‘(‘llt,rifilgnt,iorl. Phenol c,xt,raction of DNA from th(t lys:\tr: (iicl not c*hangt’ thr srdimc>nt ation l~rofilc of the DNA. Dialyzed lysatcs. 0.2 ml each, were laycrtld on 4.6 ml of linear 5?,, to 20”,, nclrlt ral su(*ros< gradients in 0.01 M-Tris .HCl buffer (pH 7.9) cont)aining 0.001 M-EDT-~ and 0.9 M-KnCl, anti t,ht> gradients were centrifuged in an SW 50.1 rotor at 38,000 revs/min for 2 h. For alkaline s~~crose gradient centrifugatioll, DNA samples in dialyzed lysatcs ww d(Xllat,rlrcd t)y mixing with an equal volume of 1 M-N&H and thrn layered on 4.6 ml of 3”,, to 20”,, stlrros3c gradients cont,aining 0.3 M-N&H, 0.7 M-NaC‘l and 0.001 M-EDT.4. In all ncxutral and alkaline gradients 0.4 ml of TO’]’0 sucrose was prescxnt, at the bottom of the cc~ntrif’upe t u hrs as a cushion. After centrifllgat’ion, gradirnt fractions UWY collrct~(l on \Vhatman no. 3 filter pa.per discs. Filter pap, IO-.” IO - 2
burnt size.
Y49 cells grow in both rich medium (T broth) and minimal medium (M9 medium) at about bhe same rate as parental DlO cells. Y49 strain appears to be normal in T7 phage adsorption and T7 DNA penet’ration into the cell. At a multiplicity of infection of 10, T7+ and T7Kphage killed 98 to 99% of bot’h DlO and Y49 cells in five minutes. When Y49 cells were infected with T7 + or T7K- pha,ge at a multiplicity of infection of about 10e3 and lysed with chloroform five minutes after infection, about 98”/; of the phages lost infectivit,y; a similar result was obtained with the DlO strain. Both T7+ and T7Kphage caused lysis of Y49 cells at about 28 minutes after infection at 3O”C, again similar to the lysis of parental strain DlO. Our preliminary experiments indicate t,hat, Y49 strain possesses an activity of protein kinase which is equivalent to that of parental strain DlO. Both strains were grown in t’he presence of [32P]orthophosphate, and hydrolysates of total cellular proteins were analyzed to assay the content of phosphoserine residues. No significant difference was found in phosphoserine content between Y49 cells and DlO cells.
HOST (a)
(a)
MUTATION (cl
Cd)
ANI) (e)
(f)
(a)
T7 (h)
PHAGE (i)
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ASSEMBLY (il
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‘I(:. 1. Gel electrophoretic profiles of proteins synthesized in u.v.-irradiated Dl0 and Y-l!) 1~11s rted with T7+ phago. u.v.-irradiated DlO and Y49 cells were infected with T7+ pbagc as *ribed in Materials and Methods. At indicated times after infection, 0.1 -ml portions of the. ,11re were labeled with [35S]methionine for 4 min, then chased with non-radioactive mc-thionilw I.5 min at 30°C. Labeled cells were processed and subjected to electrophorwis in a I:i”,, ;lamido/O~l “/A SDS slab gel at 30 m.4 as &scribed in Matwials and Mrthotls. The slab g~nthrsizc~cI were almost t#he same as t)hat in DIO cells (Fig. 1 (h) to (n)). Upon infection uit’h T7K- phage. both u.v.-irradiated DlO and Y49 cells synt,hesized T7-specific protjeins similarly as upon infect,ion w-it11 1’7 + phagt~. Tllus, it appears
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FIG. 2. (:(>I elwtrophoretic profiles of proteins synthesizrrl in u.v.-irratliatccl Y49 c~c~llsinfwtrtl Y49 ~1311s~VCW~infc~rtetl with phrtg” ((f) to (j)). u v .- irmtliatrtl wit,h T7 + phapc ((a) to (c)) or T7KTT + or T7K (Aj7, amber mutation in gcuc: 0.7) phapr: and labcle~l with 1“%]methionint~ for 1 min at indicatzd times after infkrtion and chasctl for 1.5 min. Labrlrd I)rott*ins \VPI‘P analyzrd t>>’ 10% acrylitmid~/O. I O,& SDS slab gel eloctrophorssia as described in the legerrcl for Pig. 1, u.v.-irradiatrd Y49 cplls infertctl with T7 + phage ((a) ix) (c)). (a) Iabr~ling I to 5 min aftvr infection; (h) 5 to 9 min; (c) 9 to 14 min; (d) 13 to 17 min: (e) 17 to 2 t min. u.v.-irradiated Y49 cells infwtcd with T7Kphagr ((f) to (j)). (f) IA:ttxSling I to 6 min after infection; (g) 5 to 9 min: (h) 9 t)cl 13 min; (i) I3 to 17 min: (j) 17 to 21 min.
HOST
MUTATION
AliD
TS
I’HACE:
ASREIIRLI~
IIll
that Y49 cells can synthesize T7-specific prot’eins normally lvhen infected with either T7+ or T7K- phage, as shown in Figure 2. As expected from these results, T7-specific RNA synthesis in Y49 cells is also normal. Comparison of RNA synthesis bet’ween T7+ phage-infected DlO and Y49 wlls showed no significant difference in t,he tota’ amount of radioactive uridine incorporation into RNA nor in the amount, of radioact’ive RNA hybridizable to T7 DNA. Similarlp, no difference was observed in the Ti-specific RNA-synthesizing aLlit>bet’\\een TTK-- -infected DlO and Y49 cells. Therefore. T7 phage genes all appear t,o be well expressed in Y49 cells even though the production of progeny phage is extremely poor in T7K--infectjed Y49 cells. \Vr assume that’ the mutation in Y49 strain is notI blocking the expression of '1'7 gwt~~ atIc1 t’he prodtrction of direct gene products.
(c) ,!!ynthesis of T7 I)L$‘~ in! j-d!) ds with T 7+ or T’iK- p,hcuy
irlfwted
Rates of DNA synthesis in T7+ or T7K phagr-infected DlO and Y49 cells were measured by pulse-labeling with [3H]t,hynlidinr for one mituue at various times after infect~ion. As shown in Figure 3(a), the rate of DKA synthesis in Tit-infect’ed DlO cells rapidly increased at about eight minutes after infection and reached a maximum tenfold increase about 19 minutes after infection. In 7’7 +-infected Y49 cells, init,ial rate of DNA synthesis was verv similar to t.hat observed with D10 cells. However, the increase in the rate of DNA synthesis in Y49 cells terminated sooner than in I)10 cells. These pulse-labeled DNA samples taken after ten minmes of infection \vore sl~owa to bc exclusively T7-specific h,v hybridization with Ti DEA. Total amornn of’ T7 DN_A svnthesizcd in Y49 cells was about SO’);, of that in DlO cells.
1 0
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Time after infection (al
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V‘rc:. 3. ltate of DNA synthesis in DlO and Y49 cells infwtt~tl with T7+ phugt: (a) or ‘1’7K phagr (b). Cells were grown to 3 > lO*/ml in MS-glurow medium at 30’C and infected with T7 + phage (a) or T7Kphage (b) at a multiplicity of infection of 10. At indic>ated times after infection @d-ml portions of the culture were withdrawn and pulse-labeled with PO pCi [3H]thymidinc: for I rnin at 30°C. The incorporation of [3H]thymidine was terminated by the addition of cold SO,, trivhloroacctiv acid and acid-precipitable radioactivity rr~t~ainc(! on a glass fiber filttar was tlvtwmined. (a) -0-0-, T7+ phage-infected DlO cells; -3-C’-. TT+-infected Y49 cells. (b) -e-e--, T7K(A5i) phage-infected DlO cells; -5 _- “,-, T7K--infected Y49 ~11s.
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7. DNA ligase activity in wll-free extracts from DlO and Y49 cells. Souiratrd crll-free extracts prepared from DlO and Y49 cells were assayed for DNA ligase activity as described in Materials and Methods. The assay measured acid-precipitable radioactivity due to the formation of covalently closed circular molecules from 5.8 nmol (4600 cts/min per nmol) of linear 3H-labeletl [d(A-T)], oligomers in 0.1 ml reaction mixture after 30 min at 37°C. -a--@--, Cell-free extract from DlO cells; -O-a-, cell-free extract from Y49 cells. FIG.
However, since the u.v.-sensitivity of T49 cells is the same as that of DlO cells and T7K- phage can grow well on a polA- mutant strain of E. coli, Y49 st,rain is not considered as a polAmutant. DNA ligasc activity in Y49 cells was examined in comparison with that in DlO cells. Cell-free extracts were prepared from I)10 and Y49 cells and assayed for DNA ligase activity. The result shown in Figure 7 indicat)es t)hat, the DNA ligase activities in both cell-free extracts are about the same. (c) Accumulation of concatemeric T7 DNA ill YPY cells infected u*ith other T7 mutant phages Two classes of T7 mutant phages are known to be defective in processing T7 DNA. Mutant phages in t,he first class have mutations in either gene 3 (endonuclease), gene 18 (maturation protein), or gene 19 (maturation protein). In host cells infected wit)h one of these phages, concatemeric T7 DNA remains even late in infection (Hausmann & LaRue, 1969; Striitling et al., 1973; Miller et al., 197G). The second class of phage mutant,s has mutations in either gene 8 (head protein), gene 9 (head assembiy
HOST
MUTATION
AND
T7 PHAGE
ASSEMBLY
107
protein, or scaffolding protein), or gene 10 (major head protein). This class of mutant, phages fails to produce phage head precursor structures (prohead) (Studier, 1972) and concatemeric T7 DNA accumulates (Hausmann & LaRue, 1969). Tn order to examine whether the generation of short T7 DNA in T7 phage-infected Y49 cells is due to a defect in cleaving the concatemeric DNB t,o proper genonw size DNA, Y49 cells were infected with T7 mutant phages known to accumulate concat,emeric T7 DNA in normal host. Pulse-labeling DNA wit.h [ 3H~lthvmidinc~ followt~cl by a chase was carried out similarly to that performed with ?i + and T7K--infectetl cells. Labeled T7 DNA in the lysates from DlO and Y49 cells infect,ed \vith th(w mutant phages was analyzed by sucrose gradient centrifugation. Figure 8 shows sedimentation profiles of T7 DKA in DlO and Y49 cells infectoti
6
2
Froctlon
number
FIN. 8. Sedimentation patterns of pulse-labeled DNA from T7 3- phage-infected DlO and Y49 cells. The conditions for T7 3- (3-29, amber mutation in gene 5) phage infection, pulse-labeling with L3H]thymidine followed by chase, and sedimentation of DNA samples in neutral HIWIWW gradients were the same as described in the legend to Fig. 4. (a) and (b). DNA from T7 3- phage-infected DIO cells. (a) Pulse-labeled I.5 min; (b) pula~laheled and chased for 9.5 min. (c) and (d). DNA from T7 3- phage-infc&cd 1149 cells. (c) l’ula~-labeled for 1.5 min: (cl) 1~11lne. labeled and chased for 9.5 min. -m-e-, 3H-labeled DNA; - -c)-CJ--, 32P-labeltd marker 7’7 phage DNA.
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with T7 3- phage (3-29, amber mutation in gcallr?SX rvn;;bin(~(l ;tf’tcv appeared as concatemers (Fig. 8(s)) ant1 most of concatc~riwrir chase (Fig. 8(b)). A kmall portion of ttlfa concakmcric DXA molccwlf~s \\‘as coll\~cdf~ti t,o smaller pieces but’ the cleavage appears to be non-specific. and no spc&kl sixc vtass of DNA was generated aft,cr a long chase (Fig. 8(b)). A similar result, \vas obtainrd xlith the DNA from T7 3- -inf&ecl Y49 cells as sho\vn in Figurv X(c) and ((1). i\gaill. most of t,he pulse-labeled concatemeric T7 DKA (Fig. 8(c)) remained after chase nntl only a small port,ion of concatemers n.as clraved randomly to gencratc smaller lkcc~s after ;L long chase (Fig. S(d)). Figure 9 shows sedimentation profiles of T7 D1L’A in D10 and Y49 cells infected with T7 9- phage (9-l 7, amber mutation in gene !,), represenhing bhe second class of T7 mutant phages. The results shown in Figure 9 arc similar to those obt~ainetl with
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30
number
FIG. 9. Sedimentation p&terns of pulse-labeled DNA from T7 9- phage-infected D10 and Y49 cells. The conditions for T7 9- (9-l 7, amber mutation in gens 9) phage infection, pulse-labeling with [3H]thymidine followed by chase, and sedimentation of DNA samples in neutral SU~~OSC gradients wcrc the same as described in the legend to Fig. 4. (a), (b) and (c). DNA from T7 9- phage-infected DlO cells. (a) Pulse-labeled for 1.3 min; (b) pulse-labeled and chased for 3 min; (c) pulse-labeled and chased for 9.5 min. (d), (e) and (f). DNA from T7 Y- phage-infected Y49 cells. (d) Pulse-labeled for I.5 mill; (e) pulse-labeled and chased for 3 min; (f) pulse-labeled and chased for 9.5 min. “ZP-labeled marker T7 phagc D N.4. --e--e-, 3H-labeled DNA; --c)-Cl--,
HOST
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ASSEMBLY
109
T7 3- infect,ion shown in Figure 8. In both DlO and Y49 cells, T7 9- infection caused an accumulation of concatcmeric T7 DNA even late in infection. Only a small portion of the concatemer DNA was randomly cleaved and converted to smaller size species after a long chase (Fig. 9(c) and (f)). The results presented here showed that t,he mutation in E-49 strain has no e&ct on the defect, in the cleavage of concatemeric T7 DNA resulting from either ‘I’S 3- or ‘1’7 !)- mutation. Therefore, it’ appears t,hat the T7-specific expression of tjhe host) mutation inY49 strain, resulting in a generation of homopeneonsshort DNS molecules. requires the function of T7 genes 3 and 9.
4. Discussion A new mutant, strain, Y49, which causes an abnormal processing of ‘I’7 phat#t> progeny DNA, was isolated from E. coli K12. Y49 cells synthesize T7-specific RKAand prot,eins normally when infected with T7 + or T7K- phage: t#hc amount of T7 DX.‘X synthesized in Y49 cells is only slightly less than t’hat in parental, normal host Dl.0 cells. Sucroxc gradient centrifugal analysis revealed that, TS + or T7Kphage-infcctecl Y49 cells produce concatemeric T7 progeny DNA molecules as parental DlO cells do. but labe in infection these concatemers are converted to a class of DNA molecules with a size about 30% shorter than the T7 genome. Kcplicat ion of T7 DNA was shown to involve concatemeric intermediat,e molecult\s. thrc~~~to four times longer than the size of the T7 genome. before these concatemrtrs are cleaved t,o the genome size and packaged int,o phage particles (Kelly & Thomas. 1969: Schlcgtll & Thomas, 1972). Therefore, it leas thought’ t’hat the formation of concatemeric TT progeny DNA requires host enzymes such as DNA polymerasr 1 and DNA ligase in addition to T7 gene &coded T7 DNA polymerasc (IfTatson, 1972). Since concatrmeric T7 DNA was formed in the mutant Y49 cells. t,he mut,at.ion in Y4!) strain does not seem to be involved in the concat.cmer format,ion. In fact., WV havct shown t,hat Y49 stra,in possesses a normal Irvel of DSA ligase activit!r. In addition. normal u.v.-sensit,ivity of Y49 stmin suggests that DNA polymerast~ I activit,y is not. affected by the Y49 mut,ation. It has been shown that, certain mut’atjions in T7 genes block t,he processing of ‘1’7 concat,emeric DNA to t)he genome-size DNA. Concatemeric T7 DNA remains mostl! uncleavcd even late in the infection in norma,l host cells infect’ed wit,h these mut’ant phagt,s. These mut,at,ions are eit,her in a, class of genes (genes 8. II and 70) which are involved in 7’7 phage head formation (Hausmann & LaRue. 1909: St)udicr, 1972: our unpublished results), or in a class of genes (gtbnes 3. 18 and I.!?) involved in ‘I’7 DNA processing (Hausmann $ LaRue, 1969: StrIitling rt al., 1972; Miller ct al., 1976). I &&on of the host with mutant phages of the latter class causes an accumulation of caoncatcmctr ‘I’7 DNA with a concomit’ant accumulation of phage head precursor particles, prohtlad (Studier, 1972). I\S prrsent.rd in this paper, Y49 cells infcct~ed with T7 3- or ‘I’7 Q- phagc accumulatt>d concatcmt+c T7 DNA molecules, and most of the concatrmers were not’ clt>avecl in a similar manner as in parental DlO cells. Thereforc, it appears t’hat, the abnormal processing of TT DNB in Y49 ~11s \\~hich generates short Dlt’A molecules from tlw concatemerie T7 DNA occurs after t,he formation of proheads, because the mere presence of concatemer DNA (in T7 9- phage infection) or concatemer DNA together
110
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YAhlAi,>4,
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;\NI)
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with proheads (in T7 3 - phage infection) in Y49 cells does not result’ in t!hci clenvag~* nf concatemeric DNA to generat’e the “short DNA.” Our results suggtlst t,lrat 1111’ mutated gene in Y49 strain exerts it,s effect beyond t’ho formabion of both c*oncatc>meric DNA and proheads, probably in a furt)her progressed st,age of TS DNA packaging into phage head. This prompted us to investigate t)he head assembly proc(‘ss in T7 phage-infected Y49 cells in comparison to that in DlO cells, as will be describc,cI in a forthcoming paper. Finally, we would like tjo mention that T7 DNA has a unique, non-permut,otl$ redundant region with a length about l”/ of the T7 genome at both ends of t hc double-stranded molecule (Richie et al.. 1967). Replication and processing of ‘I’7 DNA, t,herefore, involve certain specific mechanisms by which the terminall) redundant sequence is recognized and preserved (Watson, 1972). The short T7 DNA molecules which accumulated in Y49 cells infected with T7+ or T7K- phage, with a size about 307; shorter than the T7 genome, must have lost either one or both of t,hc terminally redundant regions. Electron microscopic visualization of a heteroduplcx, formed between the short DNA from Y49 cells and T7 phage DNA, t,o determine t)he missing region(s) in bhe short DNA is in progress. We thank Dr F. W. Studier for his supply of T7 mutant phages and advice. We also thank Dr M. H. Malamy for T3 phage and Dr W. S. Kelley for poZA mutants of E. ~01%. This work was supported by a U.S. Public Health Service Research Grant GM21504 (to D.N.). REFERENCES
Center, M. S. (1973). J. Viral. 12, 847-854. Enquist, L. W. & Skalka, A. (1973). ,J. Mol. Biol. 75, 185-212. Freifelder, D. (1970). J. Mol. Biol. 54, 567-577. Hausmann, R. & LaRue, K. (1969).J. Viral. 3,278-281. Hesselbach, B. A. & Nakada, D. (1975). Nature (London), 258, 354-357. Hesselbach, B. A. & Nakada, D. (1977a). J. Viral. 24, 736-745. Hesselbach, B. A. & Nakada, D. (19773). J. Tlirol. 24, 746-760. Hirsch-Kauffmann, M., Herrlich, P., Pont)a, H. & Schweiger, M. (1975). Nature (London), 255, 508-510. Kelly, T. J. Jr & Thomas, C. A. Jr (1969). J. Mol. BioZ. 44, 459-475. Maizel, J. V. (1971). Methods Viral. 5, 179-246. Miller, R. C. Jr, Lee, M., Scraba, D. G. & Paetkau, V. (1976). J. ;yloZ. Biol. 101, 223-234. Modrich, P. & Lehman, J. R. (1970). J. BioZ. Chem. 245, 3626-3631. Ponta, H., Rahmsdorf, H. J., Pai, S. H., Hirsch-Kauffman, M., Herrlich, P. & Schweigcr. M. (1974). Mol. Gen. Genet. 134, 281.-297. Rahmsdorf, H. J., Herrlich, P., Pai, S. H., Schweiger, M. & Witt,mann. H. U. (1973). AfoZ. Gen. Genet. 127, 259-271. Rahmsdorf, H. J., Pai, S. H., Ponta, H., Herrlich, P., Roskoski, K. Jr, Slchweigrr, M. & Studier, F. W. (1974). Proc. Nat. Acad. Sci., U.S.A. 71, 586589. Ritchie, D. A., Thomas, C. A. Jr, MaeHattie, I,. A. & Wensink, P. C. (1967). J. AlfoZ. Biol. 23, 365-376. Rothman-Denes, L. B., Muthukrishnan, S., Haselkorn, R. & S:tudier, F. W. (1973). Press, Virus Research (Fox, C. F. & Robinson, W. S., eds), pp. 227-239, Academic New York. Schlegel, R. A. & Thomas, C. A. Jr (1972). J. MoZ. BioZ. 68, 319-345. Striitling. W., Krause, E. & Knippers, R. (1973). T’irology, 51, 109-119. Studier, F. W. (1972). Science, 176, 367-376. Studier, F. W. (1973). J. MoZ. BioZ. 79, 227-236. Watson, J. D. (1972). Nature Nezo Biol. 239, 197-201.
HOST Yama.da, Yamada, Zillig, W., H. &
MUTATION
AND
T7 PHAGE
ASSEMBLY
III
Y. & Nakada, D. (1976). J. Mol. Biol. 100, 35-45. Y., Whitaker, P. A. & Nakada, D. (1974). Nature (London), 248, 335-338. Fujiki, H., Blum, W., Janekoviit, D., Schweiger, M., Rahmsdorf, H. J., Ponta, Hirsch-Kauffman, M. (1975). Proc. Nut. Acad. Sci., C.S.A. 72, 2506-2510.
