Valyl-tRNA synthetase modification-dependent restriction of bacteriophage T4.

0022-538X/84/07042-05$02.00/0

Copyright C) 1984, American Society forMicrobiology

Valyl-tRNA Synthetase

Modification-Dependent

Restriction of

Bacteriophage

T4t

NORMA J. OLSON* ANDGEORGE L. MARCHIN

Division of Biology(Microbiology), Kansas State University, Manhattan, Kansas66506 Received 7 November 1983/Accepted 25 March 1984

AstrainofEscherichia coli, CP790302, severely restricts the growth of wild-type bacteriophageT4. In broth

culture,mostinfections of single cellsareabortive, althoughafewinfected cells exhibit reducedburstsizes.In contrast,bacteriophage T4mutantsimpairedin the abilitytomodify valyl-tRNA synthetase developnormally

onthis strain. Biochemical evidence indicates thatthephage-modified valyl-tRNA synthetase in CP 790302 is

different from that previously described. Althoughtheenzyme is abletosupportnormalproteinsynthesis, a

disproportionateamountofphage structural protein (serum blocking power) fails to matureintoparticlesof the appropriate density. The results with host strain CP 790302 ate consistent with either a gratuitous

inhibition of phage assembly by faulty modification or abrogation of an unknown role that valyl-tRNA

synthetase might normally playinviralassembly. Shortly after bacteriophage T4 infects Escherichia coli, thehost valyl-tRNA synthetase is modified by the addition ofaphage-coded peptide calledT(12). The phagegenevsat

56 kilobases on the standard T4 genetic map is responsible for thismodification and presumably codes for theTpeptide (14). Bacteriophage T4 mutants that are deficient in the

abilitytomodify the host synthetasegrowwellontypicalE.

colilaboratory strains. The biological role for modification is thus difficultto define.

We have nevertheless continued tostudy the valyl-tRNA

synthetase modification becausewefeelthat itmight repre-senta unique kind ofregulatory eventtothebacteriophage T4, and that it might beamodel forsynthetase alterations in a number ofbiological systems(20).

Recentwork inourlaboratory has demonstratedthat

wild-type bacteriophage

T4vs'

does not formplaques ona host strain of E. coli called CP 790302 (11). A variety of phage

mutantsdeficient inmodification of hostvalyl-tRNA synthe-tase, however, plaque normally onstrain CP 790302.

Previ-ous genetic evidence had indicated that the vaiSts and relA

alleles in E. coli CP 790302 probably were responsible for

thisrestriction phenomenon.

In this article, we present evidence that the inability of T4vs+ toformplaquesonstrain CP 790302 is duetoagreatly

reducednumber of infectiouscenters,aswellastoareduced averageburstsize. Inaddition, the biochemical propertiesof thephage-modified valyl-tRNA synthetasein CP 790302are

different than those previously described foraT4-modified

valyl-tRNA synthetase (19). Thus, we hypothesize that the

novel valyl-tRNA synthetase, while continuing to amino-acylate tRNAs and participate in protein synthesis,

some-howinterferes with viraldevelopmentatalatestageinviral assembly; wedonot knowwhether this postulated

interfer-enceis direct orindirect.

MATERIALSAND METHODS

Bacteria and bacteriophage. Several strains of E. coliand bacteriophage T4wereemployedin this studyandare listed in Table 1. Cells were routinely grown in

morpholinepro-* Correspondingauthor.

tContributionno.84-208-J,Division ofBiology,Kansas

Agricul-tural Experiment Station, Manhattan, KS66506.

panesulfonic acid TGYE medium prepared according to

Neidhardtetal. (18). High-titer lysates of T4wereprepared

onE. coli NP 4aspreviously described (13),exceptthat 20-ml lots were used. Lysates were titrated on strain NP 4

unless stated otherwise.

Preparation of cell-free extracts. Cell-free extracts were

preparedasdescribed previously (16), by sonicating cultures on ice in phosphate buffer (0.006 M potassium phosphate, 0.006 M 2-mercaptoethanol [pH 7.2]). The protein

concen-trationwas determined by the method of Lowryetal. (10), andextractswere stored at4°C; they werenotusedafter 5 days.

Enzyme assays. The specific activity ofvalyl-tRNA syn-thetase aminoacylation was measured by attachment of

[3H]valinetotRNA. Reaction mixtures contained 250 ,ug of tRNA, 100 ,ug of bovine serumalbumin, 2 .mol of dipotas-siumATP, 1 ,umol of glutathione, 10 p.mol of[2,3-3H]valine

at 15

liCi/VLmol,

5 xmol of KCl, 50,umol of Tris base, and5

,umol of MgCl2, with afinalpH of 7.2. A limitingamountof valyl-tRNA synthetase (cell-free extract) was addedat 4°C

togiveafinalvolumeof 0.5 ml. Blanks contained everything butenzyme. After 5 min of incubationat 37°C, the reaction

wasstopped by cooling quickly inan ice bath and adding 3

mlofcold 5%trichloroacetic acid. After 30min,precipitates werecollectedonfiber glass filters and washed with 10 ml of

5%trichloroacetic acidand 5 ml of 67% ethanol. Filterswere

driedat 80°C for 30 min and counted in a Beckman LS-230

liquid scintillationcounterwith 5 mlof toluene base scintilla-tioncocktail. One unit ofsynthetase specific activity is the amount of enzyme that catalyze the addition of valine to

tRNAattherateof 1 p.mol/min underourassayconditions. Experiments measuring aminoacylation specific activity in CP 790302, when indicated, were performed as described

above, except that a "protective buffer" containing 0.1 M

Tris base, 0.001 M valine, 10% glycerol, and 4.2% 2-mercaptoethanol, with afinal pHof 7.3, wasused.

Serum blocking experiments. Serum blocking power was

determinedby using the standard endpointassayofDe Mars (6) and procedures thoughtfully provided by M. Snyder. Phage lysates wereprepared by adding T4vs+orT4vs2,ata

multiplicityof infection(MOI) of7,to20 ml ofE. coliNP 4

orCP790302 broth culturesgrownat30°Ctoadensityof 2x 108 cells per ml. After 90 min of incubation at 30°C with

aeration, cells were lysed by the addition of chloroform.

TABLE 1. Strains ofE. coli andbacteriophage T4

Strain Source Description

T4Bc+ Thislaboratory Wildtype; normalmodification of VRSa

T4vsl This laboratory Missense mutant; unusual modified VRS

T4vs2 This laboratory Amber mutant; nomodified VRS T4vs2 This laboratory Spontaneous revertant of T4vs2;

RFOl modified VRS

T4rII M. Snyder rdf deletion

E. coli This laboratory Wild-typeE.coli B; indicator strain NP 4

E. coli This laboratory Wild-typeE.coliKB NP 2

E. coli This laboratory valS"s NP29

E. coli CP G.Bjork F-pyrB hismetBrelAvalS'" rpsL 790302 ampA(vaiS"s by P1 fromNP

910212)

E. coli NP Thislaboratory F- recA strA valS'"pyrB trpA 910212

E. coli M.Snyder Lambdalysogen; restrictive host

G(A) for T4rII

E. coli G.Bjork leuvalS's relA+ NE 536

E. coli G.Bjork leuvalSisrelA NF 537

aVRS, Valyl-tRNA synthetase.

Lysate sampleswere incubated withT4antiserum in serum

blockingbufferat48°Cfor 12htoallowacompletereaction.

The residual neutralizing activity in each sample was

mea-sured by adding a known concentration of purified (22)

(T4vs+) tester phage and determining the inactivation after

48minof incubationat46°C. PFUproduced onE. coli G (X)

by surviving tester phage in the lysate samples were

com-pared with PFU produced by surviving tester phage in samples containing a known concentration ofCsCl-banded

T4rII (rdf deletion). Testerphage (T4vs+) form plaques on

indicator strain G (A), but T4 rII does not. Thus, serum

blockingpowerconcentrations in thelysatescanbe

convert-edtophage equivalents by using standard curvesthat relate

PFU produced by tester phage to phage equivalents ofT4 rll. Standard curves were run in eachexperiment.

RESULTS

RestrictionofT4vs+ byCP790302. Workers in our

labora-tory have demonstratedthatE. coli CP 790302 is restrictive for T4vs+ but permissive for T4vs2 (11). A pair of E. coli strains(NF536and NF 537),whichcontain adifferent valS's

allelethan CP790302,permitT4vs+ and vs-mutantstoform

plaques efficiently. Consequently, the restriction appears to

be dependent uponthe valS'sallele employed in CP790302.

We haveverified this byPlvirtransduction ofvalS+ intoCP 790302 (11), as well as by crosses with a number of Hfr

strains (P4X, KL25, KL16-99, and Hfr H). In all cases,

T4vs+ formed plaques on the valS+ recombinants at a high

frequency. In contrast, his+, metB+, and pyrB+ recombi-nants, either alone or in combination, continued to restrict T4vs+ plaqueformation (datanotshown).Theimportanceof

therelA alleletothe restriction phenomenonis more ambig-uous. Although we were unable to directly select relA+

recombinantseither via PlvirtransductionorHfrcrosses, a

related strain, E. coliNP 910212, which contains the same

valS'sallele asCP 790302 but isrelA+,allowed formation of

minuteplaques with T4vs+.

TABLE 2. Virus production in E. coli CP 790302 during single andmixedinfectionsa

Virus produced

Infection perinfectious %Reduction'

center (PFU)

T4vs+ 9

T4vs2 290

T4vs+ plus T4vs2 57 80

T6 139

T4vs+ plusT6 19 86

T4vs2 plus T6 340 0

T7 119

aCells were grown at 30°C to a density of5x107cellsper ml, and identical

cultures were infected with one or more strains of virus each at an MOI of 4. After 10min, unadsorbed phage were inactivated with phage antiserum. Five minutes later, each culture was diluted and plated on wild-type indicator strain E.coli NP 4 to determine infectious centers. Cultures were diluted, and after 90min,samples were removed and plated on the indicator strain. Experimen-taldetails were described by Adams (1).

bThe data are expressed as the percent reduction in virus produced (per infectious center)during mixed infection compared with the corresponding single infection with T4vs2 or T6.

Infection andvirus progenyproduction.Five minutes after T4vs+ or T4vs2 phage were added to actively growing

cultures of E. coli CP 790302, approximately 80% of the phage wereadsorbed, both cultures experiencedadecrease in optical absorbance, and fewer than 1% of the bacteria survivedeitherinfection (datanotshown). Adding T4vs+ to lawns of CP 790302, however, failed to produce plaques,

whereas T4vs2 formed plaques efficiently on the E. coli

strain (11). This phenomenon was further investigated by

using single-cell burst and one-step growth experiments.

When acultureof CP790302 wasinfectedwith T4vs+ and

unadsorbed phage were neutralized with antiserum, plating infectious centers on the permissive host E. coli B (NP 4)

resultedinapproximately 25% (15 of66) as manyplaquesas when the infection was conducted with T4vs2. Ninety

min-utes afterT4vs2 additionto CP 790302, an average ofmore than 200 PFU per infectious center were observed. Fewer than 10 PFU per infectious center were produced after infection with T4vs+ (Table2).Chloroformadditiontoeffect

cell lysis did not increase the burst sizes.

Furthermore,

T4vs+ phage produced on CP 790302 formed plaques with equal efficiency on CP 790302 and a standard indicator

strain, NP-4 (datanot shown).

Trans-dominant effect of infection with T4vs+. Virus

pro-duction was examined when T4vs+ and T4vs2, each at an

MOI of 4, were added

simultaneously

to cultures of CP

790302. An 80% decrease in progeny virus

production

was

observed at 90 min after this co-infection compared with virus production during T4vs2infection alone (Table 2).

Neidhardt and Earhart (19) reported the appearance of

heat-stable valyl-tRNA synthetase activity in extracts of bacteriophage T4vs+- orT6-infected NP 29 cultures. The T-odd bacteriophage were examined similarly and lacked the

ability to thermally stabilize the temperature-sensitive syn-thetase of NP 29.Theirdatasuggest thatmodifiedenzymeis formed after bacteriophage T4 or T6 infection of E. coli.

However, our results demonstrate that, unlike T4infection,

T6andT7infections of CP790302 result inplaqueformation (Table 2).

Bacteriophagemacromolecularsynthesisandassembly.The accumulation of macromolecules in CP 790302 cells

during

T4infection was analyzedbymeasuring the incorporationof

3H-labeled

precursors into trichloroacetic

acid-precipitable

[image:2.612.312.553.93.192.2]

[image:3.612.64.563.83.163.2]

TABLE 3. Proteinsynthesis andphage productionin E. coli strains at 30 and43.5°C

Relative rate of protein synthesis(%)' Burstsize on hostb

Strain 300C 43.5°C 300C 43.5°C

Uninfected T4vs+ Uninfected T4i's' T4vs+ T4vs2 T4vs+ T4vs2

CP 790302 100 96 12 10 4 254 NDC ND

NP910212 100 115 5 8 86 62 4 5

NP29 100 90 6 76 87 79 96 <.01

NP 2 ND ND ND ND 244 277 105 87

a Cultures were grown to a density of108cells per mlin a 30°C rotating water bath. Identical samples of each culture were left uninfected or were infected with T4vs+ at an MOI of 5. After 10 min, a portion of each sample (uninfected or phage infected) was either reincubated at 30°C or shifted to 43.5°C. After a10-min incu-bation,[3H]argininewas added, andits incorporation into trichloroacetic acid-precipitable material was measured by filtering and counting samples.

bBacteria weregrown at 30°C to 3x108cells per ml,diluted to 5x107 cells per ml, and infected at 30°C at an MOI of 0.1. After 10min,anti-T4antiserum was added and the cultures were diluted to tubes at 30 and43.5aC.After 5min, cultures were diluted and incubated for 100min. Samples were taken for determination ofphage production and are expressed as yield per infectious center.

cND, Not determined.

accumulated during restrictive infection (T4vs+,

T4vs2RF01) wascomparableto that measured during

infec-tions inwhich virus production was normal (T4vs2, T4vsl) (datanot shown).

In additional experiments, viral proteins were pulse-la-beled (under conditions that diminished labeling of host

proteins)andanalyzed byelectrophoresis in sodium dodecyl sulfate and byautoradiography (2, 3). Apair-wise compari-son of proteins from cultures of CP 790302 infected with T4vs+ or T4vs2 revealed nomajor differences in autoradio-graphic intensities.Thetemporalappearanceofproteinswas

similar inboth infections (datanot shown).

Phage antigen synthesis was determined by measuring

serum blocking power in

lysates

derived from E. coli cul-tures infected with T4vs+ or T4vs2. The ratio of PFU to serum blocking power measured in the lysate of T4vs+-infected CP 790302(2.4x

108 PFU/3.3

x

109

serum

blocking

power [sbp] =

7.2%)

isonly one-fifth theratio measuredin thelysate of T4vs2-infectedCP 790302(2.2 x 109 PFU/6.0x

109

sbp = 36.5%).

Although

infectious virus concentration

waslow intherestrictiveinfection,thelysate contained 55%

asmuchserumblockingpower as wasmeasuredin thelysate fromthepermissive infection.

Lysates of CP 790302 cultures infected with T4vs+ or T4vs2 weresedimentedoncesium chloride

density gradients

(Fig. 1). Althoughtwopeaks ofserum

blocking

powerwere

observed for bothlysates,agreater

proportion

ofthe

materi-al derived from the

permissive

infection sedimented at a

density typical ofassembled phage

particles. Plaque

assays

verified thepresenceof infectiousvirus. Mostofthe material

frotn the nonpermissive infection sedimentedat thedensity ofprotein. These studies suggest that

phage

maturation in

T4vs+-infected CP790302cellsis restrictedatthepositionof

virusassembly. Thefollowing experiments weredone inan attempt toidentifythe cause of the restriction.

Aminoacylated

tRNA levels. The in vivo levels of

amino-acylatedtRNAweremeasured bytheperiodate method (5).

The fraction oftotal tRNAval that was aminoacylated was

87% in uninfected, 80% in T4vs+-infected, and 72% in T4vs2-infected cultures of CP 790302. The high level of

charging of

tRNAVal

in each culture is consistent with

aminoacylationlevels

reported

by Comerand Neidhardt

(5),

who used cultures ofuninfected and T4vs+-infected E. coli B.

Proteinsynthesis and virusproduction at high temperature inT4-infectedcultures. Table 3comparesproteinsynthesisin

uninfected and T4vs+-infected cultures of CP 790302, NP 910212, and NP 29 at30°Cand when thecellswereshifted to

43.5°C. The three uninfected, temperature-sensitive strains

were unable to synthesize protein at 43.5°C because they contained a thermolabile valyl-tRNA synthetase. Thermal stabilization of the temperature-sensitive enzyme occurred

after T4 infection of NP 29, and protein synthesis continued when the infected cells were shifted to43.5°C. In contrast, protein synthesis remained depressed at 43.5°C in T4vs+-infectedcultures ofCP 790302 and NP 910212. These E. coli strainsharbor the same vaiSts allele, which is different from that in NP 29 (8, 11).

These dataareconsistent withdata obtained by comparing averageburstsizes on these strains (Table 3). Virus produc-tion in T4vs+-infected NP 29cells was the same at both 30 and43.5°C. However, T4vs+-infected NP 910212 produced progeny virus at 30°C but not at 43.5'C.

Properties of valyl-tRNA synthetase in uninfected and

T4vs'-infected

temperature-sensitive and wild-type E. coli strains. We examined valyl-tRNA synthetase activity in extractsof uninfectedandT4vs+-infectedCP790302cellsto determine whether the host enzyme was actually modified. Extracts ofuninfected, temperature-sensitive cells are

de-0

4

8

12

FRACTION NUMBER

FIG. 1. Serumblockingpower and PFU inlysates fractionated

oncesium chloridedensity gradients.Equivalentamountsofserum

blocking material from T4 vs+- and T4 vs2-infected CP 790302 lysateswerelayeredontocesiumchloridegradientsandcentrifuged for 1 hat 35,000rpm inaBeckman SW50.1rotor.Gradientswere

[image:3.612.328.561.462.660.2]

[image:4.612.55.292.68.374.2]

FRACTION NUMBER

FIG. 2. Sucrose density gradient centrifugation of valyl-tRNA synthetases.E. coli culturesgrownat30°Cwereleftuninfected or were infected with T4 vs+ at an MOI of 7for 10 min. Cell-free

extracts were prepared from the cultures, and 0.2 ml of each preparation was layered separately ontolinear 5 to 20% sucrose

densitygradients.ThesewerecentrifugedinanSW50.1rotor at4°C for 10 h at 40,000 rpm. The gradients were collected from the

bottom, andalternatefractionswereassayedforspecific activityof

valyl-tRNA synthetase aminoacylation. Closed squares represent

arginyl-tRNA synthetase specific activity, usedas amarker in the assay. The high-molecular-weight formof themodifiedenzyme is duetoitsassociationwith tRNA.

void of thisenzymeactivity when examined by the standard

in vitro 3H-amino acid attachment assay (8; N. Olson and

G. L. Marchin,unpublished data). However, whenextracts wereprepared by usinga"protective buffer" and theassays wereperformedat30°C for 15 min, in vitro specific activity ofunmodified and modified temperature-sensitive enzyme could be observed and compared. Under these modified conditions, valyl-tRNA synthetase from T4vs+-infected CP 790302 (90.6 ,umol/minper,ug of protein) exhibited approxi-matelya two-thirds reduction inspecific activity compared with enzyme from uninfected cells. This reduction (2.0 ,umol/min per ,ug of protein) is characteristically seen with modifiedenzyme(4).

Figure 2 shows difference in enzyme sedimentation

pat-ternsonsucrosedensitygradients whenextractsof uninfect-ed and T4vs+-infected cells werecompared. Extracts from

T4vs+-infected NP 29 contained a high-molecular-weight

form of the modified enzyme due to its association with tRNA (13). In contrast, T4vs+-infected CP 790302 cell

extracts contained only the lower-molecular-weight form observedinuninfected E. coli cellextracts.Thetoppanel of Fig. 2 illustrates both forms of the enzyme, which were

observed after infection of wild-type E. coli NP 2. The

absence ofahigh-molecular-weightcomplex in infected CP 790302 further implies that modification afterT4vs+

infec-tion ofthesecells is unusual, and that, in turn, leads to an alteredinteraction of the modified synthetase with tRNA.

DISCUSSION

Apreviousreportfrom this laboratory demonstratedthat

bacteriophage T4 with the wild-type vs gene is unable to

formplaquesat30°C onE. coli CP 790302 (11). Phagewith

various amber, missense, and deletion mutations in the vs geneplate efficientlyon this strain. The restriction is

attrib-utabletoinfectiouscentersthatexhibitadramatic reduction in burstsizeorfailtoproduceevenasingleinfectious virus

particle.

Despite differences intheproduction of PFU,the

accumu-lation of DNA, RNA, and protein in the twoinfections is

similarasmeasuredbyprecursor accumulation. The kind of

protein synthesized alsoappears to besimilar in the restric-tive andpermissive infections; their temporalappearanceis also the same. However, although substantial amounts of

serum blocking power are synthesized in the restrictive

infection, only a small fraction is assembled intoinfectious virus particles. The remainder sediment with a density of proteinonCsClgradients. Theseresultsindicate,therefore,

that virus maturation is blocked at some position before complete assembly.

Interestingly, bacteriophage T6 forms plaques on CP 790302. Since the

phage

is thought to

modify

valyl-tRNA synthetase, and since simultaneous infection with T4vs2

(Table2)doesnotresult insubstantially reduced burst sizes, then theinferencecanbe drawn that theT

peptide

in this T-evenphage is structurally different fromthe onein bacterio-phage T4. This conclusion is analagous tothe observations ofMoen et al. (15) that bacteriophageT4 and T6 specifya

different

repertoire

oftRNA molecules. Theability ofT7 to

plate on CP 790302 is consistent with the observation of Neidhardt and Earhart (19) that the T-odd phage do not

modifyE.colivalyl-tRNA synthetase. Thus, theyare pheno-typically like T4vs2.

Earlier studies in this laboratory (17) had indicated that, during modification ofthevalyl-tRNA synthetase inE. coli,

the T peptide is buriedin the cleft betweenthe twoglobular domains of the enzyme. The aberrant modification ofthe enzyme inCP790302described in thispaper mayproducean enzyme with a different conformation and,

therefore,

an

altered interaction with other elements in the infected cell.

The amino acid composition and molecular weight oftheT

peptide

(16) aresimilartothoseofanumberof nucleic acid

binding

proteins, suchashistoneH2A(9) and thecroprotein ofbacteriophage lambda (21).

That themodifiedenzymefromT4vs+-infected CP 790302 fails to sediment as a

high-molecular-weight

complex on sucrose

density gradients

isonedemonstrableexample ofan

altered interaction with a cellular component. This

alter-ation, however, was not detectable by any change in the

overall in vivocharging level of tRNAval in thesecells. Our

studies do not reveal more subtle alterations that may exist in the charging of different valyl-tRNA species or in the relative rates of tRNAcharging.

Theprecisemannerin whichvalyl-tRNA synthetase mod-ification arrests virus development in CP 790302 is not

understoodatthis time. The unusualmodification

apparent-ly occurs

during

T4vs+ infection of NP 910212, but tiny plaques are produced presumably because the stringent

Donini (7) has shown that T4 recruits the host relA gene function during T4 phage DNA synthesis.

In summary, wehave demonstratedthat a normal, seem-ingly mild regulatory event, i.e., the modification of valyl-tRNA synthetase, goes awrywhenbacteriophageT4 infects CP 790302. Even though the modification is apparently nonessential and normally can be dispensed with by a variety of mutations, in this particular strain its operation severely hampers virus assembly.

LITERATURE CITED

1. Adams, M. H. 1959. Methodsofstudy of bacterial viruses, p. 443-522. InA. D. Hershey (ed.), Bacteriophages. Interscience Publishers, Inc.,NewYork.

2. Bonner, W. M., and R. A. Laskey. 1974. A film detection method for tritium-labelled proteins and nucleic acids in poly-acrylamide gels. Eur.J. Biochem. 46:83-88.

3. Chace, K. V., and D. H. Hall. 1975. Characterization ofnew regulatory mutants of bacteriophage T4. II. New class of mutants. J. Virol. 15:929-945.

4. Chrispeels, M. J., R. F. Boyd, L. S. Williams, and F. C. Neidhardt. 1968. Modification of valyl-tRNA synthetase by bacteriophage inEscherichia coli.J. Mol. Biol. 31:463-475. 5. Comer, M. M., and F. C. Neidhardt. 1975. Effect of T4

modification of host valyl-tRNA synthetaseonenzymeactionin vivo.Virology 67:395-403.

6. De Mars,R. I.1955.Theproduction of phage related materials when bacteriophage development isinterrupted byproflavine. Virology 1:83-99.

7. Donini, P.1970. Amino acid controloverdeoxyribonucleic acid synthesis in Escherichia coli infected with T-even bacterio-phage. J. Bacteriol. 102:616-627.

8. Eidlic, L.,and F. C.Neidhardt. 1965. Protein and nucleic acid synthesis intwo mutantsof Escherichiacoli with temperature-sensitive aminoacyl ribonucleic acid synthetases. J. Bacteriol. 89:706-711.

9. Iwai, K., K. Ishikawa, and H. Hiyashi. 1970. Amino-acid se-quence of slightly lysine-rich histone. Nature (London) 226:1056-1058.

10. Lowry,0. H.,N.J.Rosebrough,A. L.Farr,and R.J.Randall. 1951. Protein measurement with the Folin phenol reagent. J.

Biol. Chem. 193:265-275.

11. Marchin, G. L. 1980. Mutations in a nonessential viral gene permit bacteriophage T4 toform plaques on Escherichia coli valSts relA. Science 209:294-295.

12. Marchin, G. L., M. M. Comer, and F. C. Neidhardt. 1972. Viral modification of the valyl-tRNA synthetase of Escherichia coli. J. Biol. Chem. 247:5132-5145.

13. Marchin, G. L., U. R.Muller, and G. H. Al-Khateeb. 1974. The effect of transfer ribonucleic acid on virally modified valyl transfer ribonucleic acid synthetase of Escherichia coli. J. Biol. Chem. 249:4705-4711.

14. McClain, W. H., G. L. Marchin, and F. C. Neidhardt. 1971. Phage induced conversion of host valyl-tRNA synthetase, p. 191-205.InG.E. W.Wolstenholme andMaeveO'Connor (ed.), Strategy of the viral genome. ChurchillLivingstone, London. 15. Moen, T. L., J. G. Seidman, and W. H. McClain. 1978. A

catalogue of transfer RNA-like molecules synthesized following infection of Escherichia coli by T-even bacteriophages. J. Biol. Chem. 253:7910-7917.

16. Muller, U. R., and G. L. Marchin. 1977. Purification and properties of a T4 bacteriophage factor that modifies valyl-tRNAsynthetase of Escherichiacoli.J. Biol. Chem. 252:6640-6645.

17. Muller, U. R., and G. L. Marchin. 1977. Analysis of the structureofT4bacteriophage-modified valyl-tRNA synthetase by limitedproteolysisandisoelectric focusing. J. Biol. Chem. 252:6646-6650.

18. Neidhardt, F. C.,P. L.Bloch, and D. F. Smith. 1974.Culture medium forenterobacteria. J. Bacteriol. 119:736-747. 19. Neidhardt, F. C., and C. F. Earhart. 1966. Phage-induced

appearanceofavalyl sRNA synthetaseactivity in Escherichia coli. ColdSpring Harbor Symp. Quant. Biol. 31:557-563. 20. Spadafora, C., T. Igo-Kemenes, and H. G. Zachau. 1973.

Changes in tRNAs and aminoacyl-tRNA synthetasesduringsea urchindevelopment. Biochem.Biophys. Acta312:674-680. 21. Takeda, Y.,A.Folkmanis,and H.Echols. 1977. Croregulatory

proteinspecified by bacteriophage X. J. Biol. Chem. 252:6177-6183.