Bacteriophage Tail Components

Copyright©1970 AmericanSocietyforMicrobiology Printed in U.S.A.

Bacteriophage

Tail

Components

III. Use of

Synthetic Pteroyl

Hexaglutamate for T4D Tail Plate Assembly

L. M. KOZLOFF, M. LUTE, AND L. K. CROSBY

Departmentof Microbiology, University ofColorado MedicalCenter, Denver,Colorado 80220

Received for publication 20 August 1970

The assembly of T4D tail plates occurring during in vitro complementation reactionswasfoundtobestimulatedbypteroyl hexaglutamate. Neither the pteroyl pentaglutamate northe

pteroyl

heptaglutamate substituted for the

hexaglutamate.

Asmall stimulation ofthe rate and amount of T4D tail

plate

assembly was ob-served in untreated extracts. A greaterstimulation occurred when activated char-coal-treated bacterial extracts were used. Charcoal treatment inhibited

comple-mentation only whenno

preformed

tailplateswerepresentinthe extracts, andthe inhibition was reversed by the addition of 9 X 10-6 M chemically

synthesized

pteroyl hexaglutamate. The stimulation is apparently dueto arequirement for the pteroylhexaglutamate fortailplate

assembly.

The complex T-even bacteriophage tail

struc-ture was shown

recently

by Kozloff and

co-workers(5-7)tocontainanunusualvirus-induced folic acid conjugate as a structural component.

On thebasis of itsspectrum andbehavior

during

gel permeation

chromatography,

this

compound

appearedto bea

dihydropteroyl

penta- or

hexa-glutamate. Recent developments in

solid-phase

peptide synthesis have led to the

synthesis

of folateconjugatescontaininguptoseven

glutamyl

residues (8). With these

compounds,

it has been possibleto examine therole ofthefolate

conju-gate during in vitro phage

morphogenesis.

The

need foraspecific folate

conjugate during

phage

tailplate

assembly

hasnowbeendemonstrated

by

use of the in vitro

complementation

system de-veloped by Edgar and his colleagues

(1-4,

11).

Only the

pteroyl hexaglutamate

has been found

to restore activity to systems depleted of their

endogenous folate

conjugates by

charcoal

treat-ment. Further, these systems have

permitted

mapping of the specific reaction in which the

folatecompoundisrequired.

MATERIALS AND METHODS

Preparationandpurificationofbacteriophage stocks

andextracts ofEscherichia coli Binfected with T4D

amber mutants. Mostofthebiological materials and methods wereidentical to those used earlier (6, 7).

VariousT4D amber mutants wereobtainedfrom R. S.

Edgar and colleagues (1-3). These mutants were

grownonthepermissivehost E. coli CR63 and were

purified bystandard procedures. Themutantsused

wereasfollows. (i) Defective intailplate formation:

gene6 (N102),gene7 (B16),gene 8 (N132), gene 10

(B255), gene 26 (N131), gene 27 (N120), gene 28

(A452),and gene 53 (H28). (ii)Defective intailtube

formation:gene48(N85)and gene54(H21).(iii)

De-fective in sheath formation:gene 18 (E18). (iv)

De-fective in head formation: gene 23 (B17). (v) A

quadruple mutant defective in tail fiber formation:

genes 34/35/37/38 (B25/B252/N52/B262). (vi) A

doublemutantdefectiveincompleting tail formation:

genes 11/12 (N93/N69). Extracts ofthe mutant-in-fectednonpermissivehost E. coliBwereprepared by

theprocedureusedearlier (7),exceptthat thebacteria,

at4 X 108/ml, were infected with a multiplicity of

infectionof 4:1 at30C. After 30minofincubation,

the infected cells were chilled and centrifuged at

1,400 X gfor 5 min;they were then resuspended in

cold BUM (3) plus deoxyribonuclease to 1.0% of

theiroriginal volumeandwerestoredat-70C. After

thawing at 30 C, the T4D titer was usually 101 to

2 X 109/ml, and the titer after complementation

normally increased 10- to300-fold.

Charcoaltreatmentofbacterial extracts. Two types

of activated charcoal were used, Norit A, from

PfanstiehlLaboratories, Inc.,and Darco, gradeG-60,

from Atlas ChemicalIndustries. NoritA was usedin

most experiments, but the Darco was equally

ef-fective. Three differentprocedureswereusedfor treat-ing the bacterial extracts. (i) Weighed amounts of

charcoal were added directly to an empty chilled

reaction tube; then the two complementing amber

extracts wereaddedand themixturewaskept at 0 C

for5 min. Thereaction mixture containingthe

char-coalwasthenincubatedat30 Cforvarious times. This

procedureisreferredto as treatmentwith free charcoal

inFig.2. (ii)Insomeexperiments, weighedamounts

ofNorit A were pressed on strips of cellulose tape

coatedonboth sides withadhesive.Thesestripswere immersed into individualextractsforvarious times in thecold and thenremoved. The extracts were then mixed for complementation. This method is called

the"bound" charcoaltreatment (see Fig. 2), and it 754

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permits the later addition ofreagents which might

otherwise be adsorbed bycharcoal. (iii) Various

ex-tracts werealsotreatedseparatelywithcharcoal

con-tained in washed dialysis bags. Thebagscontaining

thecharcoal (usually 100 mg suspendedin0.3 mlof

BUM) wereimmersedinthe extracts (0.5ml) for4 to 5 hr in the cold; they were then removed, and the treated extracts were mixed for complementation in

theusual manner.Thisprocedurepreventedany con-tact between the proteins in the extracts and the

charcoal, butit also removed various freely

diffus-ible substances.

Other methods and sources of chemicals.

Aminop-terin inhibition of complementation was assayed as

describedpreviously (7). Aqueoussolutions ofpteroyl

pentaglutamate, pteroyl hexaglutamate, and pteroyl

heptaglutamate, prepared by solid-phase chemical

synthesis,werethe generousgift of Carlos Krumdieck

andCharlesBaugh (8).Otherreagentswerethose used earlier (7).

RESULTS

Effect offolateconjugates oninvitro formation

ofT4D particles. Figure 1 shows that

p-amino-benzoyl glutamate,pteroyl glutamate(folic acid),

and pteroyl pentaglutamate, at approximately

10-5 M,failedto affect

complementation

between

gene 10 and gene 7 extracts. However, pteroyl

hexaglutamate at the same concentration stimu-lated complementation. The stimulation was

largestforshortperiods of incubation. However,

at2.2 X 10-5M, the

pteroyl

hexaglutamate

(and

thepteroyl

pentaglutamate)

inhibited

complemen-tation. Theseresults areconsistent withtheview

that the

pteroyl

hexaglutamate

is used for virus assembly but that it firstmustbereduced to the

dihydrocompound.

Any

excessunreduced

pteroyl

compound acts as an inhibitor of

dihydrofolate

reductase and thusofassembly (7).Therelatively low degree of stimulation (atmost

twofold)

is due presumablytothehighendogenous leveloffolate conjugates. Thisproperty and the

inhibitory

effect ofhigher concentrations oftheadded

conjugates

make itdifficultto assesstheroleofvarious folate

conjugates when the usual bacterial extracts are

used.

Inhibition of in vitro complementation of T4D

gene10andgene7 extractsbyactivatedcharcoal.

Activated

charcoal

iswellknownforits

ability

to

adsorbfolic acid

conjugates.

The effectof

adding

16mgofNorit A per mltothe

complementation

reaction between gene 7 and gene 10 extracts is

shown in Fig. 2. Charcoal addition

greatly

de-pressedthe initialrateof

phage

formationbutnot

the total amount of

phage

formed

by

300 min.

The decreasein the initialrateof

phage

formation

was found to be

proportional

to the amount of

charcoal added (Fig.

3).

"Bound" charcoal

(on

strips of

tapes)

was less effective than "free"

M N UT E S

FIG. 1. Effect of various folate conjugates on in vitro complementation between extracts made with T4D gene 10and T4D gene 7 amber mutants. The test compounds ineither water or BUM amounted to only S to 10% of

thefinalvolume. The complementation was carried out

as described in Materials and Methods. Pabaglul =

p-aminobenzoyl glutamate; pteroyl glul = pteroyl

glutamate; pteroyl glu5 = pteroyl

pentaglutamate;

pteroylglu6 = pteroylhexaglutamate.

10

~~~~~~/

o 0

xJ

n L No Chorcoal

Lu Added0

0 U_80

LU

40 16mg/ml

o ~~~0

Charcoal

z 0

60 120 180 240 300

MINUTES

FIG. 2. Inhibition by activated charcoal of in vitro

complementation between extracts of E. coli B made

with T4D amber mutant in gene 10 and a similar

ex-tract made with T4D amber mutant in gene 7. The

complementation wascarried out as described in

Ma-terials andMethods.

charcoal, but both experiments (shownin Fig. 2

and 3) suggest that charcoal adsorbs some

sub-stancefrom theextractswhichisratedetermining. Several

properties

of thecharcoal-inhibited sys-temwere studiedto determinethe compound or

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KOZLOFF, LUTE, AND CROSBY

Z 80 u

60

Charcoal

40

P0

0 20 Free

0 Chorcool \

lio

5 10 15 20 25 30

Charcool mg/ml

FIG. 3. Inhibition ofthe initial rateofformation of

newT4Dparticlesinanin vitrocomplementation sys-temby freecharcoalorbound charcoal. The initialrate

wascalculated fromthe increase inphagetiterfrom30

to60 minutesafter mixing ofbacterialextractsmade

with T4D gene 10 andT4D genp 7mutants. In the

experiment with free charcoal, various amounts of

charcoalwere added toseparate reaction mixturesat

4 C. The tubeswerethen incubatedat30 C andassayed

at various times. In the experiment with "bound"

charcoal, weighedamountsofcharcoalwerepressedon strips ofcellulose tape coatedon both sides with ad-hesive. These strips were immersed in separate tubes

containing theextractsandkeptat0 Cfor I hr. The

strips with the "bound" charcoal were then removed,

and the gene 10andgene 7 extracts weremixed and

incubated in theusualmanner.

compounds adsorbed. One possibility considered

wasthatthese levels of charcoal might be

remov-ing indole, which occurs in these extracts, and

which Nashimoto and Uchida(10) have shownto

stimulate tail fiber attachment. Results given

below clearly indicate that, although indole has

an effect oncomplementation (and might be

re-moved undercertain conditions), the removalof

indole is not the cause ofcharcoal inhibition at

levels of free charcoal of 8 mg/mlorless.

In view of thestimulationof the initialrateof

complementation shown for pteroyl

hexagluta-mateinFig. 1, it seemedmorelikelythat these low

levels of charcoalwereremovingsomebutnotall

of theendogenous folate compounds. This would

cause adecrease in the rateof phage formation,

but, since the folates are in considerable excess

over that needed for phage assembly, the total

amount of phage formed over long periods of

incubation should not be decreased. Onetest of

[image:3.490.82.230.60.262.2]

thishypothesis of charcoal inhibition is given in

Table 1. Phage assembly in vitro occurring in mixtures of T4D gene 7 and gene 10 extracts was found previously to be sensitive to the addition of

10-4 to 10-' M aminopterin, a dihydrofolate

re-ductase inhibitor (7). This compoundapparently

blocked the incorporation of the phage-induced dihydrofolate reductase into the phage tail plate.

In these previous experiments, it was observed

that the concentration of aminopterin required to

inhibit complementation was higher than that

normally needed to inhibit enzymatic activity (9).

Thiswaspresumed to be due to the high

concen-tration of folates in the extracts. Table 1 shows that charcoal treatment greatly increased the

sensitivity of these extracts to aminopterin

in-hibition and supports the view that charcoal

treatmentremovesendogenous folatecompounds

necessaryforassembly.

Mappingof the site ofcharcoal inhibition in the pathway of T4D morphogenesis. The effect of

variousamountsoffreecharcoalon threein vitro assembly reactions is showninFig.4. Thesystem

containinggene 10 and gene 7 extracts was

[image:3.490.262.454.321.565.2]

sensi-tive to aslittle as 2mg/ml. The other two systems,

TABLE 1. Effect of charcoal treatment on the

in-hibition by aminopterin of complementation with T4D gene 10 and gene 7 extracts

Initialrateof ii-Expt Prepn formation_newT4Dof bitin,_biin

particlesa %

I Untreatedextracts 2.1 X 109

+7 X 10-4M ami- 0.4 X 109 81

nopterin

+2.3 X 10-4 M ami- 1.2 X 109 43

nopterin

Treated extractsb 1.6 X 10

-+7 X 10-4M ami- 0 100

nopterin

+2.3 X 10-4M ami- 0 100

nopterin

II Untreated extracts 14.3 X 109 +2.3 X 104 M ami- 10.4 X 109 27

nopterin

Treatedextractsc 4.1 X 109

+2.3 X lOr-6 M ami- 2.1 X 109 49

nopterin

aNet new particles formed at 30 C between

30 and 60 min after mixing extracts.

',Each extract was treated separately with bound charcoal at a concentration of 20 mg/ml for25min at0 C.

cEach tube of extract was treated separately

by adding a dialysis bag containing 100 mg of

charcoalto0.5mlofextractandkeeping the tube

at0 Cfor 4 hr; thebag containing the charcoal

was then removed and the extracts were mixed.

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Charcoal Added m9/ml

FIG. 4. Effect ofcharcoalonin vitro

complementa-lionbetween extractsofE.coliBmade with different

T4Dambermutants.Thecomplementation wascarried

out in the presence ofthe charcoal as described in MaterialsandMethods.

gene 10 plusgene 54and gene 10 plus gene 48,

wereeitherstimulatedorunaffectedby the added

charcoal. At concentrations above 20mg/ml, all

complementation mixtureswereinhibited,

includ-ing those shownlaterin Table 2.This nonspecific

inhibition wasnotfurtherstudied.

It should be noted that gene 54 and gene 48

extracts both containpreformed tailplates (11),

whereas gene 10 and gene 7 do not contain this

substructure. Table 2 shows that the small

amounts of charcoal used (8 mg/ml) inhibited a

very specific site in viral assembly. All

comple-mentation systems in which either one of the

bacterial extracts contained a tail plate or more

complex tail substructure were either stimulated

orunaffectedbythe charcoal.

These results (Fig. 4 and Table 2) also show thattheinhibition dueto added free charcoalat

8 mg/ml cannot be due to removal of indole,

because the indole-sensitivereaction,theaddition of tailfiberstofiberlessparticles, occursinevery

systemgivenin Table2except thatinexperiment

15. The simplestinterpretationof the increase in

aminopterin sensitivity and the specific site of

inhibition of morphogenesis is that charcoal in-hibits tail plate formation by removing a folate conjugate.

Effectofsyntheticfolateconjugates on

comple-mentationincharcoal-treated extracts.Thehighly

charcoal-sensitivecomplementation extractsgene 7 andgene 10weretreatedseparatelywith

char-coal contained in a dialysis bag. This permitted

the later addition ofsynthetic pteroyl glutamates. The results ofadding these compounds tothese

treatedextractsareshowninFig. 5-7. The

char-coal inhibition observed afterthis type of

treat-TABLE 2. Inhibition of T4D tail assembly by acti-vated charcoala

Relative Expt Substructures Extracts

comple-no. present

men-tation

1-4 None 10 + (7 or 8, or 0.1-0.3

28 or 53)

5-8 None 27 + (6 or 8, or 0.0-0.2

26 or 53)

9-10 Tail plate 10 + (54 or 48) 0.9-1.8

11 Tail plate 27+ 54 0.7

12 Tail plate + 27 + 18 1.2

tube

13 Tailplate + 27 + 23 1.9

tube + sheath

14 Fiberless par- 10 + 34/35/37/ 0.8

ticle 38

15 Defectivepar- 10 + 11/12 1.6

ticle

aIn experiments 1-13, 0.8 mg of charcoal was added to a 0.1-ml mixture of equal portions of each of the two extracts. This mixture was in-cubated for 5 min at 0 C, a sample was removed

for assay, and the reaction mixturewas assayed again after 3 hr at 29 C. The net fold increase in T4D in the absence ofcharcoal was: expt 1, 125; expt2, 8; expt 3, 3; expt4,97; expt 5, 8; expt 6, 17; expt 7, 3; expt8, 8; expt 9, 111; expt 10, 100; expt 11, 50; expt 12,87; expt 13, 129; expt 14, 325; expt 15, 250. Inexperiments 14 and 15, 0.8 mg of charcoal wasadded toamixtureof 0.01 ml of the

purified defective particles, suspended in saline,

plus 0.09 ml of gene 10 extract. This kept the charcoal to bacterialextractratio similarto that in other experiments. The relative

complementa-tion givenisratio of net complementation in the

presenceof the charcoal to the net complementa-tion in the absence of charcoal (7).

ment was different from that shown in Fig. 2.

Both the initial rate and the level of

comple-mentation at the final point taken (300 or 180

min)weredecreased. Further,indoledialyzesinto

the bagcontaining the charcoal, and additionof

indole at aconcentration of 1.4 X 10-4 M

par-tially restored the rate ofcomplementation.

In-creasing the indole concentration to 5 X 10-4M

didnotfurther stimulatecomplementationtothe

level in the untreated extracts. Itwas also found

that indole did notstimulatecomplementation in

the untreated extracts as did pteroyl

hexagluta-mate(Fig. 1).

Figure 5 shows that 9 X 10-6 M synthetic

pteroyl hexaglutamate stimulated

complementa-tion. The addition of this compound alone to

treated extracts increased the rate of

comple-mentation, and the addition of

pteroyl

hexaglu-0o

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0.

I_

x

LJI

I c:

3:

z

[image:5.490.53.448.65.295.2]

M NUTES

FIG. 5. Effect ofpteroyl hexaglutamate (pteroyl MINUTES

glu6) andindole on in vitro complementation between FiG. 6. Effect ofpteroyl pentaglutamate (pteroyl

gene 7and gene 10extractspretreatedwith charcoal. glu5) andindole on in vitro complementation between

Theadded pteroylglu6wasat afinalconcentration of gene 7 and gene 10extracts pretreated with charcoal.

9.0 X 10-6M, and the indole wasat 1.4 X 10' M. The addedpteroylglus was at a final concentration of These two compounds were added as concentrated 9.0 X 1O6M, and theindole was at 1.4 X 10-4 M.

solutions andamounted together to only 10% ofthe These two compounds were added as concentrated

final reaction volume. The treatments and other pro- solutions and amounted together to only 10% of the

ceduresare giveninMaterials and Methods. final reaction volume. The treatments and other pro-ceduresaregivenin Materials and Methods.

tamate

plus

indole

completely

restored the

ability

of these extracts toformnewT4D

particles.

On

the otherhand, both

synthetic pteroyl

pentagluta-mate(Fig.6) and

pteroyl heptaglutamate (Fig.

7)

at9.0 X 10-6 M wereeither inactiveor

inhibitory

to

complementation.

Further, in contrast tothe results with

pteroyl hexaglutamate,

both the penta- and hepta- derivatives in the presence of indole inhibited

complementation. Higher

con-centrations of

pteroyl hexaglutamate

did not

stimulate

the

assembly

rate abovethat shown in Fig. 5for 9.0 X 10-6M.Final

phage

tailscontain

the dihydro form of this compound, and the

addedsynthetic

compound

presumably

mustfirst

bereducedtothe"dihydro" form;any excess

un-reducedcompound is inhibitory

(see

Fig. 1).This

feature of the extractspreventedany

testing

of the

possibility

that

pteroyl hexaglutamate

in the

ab-senceofindole could

completely

restorethelevel

of

complementation.

DISCUSSION

Theuseof activated charcoaltoremove

selec-tively

thefolate

conjugates

fromextractsof

bac-teria infected withT4D amber mutants has

per-mitted the direct demonstration that petroyl

hexaglutamate

stimulates assembly of the tail plate. Sincethe pteroyl hexaglutamateis known

tobeacomponent ofphage substructure

consist-ing of the tail tube andthe tailplate, it can be

proposed thatthis effect of the hexaglutamateis

due toitsincorporation intothephage structure.

Further, since dihydrofolate reductase is also directly involved in tailplate assembly, and the

inhibition of

assembly

by aminopterin and by charcoalmapatthesamesite (7),it seems

prob-able that the reductase

plus

the bound

pteroyl

hexaglutamateareincorporated together into the tailplate. Basedonwhat isknownof thesequence

of reactions involved in tail plate assembly (Edgar,personalcommunication),theaddition of

this enzymepluscofactor totheothercomponents

maybe thefinalstep in theformation of the tail

plate.

Theseexperiments do not permit a direct

de-termination of the number of folate molecules

needed for assembly, but it is obvious that the

rate of assembly is directly dependent on the

concentration ofthe folate conjugate. Each

in-fected bacterium contains about 400,000 folate

molecules (5), of which a maximum of 10% (6)

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b~~~~~~~~~~~~~~~~~~~~~~utn reoted

120

0

80

Charcool

Treated

40 Indole

z

0 60 120 180

[image:6.490.43.238.57.289.2]

MINUTES

FIG. 7. Effect of pteroyl heptaglutamate (pteroyl

glu7) and indoleon in vitrocomplementation between

gene7 andgene 10extractspretreated with charcoal.

Theadded pteroyl glu7 wasatafinalconcentration of

9.0 X10-6m,and theadded indolewasat1.4X J0-4 M.

These two compounds were added as concentrated

solutions andamounted together to only about 10%

ofthefinalreaction volume. Thetreatmentsand other proceduresaregiven inMaterials and Methods.

or40,000aredihydropteroyl hexaglutamates. The

formation of300phage particles eachcontaining

possibly six folates (6) would only require 1,800

molecules out of those present in the cell. This

large excess raises the question of whether, in

addition to participating in and influencing the

rate of phage tail plate assembly, the

phage-induced folatecompound might playsomeother

role in the formation of otherviralcomponents.

TwelveT4Dgenes, 5,6, 7, 8, 10, 25, 26, 27, 28,

29,51,and 53, have been identifiedsofarasbeing

required for tailplate formation (1, 2, 11). One

or moreofthesegenes oran asyet unidentified

genemustberesponsible for inducing the

forma-tion of thephagepteroyl hexaglutamate. In view

ofthechloramphenicol sensitivity of pteroyl

hexa-glutamate formation (6), it appears that a new

enzyme(s)mustbe formed.Themechanismof the

action of such an enzymeisnotcertain, because

the unregulated stepwise addition of glutamate

residues to the host dihydropteroyl triglutamate

might well result inthe formation of significant

amounts of pteroyl tetraglutamate,

pentagluta-mate, and possibly heptaglutamate, all ofwhich

wouldinhibit phage assembly. Other biosynthetic

mechanisms for the formation of the pteroyl

hexaglutamate deserveconsideration. Inany case,

the induction of an enzyme to make a small

critical molecule for viral assembly, such as

pteroyl hexaglutamate, represents a unique and

unexpectedexpression of the viralgenome.

ACKNOWLEDGMENTS

This investigation was supported by Public Health Service

grantsA1-06336from the National Institute of Allergy and In-fectious Diseases and 5TOI GM-01379 from the National In-stitute of General Medical Sciences.

LITERATURECITED

1. Edgar, R.S., andI.Lielausis. 1968.Some steps inthe assem-bly of bacteriophageT4.J.Mol. Biol. 32:263-276.

2. Edgar, R.S., and W.B.Wood.1966.Morphogenesis of bac-teriophageT4 in extracts of mutant-infected cells. Proc. Nat. Acad.Sci.U.S.A. 55:498-505.

3. Epstein, R. H.,A.Bolle,C. M.Steinberg,E.Kellenberger, E.Boy de la Tour,R. S.Edgar, M.Susman,G. H. Den-hardt, and A. Lielausis. 1963. Physiological studies of conditional lethal mutants of bacteriophage T4D. Cold

Spring Harbor Symp. Quant. Biol. 28:375-394. 4. King, J. 1968. Assembly of the tail ofbacteriophage T4.

J. Mol. Biol. 32:231-262.

5. Kozloff, L.M.,and M.Lute. 1965. Folicacid,astructural componentofT4bacteriophage.J.Mol.Biol. 12:780-792.

6. Kozloff,L.M.,M.Lute,L.K.Crosby,N.Rao,V. A.

Chap-man, and S. S. DeLong. 1970. Bacteriophage tail

com-ponents. I. Pteroyl polyglutamates in T-even

bacterio-phages.J. Virol. 5:726-739.

7. Kozloii,L.M.,C.Verses,M.Lute,andL.K.Crosby. 1970.

Bacteriophage tail components. II. Dihydrofolate

reduc-tase in T4D bacteriophage. J.Virol. 5:740-753.

8. Krumdieck,C.L., andC. M.Baugh. 1969. Thesolid-phase synthesis of polyglutamates of folic acid. Biochemistry 8:1568-1572.

9. Mathews,C. K. 1967. Evidencethatbacteriophageinduced

dihydrofolate reductase is a viral geneproduct. J. Biol.

Chem.241:4083-4086.

10. Nashimoto, H.,and H. Uchida. 1969. Indoleas anactivator

forin vitroattachment oftailfibers in theassembly of

bacteriophage T4D. Virology 37:1-7.

11. Wood, W. B., R. S. Edgar, J. King, I. Lielausis, and M. Hennigger. 1968. Bacteriophage assembly. Fed. Proc. 27: 1160-1166.

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