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  • JOUINAL OF VIROLOGY, Sept. 1977, p. 637-644Copyright 1977 American Society for Microbiology

    Vol. 23, No. 3Printed in U.S.A.

    Bacteriophage T4 Virion Baseplate Thymidylate Synthetaseand Dihydrofolate ReductaseL. M. KOZLOFF,* M. LUTE, AND L. K. CROSBY

    Department ofMicrobiology and Immunology, University of Colorado School ofMedicine, Denver,Colorado 80262

    Received for publication 18 April 1977

    Additional evidence is presented that both the phage T4D-induced thymidyl-ate synthetase (gp td) and the T4D-induced dihydrofolate reductase (gp frd) arebaseplate structural components. With regard to phage td it has been foundthat: (i) low levels of thymidylate synthetase activity were present in highlypurified preparations of T4D ghost particles produced after infection with td+,whereas particles produced after infection with td- had no measurable enzymaticactivity; (ii) a mutation of the T4D td gene from td I to td+ simultaneously pro-duced a heat-stable thymidylate synthetase enzyme and heat-stable phage par-ticles (it should be noted that the phage baseplate structure determines heatlability); (iii) a recombinant of two T4D mutants constructed containing bothtd t and frdts genes produced particles whose physical properties indicate thatthese two molecules physically interact in the baseplate. With regard to phagefrd it has been found that two spontaneous revertants each oftwo different T4Dfrd's mutants to frd+ not only produced altered dihydrofolate reductases butalso formed phage particles with heat sensitivities different from their parents.Properties ofT4D particles produced after infection with parental T4D mutantspresumed to have a deletion of the td gene and/or the frd gene indicate thatthese particles still retain some characteristics associated with the presence ofboth the td and the frd molecules. Furthermore, the particles produced by thedeletion mutants have been found to be physically different from the parentparticles.

    Considerable genetic and biochemical evi-dence has been presented from several labora-tories (4, 12, 15, 16, 19-21) that two early phage-induced enzymes, dihydrofolate reductase,called gp frd (25) (referred to earlier as gp dfr[12]), and thymidylate synthetase, called gp td(3, 11), were components of the T-even phagebaseplate. However, since neither the knownmissense mutations (5) nor the known non-sense mutations (17) in these genes preventedphage formation, the gene products were classi-fied by Wood and Revel (25) as nonessentialcomponents of the baseplate. This problem wascomplicated both by the failure to detect pro-teins corresponding to these enzymes uponpolyacrylamide gel analysis of phage particles(10, 24) and by isolation by viable phage parti-cles presumably containing deletions of the tdand frd genes (7). Earlier work from Mathews'laboratory (20) and this laboratory (11, 16) indi-cated that these two gene products were notrequired as intact functioning enzymes, butthat substantial portions of these polypeptidescould play the required structural role. Itshould also be noted that phage baseplates con-

    tain an unusual form of folic acid, dihydropter-oyl hexaglutamate, as a structural component,which is closely linked to the two enzyme mole-cules (15). This paper and the accompanyingreport by Mosher et al. (22), which deals largelywith gp frd in phage particles, present addi-tional chemical, genetic, and enzymatic datathat these two proteins are present in all phageparticles yet examined. The thymidylate syn-thetase and the dihydrofolate reductase mole-cules have been shown to be partially buriedwithin the baseplate structure. Furthermore,these two reports point out several anomalies inthe properties of the presumed T4D deletionmutants of the frd and td genes that suggestthat the physical and genetic mapping of thedeletion mutants may need reinterpretation.

    MATERIALS AND METHODSPreparation and purification of bacteriophage

    stocks and isolation of mutants. Escherichia colibacteriophage stocks were grown, purified, and as-sayed by standard procedures (11, 12, 15). Whenused to measure thymidylate synthetase activity,the purified phage particles were osmoticallyshocked, and the ghost particles were further puri-

    637

  • 638 KOZLOFF, LUTE, AND CROSBY

    fled on D2O-sucrose gradients (15). The T2L and T4Dstrains have been used in this laboratory for sometime. The two T4D amber mutants, one in the frdgene (frdll) and one in gene 11 (N9s), were thosedescribed earlier (15). T4D td6, a missense mutationin the td gene, was obtained from D. H. Hall (9). Theisolation and properties of a T4D tdcs mutant (strain408) were described earlier (11). The two frdts mu-tants, originally obtained from D. H. Hall, P1 andC31, and their parent T4Do strain have also beendescribed (5, 12). T4 phage bearing deletions pre-sumed to be between genes 63 and 32, originallyisolated by Homyk and Weil (7) and crossed byHomyk and Weil into a T4D strain called 1589, wereobtained from C. Mathews. The T4D 1589 straincontained a deletion in the rII gene that was used asa DNA marker (7). These deletion strains are la-beled by the names originally assigned by Homykand Weil as dell, del7, and del9. The bacterial hoststrains used included E. coli B and three mutants ofE. coli B affecting host dihydrofolate reductase ac-tivity, E. coli B TRIM 102 and TRIM 103 (12) and E.coli B RT 500, obtained from D. Baccanari (1). E. coliB201, which was td- and grew adequately when themedium was supplemented with low concentrationsof thymine, was obtained from C. Mathews. Thestandard E. coli su+ strain used was CR63. E. coliOK305 was used to assay for the "white halo"plaques produced by frd- and td- phage strains (6).The enzyme assays and other methods have all

    been described previously (12, 15). Standard re-agents were obtained from commercial sources.

    RESULTS

    Thymidylate synthetase activity in phageghost particles. Capco and Mathews (3) pre-sented the original evidence that the phage-induced thymidylate synthetase molecule wasa phage component. Later work from this labo-ratory indicated that this enzyme was a base-plate component (11). In both laboratories,early attempts to demonstrate thymidylatesynthetase activity in phage particles were un-successful. Further efforts were now made withhigher concentrations of phage ghost particlesin the highly sensitive radioactive assay (18).The final concentration of phage ghosts usedwas about 1013/ml in the reaction mixture.Those particles showing thymidylate synthe-tase activity released radioactivity from[3H]dUMP linearly for 1 to 2 h, and the enzy-matic rate was calculated per particle added(Table 1). Although the baseplate thymidylatesynthetase is known to be partially covered bygene 11 and 12 proteins (11), assays of T4Dghost particles lacking the 11 and 12 proteinsconsistently showed less thymidylate synthe-tase activity than did preparations of wholeT4D ghosts. Similar amounts of enzyme activ-ity were found in preparations of T4D, T4Dstrain 1589, and T2L, and somewhat less was

    TABLz 1. Thymidylate synthetase activity inpreparations ofphage ghost particles

    Ghost particle preparation Activity" (mol/minper 10"3 particles)

    T2L ...................... 0.07 0.01T4D ...................... 0.16 0.02T4D frdll .................... 0.03 + 0.01T4D td6 ......................

  • T4 VIRION BASEPLATE COMPONENTS 639

    sensitive. A single plaque was picked of themost heat labile of these two mutants (strain408), and, using this stock, a search was madefor a wild-type td+ revertant. This parent strain408 mutant gave normal plaques on E. coliOK305 at 30'C but gave plaques with whitehalos at 430C. Plating with E. coli B201 con-firmed that the parent 408 was td- at 430C butwas td+ at 300C. One plaque (out of 13,000 ofstrain 408 examined) was of the normal type atthe high temperature and was also td+ at 430Con B201. This revertant, presumably due to asingle spontaneous mutation, was isolated, andits properties were examined. The revertantnot only produced a heat-stable thymidylatesynthetase, as compared with parent strain 408,but also produced phage particles that weremore heat stable than the 408 and even moreheat stable than the original parent T4D parti-cles (Fig. 1).

    Preparation and properties of a T4D frdstds mutant. Evidence from earlier work (11)had shown that the baseplate thymidylate syn-thetase could physically interact with the base-plate dihydrofolate reductase. This earlier

    so

    60 - Revertantof T4D tdts

    40

    0 \*

    work involved preparing and characterizing theproperties of a double T4D mutant, using strain408, a td I mutant, as one parent and an frdemutant known as P1 (9) as the other parent. Asecond double mutant was now prepared byusing strain 408 again as one parent but adifferent frd's mutant, known as C31, as theother parent. The procedure was as describedearlier. A cross of the two mutants was madewith strain 408 as the majority parent and C31,a T4D far mutant, which is also resistant topyrimethamine (9), as the minority parent. Theprogeny of the cross were plated on bacteria onmedia containing pyrimethamine, and plaqueswere picked and tested until one recombinantwas obtained that both produced pryimetham-ine-resistant plaques and gave the td white-halo plaque on OK305 only at the high temper-ature. The recombinant was also td- on B201 atthe high temperature. The heat sensitivity ofthis double mutant was compared to the major-ity parent 408, and Fig. 2 shows the typicalresults obtained in several experiments. Asfound previously, the heat sensitivity of boththe 408 mutant and the recombinant was a

    100

    _T4D

    Revertont 0of TUD tdts

    60

    S

    40_C

    UTD tdts

    -2

    MINUTES

    FIG. 1. Heat sensitivity of various T4D particles and the T4D-induced thymidylate synthetases. (a) Thephage particles were heat inactivated in the standard way at 600C (11). (b) Extracts were made ofE. coli Binfected with the various types ofphage. These extracts were heated at 40"C, and the activity ofthe remainingthymidylate synthetase was determined. Abbreviations: P, Phosphate; ME, mercaptoethanol.

    VOL. 23, 1977

  • 640 KOZLOFF, LUTE, AND CROSBY

    0

    40xcC 31 (420)

    10.0

    4~~~~~~~~~~~~o8xC31(3 &P

    408(420)

    pH 7.0

    0.1 M P Buffer

    1.01,,,,15 30 45 60

    MINUTES

    FIG. 2. Heat sensitivity ofT4D particles producedby recombination of T4D tdt' (408) and frd' (C31)mutants. The recombinant T4D, which is tdto frdto,was prepared as described, with the td's as the ma-jority parent. Phage stocks were grown either at 28 to30C or at 42C, and the heat sensitivity at 60C ofthe phage particles was determined in the usual way.P, Phosphate.function of the temperature of assembly(whether at 42 or at 30C). Irrespective of thetemperature of assembly, the double mutantT4D tdusfrdts was more heat resistant than thetdts majority parent. These changes in heat sen-sitivity indicate again that the heat labilities ofthese two baseplate components are not directlyadditive. The results support the conclusionthat these two baseplate components influenceeach other physically, either directly or indi-rectly.

    Physical properties of revertants of T4Dfrd Is to frd+. The original frd Is phage particleswere isolated as spontaneous mutants by John-son and Hall (9) and were first characterized bytheir ability to form plaques on bacteria in thepresence of anti-dihydrofolate reductase com-pounds, such as pyrimethamine. Johnson andHall (9) showed that two ofthese drug-resistantmutants produced altered enzymes that wereboth drug resistant and heat labile. Later, Koz-loff et al. (12) additionally characterized theseT4D frdts mutants and showed that they pro-duced heat-labile phage particles. A search wasmade for revertants oftwo of these mutants, P1

    and C31, from frd's to frd+. Starting with sin-gle-plaque isolates of P1 and C31 progeny,phage particles were examined for their abilityto form normal plaques, i.e., without whitehalos, at the high temperature (370C). Two re-vertants each of P1 and C31 were found, andthe plating properties of the revertants aregiven in Table 2. All the revertants isolatedwere still resistant to the addition of pyrime-thamine to the plating media, and it can beconcluded that the change in temperature sen-sitivity of the free dihydrofolate reductase, asshown by the plaque assays, was due to a sec-ond mutation independent of the site sensitiveto pyrimethamine.The heat sensitivities of the particles pro-

    duced by the two revertants of P1 are shown inFig. 3, and the heat sensitivities of the two C31revertant particles are shown in Fig. 4. One ofthe P1 ts revertants had the same sensitivity asthe parent P1, whereas the other revertant wasmore heat resistant. Both of the ts revertants ofC31 produced phage particles with heat sensi-tivities different from the parent C31 but in thiscase the revertants produced particles thatwere more heat sensitive than the parent C31.These properties support the conclusion thatdihydrofolate reductase is a phage baseplatecomponent and indicate that any change in thepolypeptide (such as to greater heat resistanceof free enzyme) may change the heat sensitivityof the baseplate to either greater heat resist-ance, such as the Pla revertant, or more heatsensitivity, such as the C31a or C31b revert-ants.

    TABLE 2. Plating properties of various T4D frdtsrevertants to T4D frd+

    Plaques on OK305 at: Platingeffi-

    Phagestrain ~~~~~ciency onPhage strain 037C32C OK305 +3700 3200 ~(p+S)aat 37'C

    T4Do No halo No halo 0.02

    P1 Halo No halo 1.06Revertant a No halo No halo 0.70Revertant b No halo No halo 1.03

    C31 Halo No halo 0.98Revertant a No halo No halo 1.02Revertant b No halo No halo 0.92

    a Similar to the medium used by Johnson andHall (9) but containing lower concentrations of pyri-methamine (P) and sulfanilamide (S); final P = 70,ug/ml, and final S = 4.5 Ag/ml. The plating ef-ficiency is the ratio of plaques on OK305 + (P+S)to the plaques formed on OK305 without (P+S).

    J. VIROL.

  • T4 VIRION BASEPLATE COMPONENTS 641

    100.0 the frd gene, are even more sensitive to thisantiserum than is dell or parent T4D. (ii) Fur-thermore, genetic experiments by Mosher et al.

    50.0 have shown that the frd gene is not deleted inPi del7 or del9, but that the enzymatic activity isRevertont bprobably suppressed by some other, additionalmutation. (iii) A replotting of the deletions onthe T4 map (22) shows that these deletions

    0 \ must lie 2,000 nucleotide pairs more clockwise(to the left) and, therefore, would not delete the

    o2 P 1 frd gene and probably not the td gene.>lo.oM\ Additional chemical and physical propertiesP1Revertan~ae \ of these deletion mutants were characterizedPi Revertont 0 and are summarized in Fig. 5. When the dele-

    tion mutants, dell, del7, and del9, are treated50 , with pyridine nucleotides, such as NADPH and

    NADH, they are inactivated. The inactivationT. 600 reaction has been attributed to an interactionpH = 7.0 with a pyridine nucleotide binding site on the0.1 M P Buffer phage baseplate (16). It should be noted that

    NADPH inactivated these particles somewhatmore rapidly than NADH, in accord with theproperties of the dihydrofolate reductase mole-

    1.0 20 cule. The phage-binding site was also shown toMINUTES be specific for one of the two isomers of thepyridine nucleotides (16) and to include a bind-

    FIG. 3. Heat sensitivity of T4D P1 frd" and P1 1OO.efrd+ revertant particles. Two revertants of P1 fromfrdt to frd+ were isolated as described in Table 2.Phage stocks were grown at 370C, and the heat sensi-tivities at 600C were determined individually in the 50.0usual way. P. Phosphate.Properties of phage bearing deletions be-

    tween genes 63 and 32. Homyk and Weil (7)have described the isolation of three viable T4strains presumably bearing long deletions ofthe region between genes 63 and 32 on the T4linkage map, which would include the gene fortd (dell) and both genes td and frd for both del7 ,0.0\and del9. Whereas the enzymological activities aof infected extracts (3) support the view that _these genes do not produce the appropriate C31functional enzymes, other properties suggest 5.0 0that the heteroduplex physical mapping of theDNA is sufficiently inaccurate so that the for- T =600 C31mation of td and frd gene-related polypeptides pH = 7.0 Revertont bcannot be excluded. The problems of interpret- 0.1 M P Buffering the nature of these deletions are given indetail in the companion paper by Mosher et al. C31 Revertant a(22) and are summarized only briefly here.

    (i) Immunological studies have shown not .0 20 30only that these deletion mutants produce cross- MIN2TESreacting material to T4 phage thymidylate syn- MINUTESthetase and T4 phage dihydrofolate reductase FIG. 4. Heat sensitivity ofT4D C31 frdt' and C31uthtalse than t4ephage dartihydro ate redu ,' frd+ revertant particles. Two revertants of C31 frombut also that the phage particles are even more frd's to frd+ were isolated as described in Table 2.susceptible to inactivation by antisera to these Phage stocks were grown at 37C, and the heat sensi-enzymes (22) than is T4D. Whereas the sensi- tivities at 60C of the parent C31 and the two revert-tivity of dell to anti-frd serum is not surpris- ants were determined in the same experiment. P.ing, both del7 and del9, which presumably lack Phosphate.

    VOL. 23, 1977

  • J. VIROL.642 KOZLOFF, LUTE, AND CROSBY

    I-0I._

    00

    c0%I

    0C-

    FIG. 5. Effect of various inactivation treatments on different T4D strains. Stocks of the parent T4D strain1589 and the three deletion mutants ofHomyk and Weil were prepared and subjected to various treatmentsthat primarily affect the baseplate structure. Details of these procedures are given earlier for NADPH andNADH (12), conjugase (14), Cd(CN)3- (13), and L-homoarginine (23), and the heat treatment was thestandard procedure used in this work.

    ing site for the adenosine diphosphoribose por-tion of the nucleotide (19). Furthermore, it wasfound that hog kidney conjugase, an enzymespecific for hydrolyzing the y-glutamyl bonds offolate polyglutamates, also inactivated theseparticles (14). In several experiments, it wasnoted that the strain T4D 1589 (into whichthese deletions were crossed) was always inacti-vated more rapidly and to a greater extent thanthe three deletion mutants. These propertiesshow that the three deletion mutants have a

    tail structure somewhat different than T4D1589 but that they still contain the phage folateand enough of the dihydrofolate reductase mol-ecule so that the phage particles are sensitive topyridine nucleotides.Three other properties of the baseplates of

    these three deletion mutants as compared tothose of the T4D strain 1589 are also shown inFig. 5. All three deletion mutants were readilyinactivated by the reagent Cd(CN)3 (13) or byincubation with L-homoarginine (23), whereas

  • T4 VIRION BASEPLATE COMPONENTS 643

    T4D strain 1589 was highly resistant to thesetwo reagents. Further, T4D strain 1589 wasmore readily inactivated by heating at 600Cthan were the three deletion mutants. SinceCd(CN)3-, L-homoarginine, and heating at600C (12, 15) all primarily perturb the base-plate, these results confirm the observationsthat the physical properties of the baseplates ofthe deletion mutants are different from those ofthe T4D 1589 strain. However, deletions of thephage genomes between genes 63 and 32 havenot been thought to include sequences for anyessential phage structural components (25).These results indicate that these deletions doaffect structural components. The identifica-tion of the structural components in these mu-tants as polypeptides related to thymidylatesynthetase and dihydrofolate reductase mole-cules rests on the immunological, enzymologi-cal, and genetic (22) data, the inactivation ofthe phages by pyridine nucleotides, the pres-ence of phage folic acid, and the restriction ofthese strains in certain hospital strains of E.coli (22).

    Properties of deletion mutants grown onvarious strains of E. coli. Various E. colistrains were available with different amountsor types of host dihydrofolate reductases.RT500, an E. coli B strain resistant to highlevels of trimethoprim, has been shown by Bac-canari et al. (1) to overproduce the normal E.coli B frd. We isolated earlier several E. coli Bmutants that were also resistant to trimetho-prim (12) and showed that the dihydrofolatereductases produced in these mutants were sig-nificantly altered, asjudged by their heat sensi-tivity. These bacterial strains offered an oppor-tunity to compare the deletion mutant particlesassembled in different cells. Since del7 and del9are presumed to lack the T4D frd gene,whereas dell does not lack this gene, any sub-stitution of host enzymes during viral morpho-genesis would be expected to alter the heatsensitivity of del7 and del9 phage particles, ascompared with either dell or T4D 1589. Thethree deletion mutants and T4D strain 1589were grown on E. coli CR63, E. coli B RT500,and the E. coli B TRIM 102 and TRIM 103 (12)mutants. It was found that the T4D strain 1589produced in all these host cells was somewhatmore heat sensitive than the three mutants(see Fig. 5). But all three mutants, dell, del7,and del9, gave very similar heat sensitivities toeach other, irrespective of the host cells uponwhich they were grown (data not shown). Inaddition, since these particles are also sensitiveto antisera to T4D dihydrofolate reductase anddo react with the co-factors for this enzyme,NADPH and NADH, it can be assumed that all

    three deletion mutants do not, in fact, lack theability to synthesize a protein similar to theoriginal T4D dihydrofolate reductase.

    DISCUSSIONThe data in this paper and the accompanying

    paper by Mosher et al. (22) provide confirma-tion that both the T4D frd and the T4D td genesproduce components that are normal baseplatecomponents of the T-even bacteriophages. Fur-thermore, these papers support the earlierviews that these enzymes are partially buriedwithin the complex tail structure. It is not sur-prising that, whereas phage ghost preparationsdo exhibit both detectable dihydrofolate reduc-tase and detectable thymidylate synthetase ac-tivities, which have been identified as beingdue to phage gene products, the activities areextremely low. The low activities are, mostlikely, due to the occlusion of both enzymes inthe phage structure and the close affinity of thefolate, which would prevent most of these en-zyme molecules from reacting catalytically.The data also suggest that these proteins neednot be complete polypeptides to fulfill theirstructural role, since polypeptide fragmentsproduced by amber mutants in the frd or tdgene can participate in phage assembly (12).The quantitation of these two polypeptides in

    the baseplate remains a difficult problem, andMosher et al. (22) have put an upper limit ofabout six molecules of each per phage particleas the sensitivity limit of their analytical proce-dure. This value would be in accord with asimilar value for the six folates per phage parti-cle (14, 15).The question of whether the two enzymes are

    essential or dispensable gene products for as-sembling the baseplate is not completely re-solved by these experiments. The evidence forconcluding that they are essential componentsof the baseplate rests on the following data. (i)They have been detected directly or, in somecases, indirectly by some significant chemicalor genetic property in all T4 strains so far ana-lyzed, with the only possible exception beingsome partially characterized T4D deletionstrains. The properties of the deletion mutants,which are analyzed in detail for the presence offrd by Mosher et al. in the companion paper(22) and for the presence of td in this paper,suggest that these deletion mutants do containfrd- and td-like polypeptides. Earlier observa-tions with the missense and nonsense muta-tions support the view that these enzymes neednot be functional for them to play a structuralrole. (ii) The evidence on the heat sensitivity ofvarious phage frd's and td's mutants and theirrevertants, coupled with the confirmation by

    VOL. 23, 1977

  • 644 KOZLOFF, LUTE, AND CROSBY

    Mosher et al. (22) that the baseplate is the mostheat-sensitive component of the virus particle,support the idea that these two proteins areintegral parts of the baseplates and not adven-titious constituents attached to the outside ofthe baseplate. (iii) The question remainswhether there could be phage particles thatwere completely infectious but that lackedthese two components. One analogy would be tothe hoc and soc proteins, which have been re-cently shown by Ishii and Yanagida (8) to benormal but dispensable components of the T4phage head. It should be noted that, whereasIshii and Yanagida (8) readily isolated T4Dmutants lacking the hoc and soc components,the situation, at least with regard to phage frd,is different. A search was made for a T4D mu-tant (19) resistant to inactivation by the frdcofactor NADPH, and no mutants were ob-tained from 1012 T4D particles. This meanseither that the frd polypeptide with a bindingsite for NADPH is absolutely required or that aT4D mutation lacking frd is extremely rare.There is no comparable method for searchingfor the absence of the phage td in phage mu-tants. Nonetheless, since the conservation of agene product for both structural and metabolicroles is unusual, the possibility must still beconsidered that one or both of these unexpectedphage components are dispensable.

    ACKNOWLEDGMENTSThis research was supported by Public Health Service

    research grant A106336 from the National Institute of Al-lergy and Infectious Diseases.

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    Burchall. 1975. Purification and properties ofEsche-richia coli dihydrofolate reductase. Biochemistry14:5267-5273.

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    3. Capco, G. R., and C. K. Mathews. 1973. Bacteriophage-coded thymidylate synthetase. Evidence that the T4enzyme is a capsid protein. Arch. Biochem. Biophys.158:736-743.

    4. Dawes, J., and E. B. Goldberg. 1973. Functions of base-plate components in bacteriophage T4 infection. I.Dihydrofolate reductase and dihydropteroyl gluta-mate. Virology 55:380-390.

    5. Hall, D. H. 1967. Mutants of bacteriophage T4 unableto induce dihydrofolate reductase activity. Proc.Natl. Acad. Sci. U.S.A. 58:584-591.

    6. Hall, D. H., I. Tessman, and 0. Karlstrom. 1967. Link-age ofT4 genes controlling a series of steps in pyrimi-

    dine biosynthesis. Virology 31:442-448.7. Homyk, T., Jr., and J. Weil. 1974. Deletion analysis of

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    8. Ishii, T., and M. Yanagida. 1977. The two dispensablestructural proteins (soc and hoc) of the T4 phagecapsid; their purification and properties, isolationand characterization of the defective mutants, andtheir binding with the defective heads in vitro. J.Mol. Biol. 109:487-514.

    9. Johnson, J. R., and D. H. Hall. 1972. Isolation andcharacterization of bacteriophage T4 resistant to fol-ate analogues. Virology 53:413-426.

    10. Kichuchi, Y., and J. King. 1975. Genetic control ofbacteriophage T4 baseplate morphogenesis. II. Mu-tants unable to form the central part ofthe baseplate.J. Mol. Biol. 99:673-694.

    11. Kozloff, L. M., L. K. Crosby, and M. Lute. 1975. Bacte-riophage T4 baseplate components. III. Location andproperties of the bacteriophage structural thymidyl-ate synthetase. J. Virol. 16:1409-1419.

    12. Kozloff, L. M., L. K. Crosby, M. Lute, and D. H. Hall.1975. Bacteriophage T4 baseplate components. II.Binding and location of bacteriophage-induced dihy-drofolate reductase. J. Virol. 16:1401-1408.

    13. Kozloff, L. M., and M. Lute. 1957. Viral invasion. III.Release of viral nucleic acid from its protein covering.J. Biol. Chem. 228:537-546.

    14. Kozloff, L. M., and M. Lute. 1965. Folic acid, a struc-tural component of T4 bacteriophage. J. Mol. Biol.12:780-792.

    15. Kozloff, L. M., M. Lute, and L. K. Crosby. 1975. Bacte-riophage T4 baseplate components. I. Binding andlocation of the folic acid. J. Virol. 16:1391-1400.

    16. Kozloff, L. M., C. Verses, M. Lute, and L. K. Crosby.1970. Bacteriophage tail components. II. Dihydrofol-ate reductase in T4D bacteriophage. J. Virol. 5:740-753.

    17. Krauss, S. W., B. D. Stollar, and M. Friedkin. 1973.Genetic and immunological studies of bacteriophageT4 thymidylate synthetase. J. Virol. 11:783-791.

    18. Lomax, M. I. S., and G. R. Greenberg. 1967. An ex-change between the hydrogen atom on carbon 5 ofdeoxyuridylate and water catalyzed by thymidylatesynthetase. J. Biol. Chem. 242:1302-1306.

    19. Male, C. J., and L. M. Kozloff. 1973. Function of T4Dstructural dihydrofolate reductase in bacteriophageinfection. J. Virol. 11:840-847.

    20. Mathews, C. K. 1971. Identity of genes coding for solu-ble and structural dihydrofolate reductase in bacte-riophage T4. J. Virol. 7:531-533.

    21. Mathews, C. K., L. K. Crosby, and L. M. Kozloff. 1973.Inactivation of T4D bacteriophage by antiserumagainst bacteriophage dihydrofolate reductase. J. Vi-rol. 12:74-78.

    22. Mosher, R. A., A. B. DiRenzo, and C. K. Mathews.1977. Bacteriophage T4 virion dihydrofolate reduc-tase: approaches to quantitation and assessment offunction. J. Virol. 23:645-658.

    23. Shapiro, D., and L. M. Kozloff. 1970. A critical C-terminal arginine residue necessary for bacterio-phage T4D tail assembly. J. Mol. Biol. 51:185-201.

    24. Vanderslice, R. W., and C. D. Yegian. 1974. The identi-fication of late bacteriophage T4 proteins on sodiumdodecyl sulfate polyacrylamide gels. Virology 60:265-275.

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    J. VIROL.