Structural proteins of bacteriophage ∅29

\11mL0w

46, 567-57ti (1971)

Structural

Znstiiuto

G. Mnrn%n,

Cenlro

Proteins

of Bacteriophage

de Znvestigacionea Accepted

I’Plarqc~er

Biolbgicas, April

429

144.

Marl~id

6, Spawn

15, 19Yl

Bacteriophage 429, specific for some species of Bacillus, is formed of a head with fibers, a neck, and a short tail. Complete particles were dissociated by treatment. with dimethyl sulfoxide or EDTA into heads, heads with neck, and necks with tail. The head contains at least three polypeptides (molecular weights 54,000, 48,000, and 28,000). The neck consists of two collars and twelve appendages, and it is formed by three polypeptides (molecular weights, 80,000, 40,000, and 36,000). The tail contains one polypeptide (molecular weight, 71,030). Heads isolated from lysates contain. besides the two major polypeptides present in the head of normal particles, two additional polypeptide chains. The seven structural polypeptides of 629 accormt for about 60’;’ of the genetic information of phage DNA (molecular weight, 11 X 10” daltons). I?;TI
Ba&eriophage 429 is a small virus which attacks some speciesof Bacillus (Reilly and Spizizen, 1965). The genome of 429 (molecular weight, 11 X lo6 daltons) is one of the smallert double-stranded DNAs found in bact,erial viruses (Anderson et al., 1966). The part,icle consists of a head linked tJoa short t,ail through a neck. The head has a hexagonal outline and radial projections. The neck is formed by two collars; the upper collar is linked to the head base and the lower collar, located immediately below the upper one, has t,welve symmetrically attached appendages (Anderson et al., 1966). The small size of 429 DNA and the morphology of the viral particle led us to use this phage as a model to work out the morphogenesisof viruses of complexity intermediate bet#ween t,he isometric RNA-cont’aining phages (Argetsinger-St,eitz, 1970) and the more complex viruses such as the T-even pl-lagesof Escherichia coli (Wood et al., 1968; Laemmli et al., 1970). In this paper we describe methods of disassembly of +29 into simpler structural com’ Present address: Instit,ute of Neurobiology. University of (iothenburg. Sweden.

ponents and the det,ermination of the polo.peptide chains that’ serve as basic constituents of each one of these components. RIATI~:I~IAI,S

ANI)

METHODS

Naterials. Radioactive amino acids were obtained from The Radiochemical Centrr, ~4mersham. Sodium dodecyl sulfate, obtained from Sigma Chemical Co., was rccrystallized from 95 % ethanol. Acrylamidc and N ,N’-met,hylenebisacrylamide, purchased from Serva Entwicklungslabor, Heidelberg, were recrystallized as described by Loening (1967). Cesium chloride was obtained from Pierce Chemical Co. and butyl-PBD from Ciba Ltd, pancreatic DNase and RNase from Worthingt,on Biochemical Corp. and egg white lysozyme from Sigma. All other chemicals were reagent grade. Phage arrd host. Bacteriophage $29 and it’s host Bacillus amyloliquefaciens, strain H (Welker and Campbell, 1967), were obt’aincd from Dr. B. E. R,eilly. Media and assay oj fhe phage. Phage stocks were prepared by infecting cells growing in the logarithmic phase with 0.2 phage/bacterium in Id-broth (Dubnau et al., 1967) sup-

567

568

MENUEZ

plemented with 20 mM n-glucose and 0.01 mM MnClz . The infected culture was shaken for 4 hr at 37” or until lysis was complete. The suspension was incubated with lysozyme (10 pg/ml) and RNase (1 pg/ml) for 30 min and then with DNase (0.5 pg/ml) in the presence of 5 mM MgClz . Cell debris was removed by centrifugation at 15,000 g for 10 min at room temperature. The phage was assayed as described by Adams (1950). Phage dilutions were done in a solution containing 0.01 M MgClz and 0.1 M NaCl buffered with 0.05 M Tris-HCl, pH 7.8 (TMS). Bottom-agar contained L-broth supplemented with 0.01 mM MnClz and hardened with 1.6 % agar. Top-agar contained L-broth, supplemented with 20 mM n-glucose and 0.01 mM MnC12 and hardened with 0.6 % agar. Plaques were counted after having b&en incubated overnight at 30”. PuriJication of radioactive phage. The bacteria were grown at 37” in 200 ml of a defined medium containing the salts indicated by Anagnostopoulos and Spieizen (1961) supplemented with 0.1 ICI NaCI, 0.01 mM MnCln , 20 mM n-glucose, and a mixture of the twenty natural L-amino acids at a final concentration of 0.1 mM each. When the cell concentration was about 2 X lO’/ml, the cells were concentrated lo-fold by centrifugation and resuspensionin the samemedium lacking glucose and amino acids. Phage was added at a multiplicity of 50 and, after adsorption during 10 min at 30” without shaking, the cells were centrifuged and resuspendedin the samevolume of the defined medium indicated before except that the final concentration of the radioactive amino acid was 0.025 mM. The infected cells were shaken at 30” unt’il lysis. At different intervals aliquots of the culture were removed to assay for plaques and acid insoluble radioactivity as described below. Figure 1 shows the kinetics of incorporation of leucineJ4C and the appearance of ext’racellular phage. Under these conditions the latent period was about’ 40 min and the burst size 200. The lysate was treated with enzymes and the bacterial debris removed as described before. The supernatant was centrifuged at 29,000 rpm in rotor 30 of an L2-50 ultracentrifuge at 4” during 1.5 hr. The pellet was resuspended in 0.25 ml of TMS and the sus-

ET AL.

MINUTES

1. Incorporation of leucine-W and phage development in +29-infected Bacillus amyloliquefaciens. B. amyloliquefaciens in synthetic medium was infected with 429 and labeled with leucine-W (2.5 &i/ml; 0.025 mM). At the times indicated, 54 portions were assayed for trichloroacetic acid-precipitable material and for extracellular phage, respectively. O---O, 14C counts; A- - -A, extracellular phage. FIG.

pension was layered on top of a 5-20 % (w/v) sucrosegradient in TMS and centrifuged for 30 min at 35,000 rpm at 4’ in an SW 50L rotor of the model L2-50 ultracentrifuge (Fig. 2a). The fractions containing the phage were pooled and subjected to CsCl densitygradient centrifugation in a 50 Ti rotor for 16 hr at 45,000 rpm and 15”. The peak fractions, with a density of 1.45 g crnm3(Fig. 2b), were pooled and dialyzed against TMS. Disassembly of 429 into structural components. Treatment with dimethyl sulfoxide. Phage 429 in TMS (5-10 X lOI PFU/ml) was treated at 4” with 70% (v/v) dimethyl sulfoxide as indicated by Cummings et al. (1968), except that bovine serum albumin was omitted. After 30 min the dimethyl sulfoxide was removed by dialysis against TMS and the DNA was digested with DNase (1 pg/ml). Treatment with EDTA. Phage 429 in TMS (5-10 X 1012PFU/ml) was treated a$ 37” with 0.075 M EDTA for 14-16 hr in a shaking bath. The EDTA was removed bj dialysis against TMS and the DNA digested with DNase (1 pg/ml). Dissociation to polypeptide chains. An ali. quot of the sample in TMS or in water was diluted at least S-fold into a solution coni taining 5 mM sodium phosphate (pH 7.1) 1% (w/v) sodium dodecyl sulfate, 1% (v/v) 2-mercaptoethanol and 2 M urea and heated

PROTEINS

OF

PHAGE

629

.ifi!)

ments were filled with a buffer containing 0.05 M sodium phosphate (pH 7.1). 0.1% (w/v) sodium dodecyl sulfate and 0.05 ‘4 (v/v) 2-mercaptloethanol. Elect,rophoresis was carried out at, room temperature (about 22”) at a constant voltage of 4.5 V/cm for 22 hr. After electrophoresis the gels were removed from t,he tubes and fract)ionated or stained as indicated below. Determinatio?[ of radioactivity. The polyacrylamide gels were fractionat,ed with an Autogeldivider (Savant Instruments Inc.) for measurement of radioactivity after electrophoresis. The fractions were collected in FIG:. 2. Purification of phage 429 and empty glass vials (4.5 X 1.2 cm) and subjected to capsids from lysates. Bacillus am2/loliquefaciens three cycles of freezing and thawing to elute in synthetic medium was infected with $29 and the proteins from the gel. A disk of glass labeled with leucine-ilC (2.5 pCi/ml; 0.025 mM) fiber paper (Whatman GF1’A, 2.4 cm diametill lpsis. A cult,ure of uninfected cells was labeled with leucine-3H (25 &X/ml; 0.025 mM) during ter) was introduced, touching the bottom of the same period of time and lysed with lysozyme the vial, and the vials were heated in au oven (10 rg/ml) and chloroform. Equal volumes of at, 90” until dryness. A quantit,ative transfer both cttltttres were mixed and incubated 30 min of radioactivit,y from the solution to the at 37” with RNase (1 pg/ml) and Dh’ase (0.5 paper was obtained with this procedure. rg/ml.). After low-speed centrifugation, carrier I:or determination of acid-insoluble radio429 (10” PFU/ml) was added and the mixture activity, the samples were treated at room centrifuged at. 29,000 rpm in a rotor 30 at 4” durtemperature wibh 5 “< (w/v) trichloroacetic ing 1.5 hr. (a) The pellet, was suspended in TMS acid after addition of 100 pg of bovine serum and centrifuged in a 5-20’;, sucrose gradient in a albumin per sample. The- precipitate was SW 50L rotor at 5000 rpm for 30 min at 4°C. (b) Peak I from (a) was adjusted to a densit,y of collected on glassfiber paper (Whatman GI; ’ 1.44 g ~111-3 with solid C&l and centrifuged at C, 2.4 cm diamet,er) and washed with 3’; 45,000 rpm for 16 hr at. 15’ in a rotor 50Ti. (c) (w/v) trichloroacetic acid. Tot,al radioactivPeak II from (a) was layered on top of three ity was determined in aliquots pipet,ted 1ayer;i of CsCl solutions in TMS of densities 1.50, directly on glassfiber papers. The filt,crs were 1.30, and 1.10 g crns3 and centrifuged in an SW placed in small vials (4.3 X 1.2 cm), dried at 50L rotor at 45,000 rpm for 1 hr at 15”. (d) Peak 90” and covered with 3 ml of a solmion con 11 from (c) was adjusted to a density of 1.30 g taining 4 g of butyl-I’BD per liter of toluene. cm-3 with solid C&l and centrifuged in a 50 Ti Each small vial was placed inside a standard rotor at 45,000 rpm for 16 hr at 15”. Aliquots of scintillation vial and the radioactivit,y measeach fractions from the gradients were assayed for total radioactivity and phage, respectively. ured in a I’ackard TriCarb scintillation specDensities of selected fractions of gradients (b) trometer. and (d) were determined pycnometrically. The Estimation of vwleculw weightsoj’ polypepfirst fraction corresponds to the bot,tom of the peptide chaitls. The molecular weight of Yz c011r1ts; o- -0, 3H coLlIlts; tube. O---O, polypept#ide chains was determined by polyd----A, phagc; X---X, C&l density. acrylamide electrophoresis as described by Shapiro et al. (1967). After electrophoresis 5 min in a bath of boiling water followed by the gel n-asstained wit,h a 0.25 % (w/‘v) solucooling at room temperature (XIaizel et al., t,ion of Coomassiebrillant blue in met~hanol: 1968). acetic acid: water (5 : 1: 1) for about S llr and Polyacrylamide gel electyophoresis.Gel elec- destained by shaking the gel in 7.5’:; acetic trophoresis was carried out in 10% gels as acid. described by 1\Iaizel (1969). The sample Electtm 7tticroscopy. Samples of plinge or (total volume, 0.2-0.3 ml) was layered on phage components \vere dialyzed ;qainst top of :I 20 cm gel. Tlte electrode compart- 0.05 ‘11ummoniiim acetate comairring 1 null

FIG. 3. Electron scale line represents FIG. 4. Electron 0.1 /A.

micrograph 0.1 pti micrograph

of purified

429 particles

of purified

429 capsids 570

stained isolated

with

potassium

from

a lysate.

phosphotungstate. The

scale

line

The represents

PROTEINS

OF

PHA(;E:

$29

,IIgC:lr . A drop of the sample mixed with a drop of 4 c%,sodium silicotungstate, pH 7.2 or 2% potassium phosphotungstate, pH 7 was placed on carbon-coated grids and after 5 min removed with a capillary tube. The grids were examined in a Siemens Elmiskop I elf~ctron microscope.

Yurijkatiotr of’ l,-iral Capsids

Particles

and Empty

l’hage lysates gave rise t’o t,wo major components after high-speed CentAfugation and analysis by sucrose gradient sedimentation. As can be seen in Fig. 2a, peak I coincides with the peak of infectivity. The sedimentation coefficient, of $29, s~‘~~,,~, has a value of %‘7 S (unpublished observations) and a buoyant densit,y in CsCl of 1.45 g crne3 (Fig. 2b). Under the electron microscope, peak I

FIG. 6. Electron micrograph wit,h dimet hyl sulfoxide. The

of purified capsids scale line represents

FIG. 5. SIrrose gradient cr~~trifllgatioo of rapsids with Ileck. Capsid with neck were prepared by treating 429, labeled with lerlcine-IV!, with dimethyl sulfoxide as indicated in Materials and Methods. The sample wa-: mixed with complete phage and (Tmpty capaids, both labeled with le~ltine-3H, and centrifrlged ill a 15 to 3OY;> sucrose gradient in an SW 501, rot or at 36,000 rpm for 75 min at 4”. The first fraction corre-;pouds to the bott,om of the tube. a--~- ---0. II<” cvllnts; 0 -0, 3H (-orlots.

with neck 0.1 p.

prepared

by treatment

of phage

prrrtieles

572

MBNDEZ

contained intact viral particles (Fig. 3). In agreement with Anderson et al. (1966), the dimensions of the structural components of 429 are, approximately : head length, 40 nm; head width, 30 nm; length of the head projections, 14 nm; length of the neck with the tail, 32 nm. Peak II (Fig. 2a) has a sedimentation coefficient, relative to that of 429, of about 120 S. After a further step of purification in a layer gradient of CsCl (Fig. 2c), this material showed a buoyant density in CsCl of 1.30 g cmw3 (Fig. 2d). Under the electron microscope, peak II contained empty heads (Fig. 4). Preparation of Empty Headswith Neck Bacteriophage $29 is stable in the presence of dimethyl sulfoxide up to a concentration of 60 %. At higher concentrations of dimethyl sulfoxide a large fall of infectivity was observed. Figure 5 shows a sucrose gradient sedimentation of the structure produced by treatment of $29 with dimethyl sulfoxide. This component sediments slightly faster than the empty heads purified from lysates. Under the electron microscope, this material contained capsids with necks but lacking tails (Fig. 6). Disassemblyof 42~4by Treatment with EDTA During the purification of $29 it was found necessary to add magnesium ions to preserve intact phage. When purified phage was treated with EDTA, more than 90% of the infectivity was lost. Figure 7 shows a sucrose gradient of 429 treated with EDTA that reveals the presence of three peaks, A, B, and C. After an additional purification step by sucrose gradient centrifugation, peak A showed the presence of capsids with neck (like Fig. 6), peak B capsids without neck, similar to those observed in the lysates (Fig. 4) and peak C necks with tail (Fig. 8). Structural Polypeptides of $29. Bacteriophage 429 was dissociated by heating in the presence of sodium dodecyl sulfate, urea, and 2-mercaptoethanol and subjected to polyacrylamide gel electrophoresis, as indicated before. The electropherogram shown on Fig. 9 reveals the presence of seven peaks numbered I to VII in the order of increasing electrophoretic mobility.

ET AL.

FIG. 7. Sucrose gradient centrifugation of 429 treated with EDTA. Phage 429, labeled with leucine-14C, was treated with EDTA as indicated in Materials and Methods and centrifuged in a 15 to 30% sucrose gradient in an SW 50L rotor at 36,000 rpm for 2 hr at 4”.

To determine the nature of the polypeptide chains that build up the structural components isolated as indicated before, each one of these components, labeled with leutine-14C, was mixed with complete phage labeled with leucine-“H, and the mixture was dissociated and subjected to gel electrophoresis. (1) Empty heads isolated from lysates. Figure 10 shows an electropherogram of dissociated empty heads purified from lysates, indicating the presence of polypeptides III and VII and two additional polypeptides (X and Y) not present in intact phage. (2) Capsids prepared from complete phage. Figure 11 shows the polypeptide chains present in the capsids obtained by treatment of intact phage with EDTA. These particles contain polypeptides III, IV, and VII but lack the additional polypeptides X and Y present in the empty heads isolated from infected cells. (3) Capsids with neck. Capsids with neck, prepared by treatment of intact phage either with dimethyl sulfoxide or EDTA contained all the polypeptide chains present in complete particles except peak II. Figure 12 shows an electropherogram of dissociated capsidswith neck prepared with EDTA. (4) Necks with tail. The structure formed by the neck and the tail is built up by polypeptides I, II, V, and VI (Fig. 13). iMolecular Weights of the Structural Polypeptides of 429 The molecular weights of 629 structural polypeptides were determined by comparing

PROTEINS

OF

PHAGE

.iT3

429

FIG. 8. Electron micrograph of purified necks with tail prepared by with F:I)TA. The scale line represents 0.1 F. The inset is a view of the appendages of the neck; approximate magnification X G30,OOO.

treatment particles

of phagc particales showing thrx twcllvr

their elect,rophoretic mobilities with those of marker proteins of known molecular weights (Fig. 14). The estimated molecular weigllt, values of each polypept’ide shown in Table I are the averages of four determinations.

FIG. 9. Structural polypeptides of phage ~29. Bacteriophage +29, labeled with leucine-‘4C (Fig. 2), was dissociated and subjected to polyacrylamide electrophoresis as described in Materials and Methods. In this and subsequent elect,rophoretic patterns the anode is to the right and the radioactivity of each fraction is given as percentage of the total radioactivity in the gel. c?,n,,rt~~ ‘?a ml m---L WY radinn&"itv

Some of the experiment’s shown in this paper confirm and extend the elect,ron microscope studies on the structure of phage ~$29 carried out by Anderson et al. (1966). The basic morphological component,s of ~$29are the head, the neck and the tail (Fig. 3). The head has a hexagonal out’line, flaMened in the region near to t,he neck and it contains fibers. ,111 analysis of the major proteins present in this st’ructure reveals t,he exist’ence of three different, polypeptide chains (III, IV, and VII) with a molecular weight of 54,000,48,000, and 28,000, respectively (Fig. 11 and Table I).

574

Ml?NDEZ

FIG. 10. Structural polypeptides of empty capsids isolated from lysates. Capsids, labeled with leucine-W (Fig. Z), were mixed with phage labeled with leucine-3H, dissociated, and subjected to polyacrylamide electrophoresis. 14C counts: 20,813; 3H counts: 19,673. a---@, 14C radioactivity; O- - -0, 3H radioactivity.

FIG. 11. Structural polypeptides of capsids prepared with EDTA. Empty capsids were prepared by treatment of leucine-W labeled 629 with EDTA (Materials and Methods and Fig. 7). The capsids were mixed with complete phage labeled with leucineJH, dissociated and subjected to polyacrylamide electrophoresis. ‘4C counts: 14,937; 3H counts: 16,519. a---@, 14C radioactivity; o- - -0, 3H radioactivity.

ment of the phage with dimethyl sulfoxide or EDTA (Fig. 6). An analysis of the polypeptides present in tailless particles shows that the only missing protein is polypeptide II (Fig. 12). Therefore, polypeptide II (moIecular weight, 71,000) is a tail protein. The neck is formed by two collars and twelve appendages (Fig. 8). The structure formed by the neck and the tail, obtained by treat,ment of phage particles with EDTA, is

El’ AL.

FIG. 12. Structural polypeptides of capsids with neck. Empty capsids with neck were prepared by treatment of leucine-W labeled 429 with EDTA (Materials and Methods and Fig. 7). These particles were mixed with complete particles labeled with leucine-3H, dissociated, and subjected to polyacrylamide electrophoreGs. W counts: 9118; 3H counts: 10,137. @--a. 14C radioactivity; O-0, 3H radioactivity.

FIG. 13. Structural polypeptides of necks with tail. Necks with tail were prepared by treatment of leucine-14C labeled @29 with EDTA (Materials and Methods and Fig. 7). These particles were mixed with leucine-3H labeled $29, dissociated, and subjected to polyacrylamide gel electrophoresis. 14C counts: 6225; 3H counts: 12,497. 0-0, 14C radioactivity; O- - -0, 3H radioactivity.

composed of polypeptides I, II, V, and VI (Fig. 13). As polypeptide II forms the tail, polypeptides I, V, and VI (molecular weight 80,000, 40,000, and 36,000, respectively) must belong to the neck. Experiments to be reported elsewhere suggest that polypeptide I forms the twelve appendages of the neck. The empty capsids purified from lysates (Fig. 2) contain, besides the head polypep-

FIG. 14. Estimation of the molecular weights of 629 structural polypeptides. 0.25 ml of a solution contining 40 rg of bovine serllrn albumin, and human immunoglobulin G, ovalbllmin, respectively, were dissociated and subjected to polyarrylamide elect rophoresis and the gel was stained as described in Materials and Methods. Another gel loaded with 629 proteins labeled with lerlcinc-‘4C was subjcctrd t,o electrophoresis in parallel. Aft.er fractionation and determination of radioactivity, the relative mobility of each peak (migration distance/tot al length of the gel) was determined. 0, bovine serum albllmin; @, imm\moglobulil~ (;, heavy chain; ~3: ovalbumin; A. imrnut~c,glohuliII (;, light chair).

\vei:ht

Structural component

80,000 7 1 ) 000 5-t ) 000 48,001) 40,001)

Xerk Tail Head Head iSeck

3i,OOO

?;eck

28. I)!)0

Head

\loleculnr

tides III and VII, two addit,ional polypept,ides, X and Y (E‘ig. 10). These two polypeptides have a molecular weight of about 46,000 and 39,000, respect,ively. Several authors have recent.ly reported that in the assembly of T4 heads some proteins are incorporated in t,he capsid as precursors t’hat later on are cleaved to give place to the final proteins present in the normal part,icles (Kellenberger and Kellenberger-van der Kamp, 1970; Laemmli, 1970; Hosoda and

Cone, 1970; Dickson et al., 1970). This finding poses t,he quest,ion whether polypeptides X and/or Y are precursors of normal phage proteins, proteins incorporat’ed only transiently into t,he capsid during the morphogenesis of the viral particle or partially tlegraded polypeptidc(s). An analysis of the trypt,ic peptides of polypept,ides X :~nd 1 compared with those of the pol~~prptidw present in normal part’icles may shed some light, on thi;S question. Bact,eriophage $29 contains a sin& molecule of double-stranded l>NA, molecular u-eight) 11 X 10” daltons (Anderson el al., 1966; ViAuela et al., 1970). This sizcb makes the t,ask of identifying all the genes of +N DKA and t llrir functions feasible. Assuming that the complete genome of $29 is assymrtrically transcribed to messenger RN‘Z, 629 DN.1 C:LI~ code for about 5500 amino :wi(l residues. Table I shows t,hnt, the sewn lx~lypeptides isolated from 629 part,icles accounts for about 3500 amino acids or 60 ‘;; of t Ire genetic information of the virus. A stud:- of S9 tempcrat,urc-~cnsitivc mutant.s 01’ $29 has :~llow-ctl the identification of clwc~n cis trons in the genomc of @29 (T:&~vPI~, Jimknez. Sala3, and \‘ifiwla, submit ted to publication).

AJ,ALIS,

cnce,

&I. I-I. (1959). X-en- York.

~~N.\(;NOSTOI’OI~l~OS,

“Ractcriopli:~ge”. (‘.,

:lnd

Irrtc,rsci-

SI~IZIZEX,

Reqlliremcnt H for t lallsformatioll sccbfilis. J. KMfPTiOI. 81, 711L’il(i.

in

.J. (l!)til,. /1trc~i//~u

1). I,., IlICJSI\N, 1). I)., and I:EIl>J.Y, B. E. (196c,,. Ytructure of Hac+/(us s~bfil%s bacteriophage $29 a~rd the length of +29 deoxyribonucleic acid. .I. Ruc(cario/. 91, 20X1-2088. ARGETSIN~ER-STEIT~, J. (1970). Thcl rccost itlltion ASDERSOS,

576

MfiNDEZ

of RNA bacteriophage. In “Str\lcture and Replication of Macromolecules” (5. Ochoa, C. F. Heredia, C. Asensio, and I). Nachmansohn, eds.), pp. 203-212. Academic Press, New York. CUMMINGS, D. J., CHAPMAN, V. A., and DE LONG, S. S. (1968). Disruption of T-even bacteriophages by dimethyl sulfoxide. J. Viral. 2, 610-620. DICKSON, R. C., BARNES, S. L., and EISERLING, F. A. (1970). Structural proteins of bacteriophage T4. J. Mol. Biol. 53, 461-474. DUHN~U, D., GOLDTHI~AITE, C., SMITH, I., and MARMUR, J. (1967). Genetic mapping in Bacillus subtilis. J. Mol. Biol. 27, 163-185. Hosooa, J., and CONE, R. (1970). Analysis of T4 phage proteins. I. Conversion of precursor proteins into lower molecular weight. peptides during normal capsid formation. Proc. Nat. Acad. 9ci. U. S. 66, 1275-1281. KELLENBERGER, E., and KELLENBERGER-VAN DER KAMP, C. (1970). On a modification of t)he gene product P23 according to its use as a subunit of either normal capsids of phage T4 or of polyheads. FEBS Lett. 8, 140-144. LSEMML~, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. LAEMMIJ, U. K., M~LBERT, E., SHOWE, M., and KELLENBERGER, E. (1970). Form-determining function of the genes required for the assembly of the head of bacteriophage T4. J. Mol. Biol. 49, 99-113. LOENING, U. F. (1967). The fract,ionation of highmolecular-weight ribonucleic acid by poly-

ET

AL.

acrylamide-gel electrophoresis. Biochem. J. 102, 251-257. MAIZEL, J. V., Jn. (1969). Acrylamide gel electrophoresis of proteins and nucleic acids. In “Fundamental Techniques in Virology” (K. Habel and N. P. Salzman, eds.), pp. 334-362. Academic Press, New York. MAIZEL, J. V., Jn., WHITE, 11. O., and SCHARFF, M. D. (1968). The polypeptides of adenovirus. I. Evidence for multiple protein components in the virion and a comparison of types 2, 7A, and 12. ViroZog2/ 36, 115-125. REILLY, B. E., and SPIZIZEN, J. (1965). Bacteriophage deoxyribonucleate infection of competent Bacillus subtilis. J. Bacterial. 89, 782-790. SHAPIRO, A. L., VIWUELA, E., and MAIZEL, J. V., JR. (1967). Molecular weight estimation of polypeptide chains by electrophosis in SDS-polyacrylamide gels. Biochem. Biophys. Res. Commm. 28, 815-820. VIWUELA, E., M~NDEZ, E., TALAVERA, il., ORTfN, J., and SALAS, M. (1970). Structural components of bacteriophage 429. In “Structure and Replication of Macromolecules” (S. Ochoa, C. F. Heredia, C. Asensio, and 1). Nachmansohn, eds.), pp. 195-202. Academic Press, New York. WELKER, N. E., and CAMPBELL, L. L. (1967). Unrelatedness of Bacillus amyloliquefaciens and Bacillus subtilis. J. Bacterial. 94, 1124-1130. WOOD, W. B., EDGAR, R. S., KING, J., LIELAUSIS, I., and HENNINGER, M. (1968). Bacteriophage assembly. Fed. Proc., Fed. Amer. 5’0~. Ezp. Biol. 27, 1160-l 166;.