Interactions between a satellite bacteriophage and its helper

J. Mol.

(1976) 106, 683-707

Biol.

Interactions KATHLEEN

Between a Satellite Bacteriophage and its Helper J.

BARRETT?,

University (Received

L.

MARGARET

MARSH

AND

RICHARD

Department of Molecular Biology of Cali;fornia, Berkeley, Calif. 94720,

1 August

CALENDAR

U.S.A.

1975, and in revised form 10 June 1976)

P4 is a satellite phage which relies on a helper such as P2 to supply the gene products necessary for particle construction and cell lysis (Six, 1975). P4 can activate the expression of late genes from a P2 helper phage, using a mechanism different from that employed by the helper. In the presence of P4, replication of P2 DNA is not required for late gene expression (Six & Lindqvist, 1971), and the polar effects of P2 amber mutations are suppressed. Despite its small size P4 codes for two late proteins as well as two early proteins. One of the P4 early proteins is that the product of gene a. The expression of P4 late genes is stimulated by the helper phage. Thus the P2 and P4 chromosomes exhibit reciprocal transactivation. The presence of the P4 genome causes the P2 head proteins to form a head smaller than that found after infection by P2 (Gibbs et aZ., 1973). P4 late proteins associate with head-like structures and may determine the small size of P4 heads.

1. Introduction Satellite bacteriophage P4 can make plaques on Escherichia cd only if the host is lysogenic for P2 or a related phage (Six, 1963; Six & Klug, 1973). P2 can serve as helper either as a co-infecting phage or as a prophage. P4 requires all the known head, tail and lysis genesof the P2 helper phage for a productive infection, but neither of the two essential P2 early genes,A or B (Six, 1975). In ordinary P2 growth, these two P2 early genesare required for expression of the P2 late genes(Lindahl, 1970; Geisselsoderet al., 1973). However, co-infection with P4 permits P2 mutants in A or B to express late P2 functions. The ability of P4 to switch on the expression of the P2 late genes, under conditions in which P2 itself cannot, has been termed transactivation (Thomas, 1970). P4 and its helper phages appear similar in the electron microscope, as might be expected from the utilization by P4 of all of the head and tail functions of P2 (Inman et al., 1971). The tails of P2 and P4 are indistinguishable. Both phage haveicosahedral heads; however, the P4 head is only one-third the volume of the P2 head. Likewise, the DNA of P4 (7 x lo6 daltons) is only one-third the mass of P2 DNA (22 x lo6 daltons) (Inman & Bertani, 1969; Inman et al., 1971). These two phage DNA molecules have identical cohesive ends (Wang et al., 1973), but are otherwise different, since they cross-hybridize to an extent lessthan 1% (Lindqvist, 1974). Thus the P4 genome is not an extensively deleted helper genome, but a heterologous DNA molecule with a coding capacity of about ten genes. t Current address: Germany.

Max

Planck

Institut

fiir

Virusforschung,

West

683

Spemanstrasse

36,

74

Tiibingen,

684

K.

J.

BARRETT,

M.

L.

MARSH

AND

R.

CALENDAR

What functions might these genes determine. 2 One is transactivation of the helper phage. In addition, satellite phage P4 can replicate its own DNA (Lindqvist & Six, 1971) and lysogenize the host in the absence of a helper (Six & Klug, 1973). Since P4-lysogenic cells are immune to P4 superinfection and carry an inserted prophage, P4 should code for a repressor and an integration protein (Six & Klug, 1973). Still another P4 function is to determine small head size : P4-sized heads (diameter 45 nm) are not found in cells infected with P2 alone (Gibbs et al., 1973) nor are PS-sized heads (d = 62 nm) found in P4-infected, P2-lysogenic cells. These fmdings show that small head size must be determined either by P4 DNA or by a P4 gene product. To date mutants have been found affecting two P4 essential functions. All amber mutants isolated so far fall into gene u. These mutants are unable to replicate P4 DNA under non-permissive conditions, but retain the ability to activate P2 prophage helper late genes (Gibbs et al., 1973; Lindqvist, 1974). P4 gene ,6 is defined by one temperature-sensitive mutant, which is dominant over P4 wild-type (Gibbs et al., 1973). At non-permissive temperature this mutant can replicate its own DNA, but the ability of the infected cell to synthesize RNA, DNA and protein is depressedand the infected cells are killed efficiently. The function of P4 gene /3 is not presently understood. Since genetic studies have thus far yielded limited information on satellite phage P4 functions, we have turned to a study of phage-induced protein synthesis to gain more understanding of the P4 life cycle.

2. Materials (a,)

and Methods

Bacterial and phage strains of E. coli strain C (Bertani

All strains used are derivatives & Weigle, 1953) and are described in Table 1. The prophage P2 Zg cc carried by 2 of those strains is a fast-growing P2 mutant (Bertani et al., 1969). P2 car mutations block the spontaneous release of P2 phage from lysogneic strains (Lindahl & Sunshine, 1972). The P4 strains used are P4 virl, which is immunity-insensitive (Six & Klug, 1973); P4 virl aaml, P4 virl aam5, P4 virl aarnl4, and P4 virl aam (Gibbs et al., 1973). P4 virl aam and P4 virl aam are 2 mutants obtained by hydroxylamine mutagenesis which do not complement P4 virl aaml (Six & Lindqvist, personal communication), and which show less than 0.1% recombination with P4 virl aaml and with one another (L. Sousa, unpublished data). The P2 strains used are P2 virl, tail mutant P2 virl Fam4, and the early mutants P2 viol Aam (Sunshine et al., 1971). P2 virl Aaml27 and P2 virl Barn116 (Lindahl, 1971) and P2 c5 Aam BamllG, isolated by L. E. Bertani; viable deletion mutants P2 deZ22, P2 dell (Chattoraj & Inman, 1972) and P2 de22 (Bertani, 1975). All phage except P2 dell and P2 de12 carry a vir mutation to eliminate the lysogenic response. For the sake of simplicity in writing, the vir mutations are not mentioned in the text or Figure legends. (b) Media and chemicals LB broth (Bertani, 1951) was modified to contain 1% NsCl, 0.1% glucose, 1.6 mMMgCle and 0.5 mM-C&l,. TPG-AA is the Tris-based minimal medium TPG-CAA described by Lindqvist & Six (1971) with 5 rg/ml of each L-amino acid in place of casein hydrolysate. TPG-LA is the same as TPG-AA, except that L-amino acids were reduced in concentration to 0.3 pg/ml. Super TPG (Pruss et al., 1974b) contains each ~-amino acid at 33.3 pg/ml. TPG basal is the minimal salts of TPG-CAA with CaCI, at 1 mM and FeCle*GH,O at O-1 pg/ml. P2 phage were stored in P2 buffer which contains 1% ammonium acetate, 0.01 MMgCl, and O-01 M-TrisaHCl (pH 7.2). P4 phage were stored in the same buffer except that the MgCl, concentration was 0.08 M (P4 buffer).

SATELLITE-HELPER

INTERACTIONS

635

TABLET Bacterial Collection

Relevant

&rains

propertiest

Reference

HF4704

uvrA thy

Lindqvist

C-la

Prototrophic

Sasaki

or origin

& Sinsheimer t Bertani

c-117

Prototrophic

(P2)

Bertani

C-339

Prototrophic

(P2 Zg cc)

From a gift

C-620

supD

C-1056

Polyauxotrophic,

str, standard

indicator

for P2

Wiman

c-1749

Polyauxotrophic, P4

str (P2 cozl),

indioator

for

Gibbs

et al. (1973)

C-1748

Prototrophic

Gibbs

et al. (1973)

c-1757

Polyauxotrophic, aupD, standard most P2 a171 mutants

C-1758

Polyauxotrophic, for P4 am mutants

C-1766

Prototrophic,

C-1792

Polyauxotrophio, assay of P2

C-1896

supD (P2 zg cc)

t The

abbreviations

(1964) C-la from

Sunshine

(P2 ~0~1) indicator

for

aupD (P2 co24), indicator

supD (P2 ~024) aupB used for growth virl Fanad

and

used are in accordance

with

Taylor

by lysogenization; Erich Six et al. (1971)

et al. (1970)

Sunshine

et al. (1971)

Gibbs

et al. (1973)

Gibbs

et al. (1973)

Sunshine From & Trotter

et aZ. (1971)

C-620

by lysogenization

(1972).

The chemicals used for radioactive labeling and electrophoresis are the cribed by Lengyel et al. (1973) except that electrophoretic grade acrylamide from Biorad and used without recrystallization. 14C-reconstituted algal protein ( 1 mCi/ml) was purchased from Schwarz-Mann or New England Nuclear.

(c) Phage growth and

(1967)

(1966)

same as deswas bought hydrolysate

puri&ation

P4 was grown in 10 1 of the modified LB broth described in section (b) above, supplemented with O.Olo/o antifoam. The medium was placed at 37°C under forced aeration. When the host strain C-339 has grown to 0.D.600 = 0.07, lOlo phage were added. When lysis began about 2 h later, 500 ml of 4% EDTA (pH 7) were added to block readsorption. After lysis was complete, cell debris was removed by centrifugation and 1 1 of 1 M-MgCl, was added to stabilize the phage (titer 3 x 10IO/ml). P4 uam mutants were grown on C-1766 or C-1895 as described by Gibbs et al. (1973) or in the LB broth described in section (b) above. P2 phage stocks were grown as described by Lengyel et al. (1973). P2 Am81 was grown on strain C-1757. P2 Faml was grown at 32°C on C-1792. Phage lysates were concentrated with 0.5 M-Nacl and 7% polyethylene glycol6000 as described by Yamamoto et al. (1970). The resuspended pellets were treated with 20 pg of DNase I/ml at 37’C for 20 to 30 min and the solutions were centrifuged at 7700 g for 20 min. The pellet was resuspended in appropriate buffer and the DNase treatment was repeated twice. The three resultant supernatants and the final pellet were assayed for phage. The phage were usually found in the supernatants of the first and second DNase extractions. Those fractions containing the bulk of the phage were used for experiments described here. Some phage preparations were further purified by sedimentation to neutral buoyancy in CsCl; others were extracted from the polyethylene glycol pellet without the use of DNase (see growth of labeled phage, below).

686

K.

J.

BARRETT, (d)

Growth,

M.

L.

pur$cation

MARSH and

AND analys/sis

R.

CALENDAR

of labeled

phage

C-la and C-117 were grown in super TPG to a density of about 3 x 10” cells/ml and concentrated lo-fold in the same medium by centrifugation. The C-la and C-l 17 cultures were infected with P2 and P4, respectively, at a multiplicity of about 10 each. Adsorption was allowed for 10 min at 37°C and the cells were diluted IO-fold into 37°C super TPG containing 5 PCi of 14C-reconstituted algal protein hydrolysate/ml. After 20 and 40 min shaking at 37°C EDTA was added to a final concentration of 2 mM to the P2 and P4infected cells, respectively. At 65 and 90 min bacterial debris was removed by centrifugation at 10,000 g for 20 min. The supernatant from the P4 lysate was brought to 0.09 M-MgCl, and both lysates were concentrated with 10% polyethylene glycol 6000 and 0.5 M-NaCl. The resuspended pellets were extracted as described above except that DNase was omitted. All 3 supernatants were pooled and the phage were sedimented to equilibrium in CsCl density gradients (19.1 g of CsCl per 30 ml of P2 lysate; 16.4 g of CsCl per 30 ml of P4 lysate) at 22,000 revs/min for 3 days in an SW25 rotor. Four out of six preparations exhibited a peak of radioactivity in the bottom third of the tube which is DNA-containing phage heads (full heads), in addition to a phage band in the upper middle of the tube. The peaks of heads and whole phage from each gradient, were pooled and rebanded separately for 36 h at 32,000 revs/min in an SW50.1 rotor. Alternatively, purification of P4 heads and phage was accomplished by sedimenting the pooled supernatants through 10% to 30% glycerol gradients in P4 buffer at 22,000 revs/mm in an SW50.1 rotor for 200 min. Fractions were collected from the tube bottom, counted for radioactivity and assayed for phage plaque-forming units. Sedimentation constants were determined relative to P2 phage (s~~,~ = 280 to 285, Bertani & Bertani, 1970). The sedimentation constants are: P4 phage, 175 & 5 S; P4 heads, 220 & 7 S; and P2 heads, 390 & 10 S. These heads contain DNA as determined by their buoyant density in CsCl, and by their content of 3H-labeled thymidine. The samples from CsCl or glycerol gradients were prepared for SDSt/acrylamide gel electrophoresis by precipitation of a suitable portion with 5 to 10% trichloroacetic acid overnight in the cold; they were centrifuged for 20 min at 17,500 g and washed with cold acetone as described by Maize1 (1972). The pellet was taken up in sample buffer (Lengyel et al., 1973) and boiled for 2 min to dissolve the pellet and dissociate the phage particles. (e)

Phage-induced

proteins

P4 does not shut off host protein synthesis. HF4704 cells were treated, therefore, with U.V. light prior to infection in order to decrease the synt,hesis of host, proteins. Cells were labeled, and the radioactive proteins were analyzed on SDS gels as described by Lengyel et al. (1973) with the following modifications. The cells were grown in TPG-AA, washed in TPG basal and irradiated for 4.5 min at 5.5 ergs/mm2 per s (1500 ergs/mm2). Irradiated cells were concentrated lo-fold and infected with phage at an m.o.i. of 10 in TPG basal. After incubation for 10 min at 37°C to allow adsorption, the cells were diluted IO-fold into 37°C TPG-LA (t = 0). Pulse-labeling was carried out by adding 3 ml of cells to a prewarmed flask containing 20 pCi of 14C-reconstituted algal protein hydrolpsate. The pulse was stopped 2 min later by the addition of cold L-amino acids (25 pg of each amino acid) followed by pouring the culture onto an equal volume of ice. Extracts were made by boiling the cells in sample buffer; the radioactively labeled proteins were separated on SDS/acrylamide gels and visualized by autoradiography (Laemmli, 1970; Lengyel et al., 1973). The molecular weight of each protein band was determined by comparison with the known molecular weights of P2 proteins (Lengyel et al., 1973,1974). The amount of protein synthesized was determined by tracing the aut,oradiograms with a densitometer and weighing the peaks obtained (Lengyel, 1972). The arbitrary units used (relative number of molecules) correspond t’o the mg of paper that would be obtained from a sample containing lo8 cells labeled for 2 min at a specific activity of 13 &X/pg, if the gel were exposed for 100 h and traced with a Joyce-Loebl densitometer using an F wedge and an arm ratio of 5 :l. The relative number of molecules is defined as lo5 times this number divided by the molecular weight of the proetin. t Abbreviations

used

: SEW,

sodium

tlodrcyl

sulfate;

m.r,.i..

multiplicit,y

of infection.

SATELLITE-HELPER (f)

INTERACTIONS

687

Electron microecopy

Fractions from glycerol gradient sedimentation experiments were dialyzed against 0.12 M-ammonium acetate, 1 mM-MgCl, (pH 7.5) and analyzed in a Joel 1OOB electron microscope at magnification of 42,600 x . Collodion-covered platinum grids, which had been carbon-coated and treated with a glow discharge were used. One drop of sample was placed on the grid for 1 min, after which excess liquid was removed, leaving a thin film (Anderson, 1961). Next, a drop of bushy stunt virus at 3.4~ 1012/ml was similarly applied and dried, followed by a drop of 2% uranyl acetate stain. Excess stain was removed by touching filter paper to the drop on the grid, and the grid was allowed to air dry. Polystyrene latex spheres of known diameter were sprayed onto the other side of the grid using a nebulizer. These beads were used for particle size determination.

3. Results (a) P4 early and late proteins In order to examine the proteins induced by P4, non-lysogenic cells were WV.treated to depress host protein synthesis, infected with P4 (or mock-infected), and labeled with 14C-labeled amino acids. Cell extracts were made, and the labeled proteins were analyzed by SDSjacrylamide gel electrophoresis and autoradiography. Extracts made from P4-infected cells contain four labeled proteins that are absent in extracts made from uninfected cells (Fig. 1). P4 protein 1 has a molecular weight of 88,000 f 1000; protein 2, 45,000 & 1000; protein 3, 27,000 f 1000 and protein 4, 19,900 + 500. In P4-infected cells a fifth labeled protein can be seen slightly above P2 protein 0 ; however, this protein was not observed reproducibly and will not be discussed here. The P4 proteins were classified as early or late by pulse-labeling cells for two minutes at various times after infection. The autoradiograms from each gel were traced with a densitometer and the amount of radioactivity in each P4-induced protein peak was determined by weighing the peak (Fig. 2). P4 proteins 1 and 2 are labeled by the tenth minute after infection (Fig. 2(a)), and have been detected as early as two to four minutes after infection (data not shown). The rate of labeling of P4 protein 1 declines after ten minutes, while that of protein 2 continues undiminished throughout infection. P4 proteins 3 and 4 are not detected until 40 to 50 minutes after infection (Fig. 2(b)). Therefore P4, despite its small genome size, codes for both early and late proteins. (b) Suppression

of polarity

by P4

Our electrophoresis procedure does not separate P4 proteins 2 and 4 from P2 tail proteins FI and FII, respectively (Fig. l(b) and (c)). In order to study the synthesis of these two P4 proteins in infections where P2 is also present, we employed the P2 polar tail mutant Fam4, which does not synthesize the products of the FE transcription unit : FI, FII and T (Lindahl, 1971; Lengyel et al., 1974). This procedure led to the unexpected finding that cells infected by P2 Fam4 and P4 can synthesize protein T, although this synthesis is delayed relative to other P2 late proteins (Fig. 3(b)). Thus the polar effect of the Fam4 mutation is relieved by P4. This experiment confirms an earlier finding of M. Sunshine (see preceding paper). (c) Reciprocal

transactivation

of P2 and P4 late genes

P4 transactivation of helper late genes was studied using P2 mutated in early genes A and 3, which cannot synthesize DNA (Lindqvist, 1971) or late mRNA (Geisselsoder et al., 1973). When such a double mutant infects u.v.-treated E. coli

Fro. 1. Proteins made after infection with P4. A culture of HF4704 was u.v.-irradiated, infected with phage or subjected to a mock infection and pulse-labeled from 70 to 72 min. The radioactive proteins were displayed on a 10% SDS/acrylamide gel aa described in Materials and Methods. (a) Uninfected cells; (b) P4-infected cells; (c) cells co-infected with P2 Barn116 end P4. The P4induced proteins are identified on the left, while P2 proteins (Lengyel et al., 1973,1974) are identified on the right.

SATELLITE-HELPER

.1QJ

0

20

INTERACTIONS

40

60

689

00

20-

0

0

AA - 20 Time

40 after

60 infectio:r

FIG. 2. Kinetics of synthesis of the P4 proteins. with P4 and pulse-labeled for 2 min at the indicated acrylamide gels. The amount of protein which was tated by densitometry as described in Materials and in parallel and the background of host proteins has (a) P4 early proteins 1 (-@-a-) and 2 (-O-O-); and 4 (-O-O--).

SO (min)

Ultraviolet light-treated cells were infected times; the extracts were analyzed on 10% synthesized in each 2-min pulse was quantiMethods. An uninfected culture was labeled been subtracted. (b) P4 late proteins 3 (-A-A-b

no late P2 protein synthesis can be detected (<2% of the H protein and 10.5% of the 0 protein synthesized by P2 Faml, data not shown). In the presence of P4, however, P2 Aaml27 Barn116 synthesizes its own late proteins (Figs 3(c) and 4(c)). Thus the transactivation of P2 late genes by P4 can be easily observed by our methods. The most interesting feature of these experiments is the striking effect the P2 genomehas on the expression of P4 late genes.Synthesis of the P4 early gene products is not alteredt (compare Fig. 3(b) to Fig. 2(a)), but P4 protein 3 synthesis is clearly t The analysis of the synthesis of P4 early suppresses the polarity of P2 Pam4 and might suppression (assuming that FII is the product gene). The synthesis of co-migrating FI by rate of synthesis of P4 protein 2. Since this is activated by the P2 helper.

protein 2 is potentially ambiguous, because P4 cause the synthesis of co-migrating FI by polarity of gene F and that FI is the product of an adjacent polarity suppression would increase the apparent not the c&Be, it is clear that protein 2 is not ~WJV,+

K.

690

J.

BARRETT,

M.

L.

MARSH

AND

R.

CALENDAR

)-

)-

/’

Time

after

mfectlon

hn)

(cl

(b)

(a)

Fro. 3. Comparison of the kinetics of synthesis of the 2 P4 early proteins to a minor P2 late protein. Irradiated cells were infected with (a) P2 Fam4, (b) P4 and P2 FamQ, and (c) P4 and P2 Aam Barn116 and pulse-labeled for 2 min at the time indicated. Extracts were analyzed on 10% gels. P2 protein H is a tail protein which was chosen because it is synthesized at a rate oomparable to that of the P4 early proteins 1 and 2. P4 protein 2 is obscured by P2 tail protein FI in the infection with P2 Aam Barn116 and may include some FI in the infection with P4 and P2 Pam4 (see text). -0-o-, P2 protein T; -O-O-, P2 protein H; -A-A--, P4 protein 1; -m-n--, P4 protein 2.

0 .. Ml!! IO

20

3c

Time

(a)

ofter

InfectIon (b)

(mln) (cl

4. Comparison of the kinetics of synthesis of P4 (a) P2 Fam4, (b) P4 and P2 Fam4, (c) P4 and P2 Aaml27 Bamll6, protein 3 to 2 major P2 late proteins. The procedures are the same as those described in Fig. 3. - 0 - 0 -, P2 protein 0; - l - l -, P2 protein N; - n - n -, P4 protein 3. FIG.

SATELLITE-HELPER

INTERACTIONS

691

stimulated. Cells infected only with P4 do not produce protein 3 until 40 minutes after infection (Fig. 2(b)); however, cells infected with P4 and PZ Fam4 or P2 Aaml27 Bum116 can synthesize protein 3 between 10 and 15 minutes after infeotion (Fig. 4(b) and (c)). The rate of protein 3 synthesis is five- to tenfold higher in the presence of P2 than in the absence of P2 and is approximately equal to the rate of synthesis of the P2 major head proteins, N and 0. P4 protein 4 is also transactivated by a P2 helper (data not shown). Thus, transactivation is reciprocal in the P2-P4 system: P4 transactivates the helper late genes and P2 transactivates the P4 late genes. We asked whether the transactivation of P4 protein 3 was due to any of the known P2 early gene products. The P2 double early mutant P2 Aaml27 Barn116 trarw activates P4 protein 3 at the same time as does the tail mutant P2 Fam4 (Fig. 4). Thus the early, increased rate of synthesis of this P4 late gene is not under the control of the two known essential P2 early genes. We also examined mutations which are deleted in the three known non-essential regions of P2 : dell, de12 and vir22 (Bertani, 1975). These P2 mutants also trunsactivate P4 protein 3 (data not shown), as might be expected from their ability to serve as helpers for P4 (E. W. Six, personal communication). These results suggest that the tralasactivation of the P4 genes must be accomplished by a P2 gene which has not yet been defined by a conditional lethal mutation or by a viable deletion mutation. (d) Adysis

of P4 a amber mutant8

In order to determine the functions of the P4-induced proteins, we first asked which of these proteins are affected by P4 mutations in gene a. These mutants should prematurely terminate the a gene product at the site where the amber mutation occurs (Sarabhai et al., 1964). We examined the synthesis of P4 early proteins in cells infected with various P4 aam mutants by pulse-labeling from 10 to 12 minutes after infection (Fig. 5). Synthesis of protein 2 can be seen, but no band is detected at the position of protein 1. Instead, a series of amber fragments of different sizes are observed. The two P4 amber mutants obtained by hydroxylamine mutagenesis, aam and aam51, make the largest and smallest fragments with molecular weights of 65,500 and 21,500, respectively. Three nitrosoguanidine mutants make amber fragments of 27,500, while the fourth such mutant makes a slightly smaller fragment of 27,000. These results show that protein 1 is the product of P4 gene a and also permit the ordering of these mutations on the genome : NH, terminus, aum51, aam26, aam1,5,14, aam52, COOH terminus. This order is consistent with genetic mapping data (Souza et al., 1976). Examination of the kinetics of synthesis of P4-induced proteins in cells infected with P4 amber mutants showed, as expected, that the amber fragment was synthesized with the same kinetics as protein 1 (Fig. 6 (a)). P4 early protein 2 is synthesized in cells infected with P4 amber a mutants (Fig. 6(b)); however, the rate of synthesis is less than that seen in P4 am+-infected cells. This may be a gene dosage effect, since P4 aam mutants cannot replicate their DNA in a non-permissive host. P4 aam mutants synthesize little or no P4 late protein in the absence of a helper phage (Fig. 7(c) ; unpublished data), indicating that P4 DNA replication may be needed for P4 late gene expression. This requirement for DNA replication is relieved in the presence of a P2 Bum mutant helper (Figs. 8(g) and 9(c)). To be sure that this expression of P4 late proteins was replication-independent, we measured the

692

K.

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BARRETT,

M.

L.

MARSH

AND

R.

CALENDAR

FIG. 6. Proteins synthesized by P4 OLamber mutants. Irradiated cells were infected with various C( amber mutants, labeled from 10 to 12 min after infection and electrophoresed on a 10% SDS/ aorylamide gel. (a) P4 + P2 dell (labeled from 20 to 40 min after infection with 20 p’c of algal hydrolysate per 6 ml of cells); (b) uninfected cells; (c) P4; (d) P4 aam51; (e) P4 aam26; (f) P4 ctaml4; (g) P4 aam5; (h) P4 ctaml; (i) P4 uam52.

of [3H]thymidine into acid-insoluble material, and found no stimulaover the background observed in these u.v.-treated, uvrA cells (data not shown). While performing these experiments, we noted that P2 Aam and P2 Aam Barn mutants are less efficient than P2 Barn116 in transactivating synthesis of P4 protein 3 from P2 aam mutants (Figs 7 (c), 8(f) and 9). P4 uam5.2 does not transactivate P2 Aam or P2 Aam Barn mutants well, although P2 late gene expression is clearly stimulated above background (Figs. 7(a) and (b) and 9(a) and (b)). This effect is probably due to gene dosage. When P4 aam is co-infected with P2 BamllG, however, P2 late proteins are made at a high level, even though DNA replication is not detected (see above). incorporation tion

SATELLITE-HELPER

.c

INTERACTIONS

Time

0

after

20 Time

infection

40 after

hid

60 infection

80 (mid

Fm. 6. Kinetics of Bynthesis of proteins in cells infected infected with P4 or P4 aaml. The kinetic8 of synthesis of the determined by comparison with an uninfected aontrol as (a) P4 protein 1 after P4 infection (-a--•-); P4 tcam (--O--O--). (b) P4 protein 2 after P4 infection (-W-w-); infection (-n-n-). We sometimes observe that the rate stant after 40 min; in other experiments its rate of synthesis the time observed (compare Fig. 2(a) and Fig. 6(b)).

(e) Function

693

with a P4 P4 proteins described fragment P4 of synthesis continues

d~o?n mutant. Cells were and cwm fragment were in the legend to Fig. 2. after P4 ~~3~31 infection protein 2 after P4 awn1 of P4 protein 2 is conto increase throughout

of P4 late proteins

In a double infection with P2 and P4, the P4 late proteins are synthesized in concert with the P2 late proteins and in amounts comparable to the major components of the P2 head (seesection (c) above). Phage proteins synthesized late after infection are usually components of the phage virion or are required for the assembly of infective particles. Thus we infer that the P4 late proteins are also involved in P4 virion assembly. In order to test this hypothesis, we looked for P4 late proteins in phage-related particles. P2 lysogenic cells were infected with P4 and continuously labeled with radioactive amino acids. The lysate was concentrated with polyethylene glycol 6000 and the P4 phage and P4 heads were purified on CsCl gradients and analyzed on 10% acrylamide gels (seeMaterials and Methods). They were compared to P2 phage and heads which were similarly labeled and purified (Fig. 10). P4 phage contains a11 the proteins previously found to be components of P2 phage, as is expected from the

694

K.

J.

BARRETT,

M.

Time (a)

FIG. 7. Comparison of P4 protein 3 in single and Bamll6. The procedures -A-A-, P4 + P2 -O-O-, P2 Aaml27

L.

ofter

MARSH

InfectIon (b)

AND

R.

CALENDAR

(mln) (cl

the kinetics of synthesis of (a) P2 protein H, (b) P2 protein 0 and (c) double infections of irradiated cells with P4, P4 aam and P2 Aam are the same as those described in Fig. 3. Aam BamllG; --O-O-, P4 aam52 + P2 Anml27 BamllG; Bam116; -v-O-, P4; -n-n--, P4 mm52.

requirement of P4 for the P2 head and tail genes (Six, 1975). P4 protein 1 is not present in these purified phage particles (Fig. 10). P4 proteins 2 and 4 are obscured by two of the major tail components, FI and FII (Fig. 10) ; their presence can best be determined by analysis of phage heads, which lack these tail proteins. P4 phage heads contain all of the proteins of P2 phage heads, but contain two proteins which are not found in P2 heads. There is a small amount (3 to 6 molecules/ head) of a protein (M, N 45,000) which migrates slightly ahead of P2 tail protein FI, at the position of P4 protein 2 and P2 protein N, the precursor to the major capsid protein N* (Fig. 10; Table 2). Since P4 heads contain elevated amounts of proteins hl and h2 (Fig. 10; Table 2), which are thought to be intermediates in the cleavage of N to N* (Lengyel et al., 1973), it may be that P4 heads also contain a small amount of totally uneleaved N protein. In addition, P4 heads contain a protein band at molecular weight about 19,000 which is absent in P2 phage heads. There are two major proteins of this molecular weight in cells infected with P4 and P2: P4 late protein 4 and P2 tail protein FII. Since there is no detectable FI present in these preparations and therefore little or no contamination with tails, this protein must be P4 protein 4. We have previously reported that there are approximately 45 copiesof P4 protein 4 in P4 phage heads(Pruss et al., 1974a; Goldstein et al., 1974). Analysis of all the fractions from a CsCl gradient of P4 phage heads showed that the peaks of N* and P4 protein 4 do not band at exactly the samedensity. P4 protein 4 is deficient on the heavy side of the N* peak and elevated on the light side (Fig. 11). In fact it is possibleto isolate P4 heads which contain little or no P4 protein 4 (Table 2). This suggeststhat protein 4 is either not a component of the phage head or that it can be dissociated from phage heads during the equilibrium centrifugation in approximately 4 M-CsCl,as has been observed for certain proteins in E. co.5ribosomes (Meselsonet al., 1964). In an attempt to distinguish between these possibilities, P4 phage and phage heads were concentrated by polyethylene glycol 6000 precipitation

SATELLITE-HELPER

INTERACTIONS

Fra. 8. Proteins synthesized by P2 AamlBY, P2 Bam116, P4 and P4 aam52. were infected with these mutants in single and double infections, labeled from after infection (except for (j)) and electrophoresed on & 10% SDS/aarylamide gel. indicated on the left and P4 proteins on the right. (a) Uninfected cells; (b) P2 BamllG; (d) P2 Aam12Y + P4; (e) P2 Barn116 + P4; (f) P2 AamlZY + P4 (h) P4 aam52; (i) P4; (j) P4, 60 min. Barn116 + P4 aam52;

696

Irradiclted cells 60 to 62 min P2 proteins are Aam12Y; (c) P2 aarn52; (g) P2

and sedimented through glycerol gradients without prior purification on CsCl gradients. This allows purification of the phage in a few hours at low ionic strength and should minimize dissociation of protein 4 from phage heads. Fractions were collected and analyzed for phage and radioactivity (Fig. 12). The fastest sedimenting peak (about 220 S) consists of heads which contain DNA (full heads) ; the peak which sediments slightly less rapidly (180 5) is phage particles. The major peak which sediments at 120 S is discussed below. Each fraction was analyzed on a 10% acrylamide gel. The phage heads are slightly contaminated with phage, as is indicated by the presence of the tail protein FI (Fig. 13; Table 3), and therefore a small amount of FII must also be present. When the FII + protein 4 band is corrected for the presence of FII,

K.

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MARSH

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CALENDAR

t IOO-

60-

Time (0)

offer

lnfectlon

(mm) (b)

FIU. 9. Comparison of the kinetics of synthesis of P2 protein 0 and P4 protein 3 in single and double infections of irradiated cells with P4, P4 aam52, P2 Aaml27 and P2 BamllG. The procedures are the same as those desaribed in Fig. 3. (a) P4 + P2 Aam, protein 3 (-A-A-); P4 + P2 Aam, protein 0 (-A-A-); P4 aam 0 (-m-M-); P4, protein 3 + P2 Aam, protein 3 (-n-O-); P4 aam + P2 Aam, protein (-O-O-); P2 Aam, protein 0 (-+-+-); P4 cLa?n, protein 3 (-V-V--). (b) P4 + P2 Barn, protein 3 (-a-A-); P4 + P2 Barn, protein 0 (-A-A-); P4 ctam + P2 Barn, protein 3 (-n-n-); P4 aam + P2 Barn, protein 0 (-¤---); P2 Barn, protein 0 (-+--+-).

these phage heads are found to contain about 25 molecules of P4 protein 4 per head (Table 3). This result suggests that P4 protein 4 is in the P4 phage head, but is lost to a variable degree during prolonged centrifugation in high concentrations of CsCl. P4 protein 3 is sometimes found as a minor component of P4 phage purified by CsCl gradient centrifugation (Table 2), and this finding is difficult to interpret. However, large quantities of protein 3 are always found associated with head-like particles containing no DNA. When polyethylene glycol precipitates from phageinfected cells are fractionated by velocity sedimentation, protein 3 sediments at 120 8, at a position similar to that occupied by most of the P2 major capsid protein (Fig. 13). When the peak fraction of the 120 S material was examined in the electron microscope, 5x lOlo head-like particles/ml of about 40 nm diameter were seen (Fig. 14). These particles stained as though they were devoid of nucleic acid, when compared to a bushy stunt virus control. Twenty-five per cent of these head-like particles appeared to contain cores. These cores could be artifacts of staining, although the regularity of some of them suggests that they may be real (Fig. 14, inset). It is not possible to say with certainty whether P4 protein 3 is part of the empty heads or a

SATELLITE-HELPER

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697

Fm. 10. Analysis of the protein components of P4 and P2 phage and head particles. (a) to (d) Radioactively labeled phage were prepared as described in Materials and Methods except that DNase was added to the crude lysate to a concentration of 20 agglml. The phage and heads were purified on 3 successive CsCl equilibrium density gradients and electrophoresed on 10% aorylamide gels. The head proteins are indicated on the left of the Figure and the tail proteins on the right. The P2 head and tail protein identikations are from Lengyel et al. (1973, 1974). (a) P2 heads; (b) P4 heads; (0) P2 phage; (d) P4 phage. (e) to (g) A culture of HF4704 was treated as described in Fig. 1 and pulse-labeled from 70 to 72 min after infection. The radioactive proteins were displayed on a 10% SDS/acrylamide gel as described in Materials and Methods. (e) Uninfected cells; (f) P4-infected cells; (g) cells co-infected with P4 and P2 BamllG.

component fractionated. precipitated 46

of the cored heads until the 120 S glycerol gradient peak has been further In experiments with two other preparations of polyethylene glycolphage, P4 protein 3 sedimented slightly faster than N*, suggesting that

698

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TABLE 2 Composition

Protein

M,

of P2 and P4 phqe and heads: number molecules per particle P2 Phage

P4 Phage

512

T

94,000

H

71,000

t3

51,000

FI

46,000

181+18

2+N

44,000

ND

hl

42,200

1.7f

h2

40,700

N*

18&3

P2 Heads

of

P4 Heads

6+2

ND

ND

18&3

ND

ND

4*1

ND

ND

~180

ND

ND

ND

ND

6t

4&2

23*4

3+lij

2313

1013

46+7

13+2

531-4

36,000

484527

210*19

5480

1210

h6

32,300

21&5

2113

2512

23-14

t5

31,000

22h5

22&3

ND

ND

3

26,500

ND

ND

ND

4 + FII

19,600

273f22

ND

26~167

h7

17,800

87&13

44*14

813§ 265&52

95+13

3356

P2 and P4 phage and heads were labeled with radioactive amino acids and purified through 2 or 3 C&l gradients as described in Materials and Methods. The data was obtained from 2 separate preparations of labeled phage and phage heads. The peak tube or tubes of each preparation were analyzed on a minimum of three 10% aorylamide gels; the amount of protein in each band was determined by densitometry and weighing. The numbers given are the average and the standard deviation. The number of molecules per particle was determined by assuming that P2 phage contains 3.6 x lo7 daltons of protein (Inman & Bertani, 1969; Bertani & Bertani, 1970). To calculate the number of molecules per P4 phage particle we assumed that the number of copies of tail protein FI in a P4 phage is the same as in a P2 phege since the tails of the 2 phage appear identical. The composition of P4 and P2 heads was determined by assuming that the number of copies of N* in each phage head is the same as the number of copies in each phage particle. ND, not detectable. t Determined from one measurement. Six other determinations were 5 4. $ Determined from one measurement. Six other determinations were 12. $ Determined from 5 measurements. Two other determinations were 2 5. I/ Determined from 2 measurements. Four other determinations were < 4. 7 Determined from 5 measurements. Two other determinations were 22.

protein 3 is more likely to be associated In one of these experiments, P4 protein protein.

with cored heads than with empty heads. 3 sedimented with a peak of uncleaved N

4. Discussion (a) P4-induced

proteins

We have shown that satellite phage P4 codes for at least four proteins (Fig. 1). Proteins 1 and 2 are early proteins whose synthesis begins within four minutes after infection. Proteins 3 and 4 are late proteins whose synthesis begins about 40 to 50 minutes after infection in the absence of a helper (Fig. 2). Together these four proteins represent about half the coding capacity of the P4 genome.

gel analysis of CsCl gradient fractions of P4 heads. A radioactively labs 3led FIG. 11. Acrylamide and purified as described in Materials and Methods. The phage h cad phage lyss bt,e was prepared was sedimented to equilibrium in a second gradient (see Mate1 Bials peak from the first CsCl gradient as well as the fractions on both sides of the peaks were anely xed and Metho Ids). The peak fractions on 10% *< zylamide gels. Approximately 6000 cts/min or when necessary, all of the fraotion, W8S The fraotion more dense than La) analyzed om the gel. The left most panel is the densest fraction. had too lil ;tle material for analysis.

K.

700

J.

BARRETT,

M.

L.

MARSH

AND

3007 Empty

/

250-

P4 phage

CALENDAR

heads

t

“g zoo* \E E 1502 z : 2 IOO-

R.

”

50-

Oh 0

4

.3ottom

8

12

16

20

24 TOP

FIQ. 12. Glycerol gradient sepctration of particles from P4-infected cells. E. cc& C-117 w&s infested with P4 and grown in “C-18beled cunino ecid-aontaining medium 8s described for preparation of radioactive phage. After cell lysis the p8rticle fraction wss pelleted with 10% polyethylene glyool 6000 at 0.6 ~-Nacl and fmctionated on a glycerol gradient as described in Materi8ls and Methods. The position of the P4 phage peak was determined by essays for plaque-forming units.

TABLE 3

Amount of protein 4 in P4 p?wgeheadspuri@d by glycerol gradient sedimentation

Fraction number

Relative number of molecules in gradient fraction FI N’ FII + 4

6

1.7

47

7

2.6

331

8

4.2

166

7.3

Number of molecules in contaminating whole phage in fraction N* PI1

Number of phage heads in fraction N* 4

Number of moleoules per ph8ge head 4

2

2.6

43

2.9

3.8

328

39

26

26

4.9

6.3

161

19

25

46

4.7

22

The procedures used are the same as described in T8ble 2 except that the radioactively labeled ph8ge were purified by sedimentetion through glycerol gradients rather than by equilibrium sedimentation in CsCl (Figs 9 end 10). The presence of some tail protein FI shows thet these heads are slightly contaminated with whole phage (Fig. 10). The amount of FII and N* due to the presence of phege ~8s calculeted as 1.6 8nd 1.17 times the amount of FI, respectively (taken from Teble 2). The emount of FII calculated in this way was subtracted from the tot81 amount of protein in the FII + 4 band to give the amount of protein 4 in the phage head. Similerly, the Bmount of N* in the phage w&s subtraoted from the tot81 amount of N* to give the emount of N* in the phage he8d. The number of molecules of protein 4 per phe@;e heed was determined by Bssuming that each P4 phage head contains 210 copies of N* (Table 2). This same preparation w8s anelyeed on 8 seoond set of gradients with similar results.

E

FI

2X)0-+;hoge

“Fti 4

N* P2 head protein

n 2DDo-

IWO-

IOOD-

n

p

n

50-

P4 protein 3

P4protein4 P2 toil pmteil

P2 toil protsb h

8

8 lo12l4416l82022241 Fmction numbers Direction of sedimentation

Fro. 13. Distribution of P4 proteins in particles from P4-infected cells. Fractions from the glycerol gradient shown in Fig. 9 and from an identical gradient were pooled and preoipitated with trichloroacetic acid and the pellets taken up in sample buffer as described in Materials and Methods. 2 x 10’ ots/min from each fraction were then loaded onto 10% SDS/acrylamide gels, except when the total fraetion contained less than this amount, in which case the entire fraction was used. Electrophoresis, autoradiography and protein quantitation were carried out as described in Materials and Methods. This same analysis was performed with a second set of gradients with simil8r reeults.

Fm. 14. Electron micrograph of particles sedimenting at 120 S. The 120 S peak fraction from a glycerol gradient similar to the one shown in Fig. 9 was observed under the electron microscope in the presence of bushy stunt virus and uranyl acetate stain (see Materials and Methods). The bar represents 260 nm. A, P4 petit head (empty); B, bushy stunt virus (full); C, P4 petit head containing core. Inset: a head with a centered core, and external capsomers arranged in &fold symmetry.

SATELLITE-HELPER

(b) Multiple

INTERACTIONS

703

mechanisms for P2 late gene expression

We have observed transactivation of P2 late genes by P4, using P2 mutants in the early genes A and B to block the normal mode of P2 late gene expression (Figs 3 and 4). The simplest interpretation of this transactivation is that P4 replaces the products of P2 genes A and B with two functionally equivalent proteins. We do not believe that this simple model is correct, since P4 suppresses the polarity associated with certain P2 amber mutants (Fig. 3). In the accompanying paper, Sunshine et al. (1976) describe P4-induced polarity suppression in all four late transcription units. These observations lead us to believe that P4 alters the mechanism by which P2 late genes are expressed. Presumably this alteration requires the inhibition of transcription termination factor rho, which is defective in polarity suppressing mutant strains of E. coli (Richardson et al., 1975). The idea that P4 alters the mechanism of P2 late gene expression finds further support in the observation that normal P2 late gene expression is coupled to DNA synthesis (Lindahl, 1970; Six & Lindqvist, 1971; Geisselsoder et al., 1973), while P4-induced P2 late gene expression can proceed in the absence of P2 DNA replication. For example, no P2 prophage DNA replication is detected when P4 infects a P2 Aam lysogenic strain, although the P2 late genes are efficiently expressed (B. H. Lindqvist, personal communication). Similarly, in rep- host cells, P4-induced P2 late gene expression proceeds in t’he absence of P2 DNA replication (Six & Lindqvist, 1971). The term transactivation may not be appropriate for describing the activation by P4 of P2 late genes from P2 Bamll6, as E. W. Six has found that P2 B gene mutants can be complemented by P4 ccam mutants to yield a burst of P2 B mutant progeny. In these experiments P2 DNA must be replicating, since the burst of progeny is ten times larger than the number of input phage. Thus, in this case, the activation of P2 late gene expression may be occurring by the ordinary replication-coupled P2 mechanism, and not by the P4 transactivation mechanism, which does not require P2 DNA replication. In our own experiments, we have used u.v.-treated, uvrA cells for co-infection by P2 Barn and P4 uam mutants. We detect no DNA replication under these conditions, although P2 late gene expression occurs at about half the normal rate. Although P4 may replace the P2 gene B product, it does not replace the P2 gene A product. The P2 gene A product is needed for P2 DNA replication, and P4 does not cause P2 Aam DNA to replicate (B. H. Lindqvist, G. Pruss & A. Nolte, unpublished data). (c) Reciprocal

transactivation

Further evidence for the complexity of the interaction between the P4 and P2 genomes comes from our finding that P2 causes the transactivation of the P4 late proteins (Figs 2 and 4). This transactivation may occur at the transcriptional level, since the rate of P4 transcription is increased by the presence of P2 (Lindqvist, 1974). The transactivation of P4 late proteins is not abolished by amber mutations in P2 genes A or B, nor by deletions of certain non-essential regions (Fig. 4; unpublished data). A P2 early gene, as yet unidentified, probably causes this effect, since the synthesis of P4 protein 3 begins at the same time that P2 late protein synthesis normally begins. This unidentified P2 early gene may map within certain deletions

704

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MARSH

AND

R. CALENDAR

of the P2 prophage early region, since such deletions cause the P4 latent period to be extended threefold (S. Barclay, manuscript in preparation; partial data presented in Barrett et al., 1974). We have not studied these prophage deletion mutants, because the severe damage of U.V. to the prophage DNA could render it non-functional. However, the isolation of conditional lethal mutants in this P2 gene would allow clarification of its role in P4 growth. (d) The role of P4 gene u P4 protein 1 is the product of P4 gene a, since amber mutants in this gene do not synthesize P4 protein 1, but rather a series of amber fragments of varying molecular weight (Fig. 5). Mutants in P4 gene a do not replicate P4 DNA (Gibbs et al., 1973) and do not synthesize a rifamycin-resistant P4-induced transcribing activity (Barrett et al., 1972). This P4 enzyme is not the sole component responsible for transcription of the P4 or P2 late genes, because both P4 and P2 RNA synthesis is sensitive to rifamycin throughout infection (Lindqvist, 1974). Instead we think the transcribing activity is involved in P4 DNA replication. Since P4 uam mutants are missing P4 protein 1 and the P4 rifamycin-resistant transcribing enzyme, it may be that P4 protein 1 is the P4 RNA polymerase. The sedimentation coefficient of the P4 enzyme (5 S) is compatible with the molecular weight of P4 protein 1 (about 90,000) (Barrett et al., 1972; Fig. 1). Mutants in P4 gene u synthesize P4 early protein 2, although the rate of synthesis of this protein is less than in a wild-type infection. This is thought to be due to a gene dosage effect, since P4 aam mutants do not synthesize P4 DNA. It is not possible to assign a function with certainty to P4 protein 2, since there are at present no amber mutants affecting this protein. However, P4 presumably has at least three other early functions : (1) a repressor, (2) an integration protein and (3) a protein(s) responsible for the transactivation of P2 late genes. We favor a role in transactivation for P4 protein 2. We would not expect the repressor (Ptashne, 1970) or integration protein to be made in sufficient amounts to be detected in our experiments. P4 uam mutants express P4 late genes very poorly in the absence of a helper (Fig. 7(c)), although they can be made to express their late genes in the presence of a P2 Barn helper (Figs 8(g) and 9). Thus there may be two modes of P4 late gene expression: a replication-dependent mode observed in the absence of a helper, and a replication-independent mode observed in the presence of a P2 Barn helper. Replication-independent expression of P4 late proteins is also observed in the presence of a P2 Aam helper, but at a decreased level (Figs 8(f) and 9(a)). P2 Aam Barn did not stimulate expression of P4 late genes from a non-replicating P4 uam helper (Fig. 7(c)). These effects of the P2 Aam and Aam Barn helpers remain to be explained. Since the P2 A gene product does not act in trans (Lindahl, 1970), it cannot be used directly in transactivating P4 late gene expression. Instead, the P2 A gene product must be needed for synthesis of some other gene product which turns on the late genes of P4 aam mutants. P2 gene A is needed for expression of all P2 late genes (Lindahl, 1970), and may also be needed for expression of a P2 gene (ogr) which is needed for late P2 gene expression (Sunshine t Sauer, 1975; M. Sunshine, personal communication). We have discussed the pleiotropic negative effects of the P4 aam mutations as

SATELLITE-HELPER

INTERACTIONS

765

resulting from the inability of these mutants to replicate their DNA; however, other interpretations deserve consideration and cannot be excluded. Since transactivation of P2 late gene expression is very poor in P2 Aam + P4 uam mixed infections (Fig. 9), the a gene product could be functioning in both DNA replication and transactivation of the P2 late genes. In support of the idea that a phage gene product may be involved both in DNA replication and transactivation is the recent report that the product of T4 gene 45 is required both for DNA replication and late mRNA production (Wu & Geiduschek, 1975). The isolation of ts mutants in P4 gene a, as well as biochemical and genetic studies of the complex interactions between components specified by P4, P2 and E. coli, will be necessary for understanding the role of the a protein. (e) Head size determination

It has been shown that P4 DNA cannot determine small head size in an in, vitro DNA packaging system (Pruss et al., 1974b). When P4 DNA is added to extracts from P2-infected cells, trimers of P4 DNA are packaged into P2-sized (large) heads (Pruss et al., 1975). Thus we believe that the two P4 late proteins are needed for synthesis of a small (P4-sized) phage head composed of P2 head protein. In support of this idea, we have found P4 protein 4 associated with P4 heads as a minor component (about 25 copies per head). Very little 94 protein 3 is found in P4 heads or phage purified by CsCl densitygradient centrifugation (Figs 10 and 11). However, large amounts of this protein are found sedimenting at 120 S along with P4-sized head-like particles, devoid of DNA. Some of these head-like particles contain apparent cores. It is attractive to think of protein 3 as a component of a size-determining core. Such cored structures are thought to be needed for proper head formation in phages P22, T4 and h (Botstein et al., 1973 ; Showe & Black, 1973; Hohn et al., 1974; Hendrix & Casjens, 1975). Under slightly different experimental conditions, we have found P4 protein 3 in a 150 S peak which contains equal amounts of N and N*, although we do not yet know what kind of head structures this peak may contain. A better separation of cored and non-cored empty heads will be needed to reach a more detailed conclusion. The isolation of mutants in the genes coding for P4 late proteins would provide a rigorous test for the role of these proteins in morphogenesis. P4 heads contain elevated amounts of two proteins which are intermediates in the cleavage of the P2 N gene product to N*, the major capsid protein (Fig. 7; Lengyel et al., 1973). It is possible that P4 proteins 3 or 4 protect some molecules of P2 X protein from undergoing complete cleavage. We are grateful to Audrey Nolte and Roberta Wiener for technical assistance. We thank Erich Six and BjBrn Lindqvist for providing their P4 aanz mutants and Melvin Sunshine, Erich Six and Bjorn Lindqvist for communicating their results before publication. This investigation was supported by National Institutes of Health research grant AI08722 from the National Institute of Allergy and Infectious Diseases, by training grant CA05028 and research grant CA14097 from the National Cancer Institute, by National Science Foundation Grant BMS74-19607, by American Cancer Society Research Grant VC-188 and by Cancer Research Funds of the University of California. The electron microscopy was performed by Joseph Toby under National Institutes of Health research grant AI10845 to Professor Robley C. Williams, whose advice and encouragement have been essential.

706

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J. BARRETT,

M. L. MARSH

AND

R. CALENDAR

REFERENCES Anderson, T. F. (1961). Proc. Eur. Regiotial Conf. on. Electron Microscopy, D&t, vol. 2, pp. 1008-1011. Barrett, K., Gibbs, W. & Calendar, R. (1972). Proc. Nat. Acad. Sci., U.S.A. 69, 29862994. Barrett, K., Barclay, S., Calendar, R., Lindqvist, B. & Six, E. (1974). In Mechanism of Virwr Dkewe (Robinson, W. S. & FOX, C. F., eds), pp. 385401, W. A. Benjamin, Inc., Menlo Park, California. Bertani, G. (1951). J. Bacterial. 62, 293-300. Bertani, G. (1975). Mol. Gen. Genet. 136, 107-137. Bertani, G. & Weigle, J. J. (1953). J. Bacterial. 65, 113-121. Bertani, G., Choe, B. K. & Lindahl, G. (1969). J. Ben. Viral. 5, 97-104. Bertani, L. E. (1964). Biochim. Biophys. Acta, 87, 631-640. Bertani, L. E. & Bertani, G. (1970). J. Gen. Viral. 6, 201-212. Botstein, D., Waddell, C. & King, J. (1973). J. Mol. Biol. 80, 669-695. Chattoraj, D. K. & Inman, R. B. (1972). J. Mol. Biol. 66, 423-434. Geisselsoder, J., Mandel, M., Calendar, R. & Chattoraj, D. K. (1973). J. Mol. BioZ. 77, 405415. Gibbs, W., Goldstein, R. N., Wiener, R. W., Lindqvist, B. & Calendar, R. (1973). V&Zogy, 53, 24-39. Goldstein, R., Lengyel, J., Pruss, G., Barrett, K., Calendar, R. & Six, E. (1974). Curr. Top. Immunol. Microbial. 68, 59975. Hendrix. R. W. & Casjens, S. R. (1975). J. Mol. Biol. 91, 187-199. Hohn, B., Wurtz, M., Klein, B., Lustig, A. t Hohn, T. (1974). J. SupramoZ. Struct. 2, 302-317. Inman, R. B. & Bertani, G. (1969). J. Mol. Biol. 44, 533-549. Inman. R. B., Schnos, M., Simon, L. D., Six, E. W. & Walker, D. H. (1971). ViroZogy, 44, 67-72. Laemmli, U. K. (1970). Nature (London), 227, 680. Lengyel, J. A. (1972). Ph.D. Thesis, University of California, Berkeley. M. G. & Calendar, R. (1973). Lengyel, J. A., Goldstein, R. N., Marsh, M., Sunshine, V7iroZogy, 53, l-23. Lengyel, J. A., Goldstein, R. N., Marsh, M. & Calendar, R. (1974). Virology, 62, 161-174. Lindahl, G. (1970). Virology, 42, 522-533. Lindahl, G. (1971). Virology, 46, 620-633. Lindahl, G. t Sunshine, M. G. (1972). V&oZogy, 49, 180-187. Lindqvist, B. H. (1971). Mol. Gen. Genet. 110, 178-196. Lindqvist, B. H. (1974). Proc. Nat. Acad. Sci., U.S.A. 71, 2752-2755. Lindqvist, B. H. & Sinsheimer, R. L. (1967). J. Mol. BioZ. 28, 87-94. Lindqvist, B. H. & Six, E. W. (1971). Virology, 43, 1-7, Maizel, J. V. Jr. (1972). In Methods in Virology (Maramorsch, K. & Kaprowski, H., ads), Vol. 5, pp. 179-246, Academic Press, New York. Meselson, M., Nomura, M., Brenner, S., Davern, C. & Schlessinger, D. (1964). J. Mol. BioZ. 9, 696711. Pruss, G., Barrett, K., Lengyel, G., Golstein, R. & Calendar, R. (1974u). J. SupramoZ. Struct. 2, 337-348. Pruss, G., Goldstein, R. N. & Calendar, R. (19743). Proc. Nat. Acad. Sci., U.S.A. 71, 2367-2371. Pruss, G., Wang, J. C. & Calendar, R. (1975). J. Mol. BioZ. 98, 465-478. Ptashne, M. (1970). In The Bacteriophage Lambda (Hershey, A. D., ed.), pp. 221-238, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Richardson, J. R., Grimley, C. & Lowery, C. (1976). Proc. Nat. AcadSci., U.S.A. 72, 1725. Sarabhai, A. S., Stretton, A. 0. W., Brenner, S. & Bolle, S. (1964). Nature (London), 201, 13-17. Saaaki, I. & Bertani, G. (1965). J. Gem. Microbial. 40, 365-376. Showe, M. $ Black, L. (1973). Nature New BioZ. 242, 70-75. Six, E. W. (1963). BacterioZ. Proc. p. 138. Six, E. W. (1975). Virology, 67, 249-263.

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INTERACTIONS

707

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