Hershey & Chase, i952 ). .... The protein patterns were reproducible from experiment ... In most experiments synthesis of host proteins of tool. wt. greater than.
309
J. gen. Virol. (I976), 33, 3o9-319 Printed in Great Britain
Polypeptides Specified by Bacteriophage T1 By D. T. M. M A R T I N , C A T H E R I N E A. A D A I R AND D. A. R I T C H I E
Institute of Virology, University of Glasgow, Glasgow GI I 5JR, Scotland (Accepted I6 July I976) SUMMARY
The proteins synthesized during the replication of phage TI in u.v.-irradiated Escherichia coli strain B have been examined by polyacrylamide gel electrophoresis of polypeptides pulse-labelled with l~C-amino acids. Up to 5o discrete bands were identified of which about 3o were sufficiently distinct to be classified in terms of time of synthesis. Three polypeptides were synthesized only during the first 6 to 8 rain post infection (Class I, Early); i6 or I7 were synthesized predominantly during the later stages of replication starting from 6 to 8 min after infection (Class III, Late); three classes of proteins were made continuously, two at constant rate (Class II, Continuous), five at decreasing rate (Class IV, Earlycontinuous) and five at increasing rate (Class V, Late-continuous). Of the I4 polypeptides identified as structural components of the virion, three (P7, Pro and PI I) account for about 85 % of the particle weight with P7 comprising 5o % of the particle. P7 and PIo appear to result from the cleavage of larger polypeptides. Preliminary studies with amber mutants suggest that normal levels of Tr DNA synthesis are not required for the manufacture of late proteins and that a phagecontrolled function may control the switch-off of proteins made early and the switch-on of proteins made late.
INTRODUCTION
To date, complementation analysis of temperature-sensitive and amber mutants of phage TI has identified 25 genes with functions essential for replication in wild type E. coli (Michalke, I967; Figurski & Christensen, I974; J. R. Christensen, D. A. Ritchie & D. T. M. Martin, unpublished results). The functions of two genes are required for the synthesis of normal amounts of Tt DNA. Eight of eighteen partially characterized gene functions are required for the completion of phage heads and eight others are required for tail synthesis. One of the head-defective mutants also produces DNA arrest under non-permissive conditions and the two DNA-negative mutants mentioned above show pleiotropic effects, being additionally unable to inhibit host DNA synthesis or produce lysis (Figurski & Christensen, I974). The genes coding for the I8 partially characterized functions are grouped on the linear genetic map with the sequence: DNA genes, tail genes, head genes. Very little is known either of the products of these essential genes, or of the kinetics of synthesis and regulation of the gene products. As part of a study of these aspects of Tr protein synthesis we have used polyacrylamide gel electrophoresis to analyse both the polypeptides present in the page particle and the polypeptides synthesized following infection of E. co8 by wild type phage TI. Some data from our studies with TI amber mutants are also considered. The results are compared with those of a similar study published recently by Toni, Conti & Schito (I976).
3IO
D.T.M.
MARTIN
AND
OTHERS
METHODS
Phage and bacteria. Phage TI wild type (originally described as TI Ds ++ by Dr C. Bresch) was a gift from Dr L. A. MacHattie and the TI amber mutants were isolated in our laboratory. E. coli strain B, non-permissive for amber mutants, was the host strain for all growth experiments. Amber mutants were propagated on the suppressor strain E. coli KB-3 (a gift from Dr E. R6ttlander). Growth and purification of phage. Unlabelled phage stocks were prepared by the confluent lysis method on nutrient agar plates with top layers seeded with Io 8 cells and IO3 to Io ~ phage. The plates were incubated until lysis was confluent and the phage harvested by removing the top agar layer with 2. 5 ml phosphate buffer, pH 7 (adsorption medium of Hershey & Chase, i952 ). The agar was removed from the lysate by centrifugation and the supernatant fluid dialysed 2 to 3 times against phosphate buffer, pH 7, to remove traces of amino acids. 14C-labelled TI + stocks were prepared on E. coli strain B growing in M9 medium (Anderson, I946) at 37 °C. A 30 ml culture of logarithmically growing cells (2 × Io 8 cells/ml) was infected with 5 to io phage/cell. Following incubation for 7 min, I5o #Ci 14C-protein hydrolysate (CFB.25, 5o #Ci/ml > 45 mCi/mAtom of carbon, Radiochemical Centre, Amersham, U.K.) was added and 3 rain later the culture was supplemented with a further I 5 o # C i of the ~4C label. After lysis about 2 x io n particles of purified non-radioactive T1 ÷ phage was added and the phage precipitated by the polyethylene glycol-dextran sulphate phase partition method of Albertsson (I967). Finally the phage were banded by equilibrium zonal centrifugation in a pre-formed CsC1 step gradient (Matthews, I96o) centrifuged in the Beckman SW 5o L rotor at 35 ooo rev/min for 60 rain. The phage band was collected and dialysed 2 to 3 times against phosphate buffer, pH 7. Preparation of labelled intracellular proteins. Cells were irradiated with ultraviolet (u.v.) light before infection to reduce host cell protein synthesis and facilitate identification of polypeptides synthesised after infection. Because TI adsorption is dependent upon cellular metabolism (Stent, I963) it was necessary to infect cells immediately following irradiation. With the conditions described below, u.v. irradiation reduced the rate of protein synthesis for uninfected cells to IO ~ of normal whem measured immediately after irradiation and Ten 5 ~ by Io min later; after TI infection these rates rose to I5 ~ and 3o ~ respectively. In uninfected cells D N A synthesis was abolished but Tt infected cells synthesized about 1o % of the unirradiated control level. Cell lysis was delayed and often incomplete. Cultures of strain B were grown in M9 medium with aeration at 37 °C to 2 x lO 8 cells[ml. Ten ml volumes were irradiated with agitation for 3° s at a distance of i2 cm from a 3 ° W u.v. lamp (total dose about Ioooo ergs/cm 2) and immediately infected with 5 to Io p.f.u./cell and aerated at 37 °C. At I rain intervals during infection, I ml samples were removed and the proteins labelled during a 2 rain pulse with Io #1 l~C-protein hydrolysate (o'5 #Ci/ml final concentration). Incorporation of radioactive label was terminated by the addition of 0.2 ml of a 5 ~ (w/v) solution of unlabelled casamino acids (Difco Ltd.) and each sample was incubated for a further 3 min to permit completion of the polypeptide chains initiated during the pulse. The cells were killed by addition of an equal vol. of 2o ~ (w/v) trichloracetic acid, harvested by centrifugation and washed twice with I ml volumes of 5o ~ (v/v) ethanol. Prior to electrophoresis the samples were suspended in o.2 ml of a solution containing 5 (w[v) SDS, 20 ~o (w/v) 2-mercaptoethanol and IO ~ (v/v) glycerol dissolved in the stacking gel buffer used for electrophoresis (see below) and the proteins solubilized by heating at ioo °C for 2 min.
Phage TI-specified polypeptides
3r
Gel electrophoresis. Polypeptides were separated by electrophoresis in polyacrylamide gel slabs with a IO to W'5 % linear gradient of acrylamide and a discontinuous SDS buffer system. The apparatus and procedures were as described by Maizel (1969) with the modifications reported by Marsden, Crombie & Subak-Sharpe (I976). Stacking and resolving gel buffers were as described by Laemmli (I97O), other buffers have been reported by Marsden et al. (1976). After electrophoresis the gels were dried under vacuum using a procedure based on that of Fairbanks, Levinthal & Reeder (1965) as modified by T. H. Pennington (in preparation) and autoradiographs were made using Kodirex KD 54T X-ray film. When required, gels were stained with Coomassie brilliant blue and then destained prior to drying. Polypeptide mol. wt. were calculated by comparison with the migration rates of polypeptides of known tool. wt. These markers and their mol. wt. were: myosin heavy chain (22oooo); E. coli RNA polymerase ~ (4oooo), fl (I57OOO) and fix (15oooo) subunits; phosphorylase A (94ooo); beef liver catalase (6oooo); fumarase (49 ooo); yeast glyceraldehyde3-phosphate dehydrogenase (36o00); bovine pancreas chymotrypsinogen A (257oo); bovine haemoglobin (I 5 ooo); lysozyme (I4 30o); bovine pancreas ribonuclease A (I 3 7oo); horse heart cytochrome c (I 17oo). Before the gel had set it was overlaid with acrylamidefree buffer and as a result of slight mixing at the interface the top of the gel, for a distance of about 2 mm, had a steep concentration of acrylamide running from o to IO %. Consequently all marker polypeptides entered the gel. However, the three largest, of tool. wt. 22oooo, 157ooo and ~5oooo, were not resolved from one another although they were measurably slower than the 94ooo tool. wt. marker. The three largest T1 particle polypeptides (PI, Pz and P3) consistently migrated to a position between that of the three slowest marker polypeptides and that of the 94ooo mol. wt. marker. RESULTS
The proteins of the mature phage particle Purified a4C-amino acid labelled TI + phage particles were disrupted by boiling with SDS and 2-mercaptoethanol and electrophoresed on SDS-polyacrylamide gels together with a set of polypeptide markers of known tool. wt. Autoradiography of the radioactive particle polypeptides showed at least I4 distinct bands. Those which appeared consistently have been labelled PI to P13 in order of decreasing mol. wt. (Fig. I). These polypeptides range in mol. wt. from about 152ooo for PI to about I35OO for PI3 although there is a little uncertainty over these extreme values since P1 barely entered the gel and PI I, PI2 and PI3 migrated to a region of the gel where the mol. wt. standards are not completely reliable. The sum of the tool. wt. of these particle proteins is about 70ooo0, a value requiring the coding capacity of some 13 x io * daltons of double-stranded DNA. The relative amount of each particle polypeptide was estimated from the peak areas of a suitable autoradiogram (similar to that shown in Fig. I) and calculated as a percentage of the total protein content of the virion (Table I, column 3). Three polypeptides, P7, P IO and PI I account for over 85 % of the total with P7 contributing about 5o % of the particle protein, they must therefore be regarded as the major structural proteins of the virion. Calculations of the approximate number of molecules of each polypeptide present in a phage particle suggest that P7, PIo and PII are also present in the greatest number of copies (Table I, column 4).
21
vlR
33
312
D . T . M . M A R T I N AND OTHERS
I
/It \
P13 B A P12
Pll
1
II
Pl0
P9
II II P8
P7
II
P6 /?5 P4
/1\
P3 P2 P1
Fig. I. Autoradiogram and the corresponding densitometer tracing of electrophoresed polypeptides from 14C-amino acid labelled TI + phage particles disrupted by boiling with SDS and. mercaptoethanol. Polypeptides are labelled PI to P~3 in order of decreasing tool. wt. Band PI2 is a double band and has been labelled A and B. Bands which are marked above the line are not consistently observed in particle preparations. Migration is from right to left.
Time course of polypeptide synthesis during TI infection The pattern o f protein synthesis in T1 infected cells was followed by polyacrylamide gel electrophoresis o f samples pulse labelled with 14C-amino acids for 2 rain periods at I min intervals during the growth cycle. The protein patterns were reproducible f r o m experiment to experiment with a typical example shown in Fig. 2. The extreme left-hand sample (marked U) shows that very little protein synthesis occurred in uninfected host cells when the pulse was given after u.v. irradiation and that most o f the polypeptides labelled were o f low mol. wt. This makes analysis o f the low mol. wt. regions o f the gels o f infected samples rather difficult. I n most experiments synthesis o f host proteins o f tool. wt. greater than 15 ooo was almost negligible and for practical purposes could be ignored. F r o m the infected samples approx. 50 bands could be identified representing polypeptides with mol. wt. ranging f r o m about 15o0oo down to 1oo00. O f these some 30 or so were sufficiently pro-
Phage TI-specified polypeptides Table Protein band PI P2 P3 ]?4 P5 P6 P7 P8 P9 PIO PI1 PI2 A PI2 B
PI3
313
I. Proteins of the T1 phage particle Mol. wt.* ( x 10 -3) I52_+22'5 117_+ 6"2 lO3_+ 4"7 57+ o'8 50_+ 1'3 42'5_+ o'7 33_+ o'3 29"5_+ 0"4 29_+ 0"3 26_+ 0"4 I6_+ 0"3 t4_+ 0'8 | I4+ 0"8 ~
of total particle proteint
Number of molecules per particle:~
I'5 I'O I'O I-2 I'5 o'7 49"6 1'5 3"5 I6'3 20"1
3 3 3 7 IO 6 5o5 17 4I 2II 422
2'I
52
13'5_+ o'4 J
* Average of three independent determinations. t Estimated by scanning suitable autoradiographs with a Joyce-Loebl microdensitometer with punch tape output and computing the peaks with a Wang calculator programmed to correct for non-linearity in the response of the film to increased exposure. :~ Calculated from the data given in columns 2 and 3 using band P6 as having unit frequency. nounced and distinct to permit classification on the basis of the time course of synthesis. Our analysis of the patterns indicates the existence of five classes of polypeptide. Eight polypeptides were predominantly made during the early stages of infection (marked E and EC); the three marked E (early) stopped being synthesized by 6 to 8 min after infection and the five EC (early-continuous) polypeptides continued to be synthesized at reducing rates. Sixteen or I7 were synthesized predominantly during the later stages of infection (marked L), i.e. starting from 6 to 8 min after infection and continuing at increasing rates until lysis. Two polypeptides were made continuously throughout the growth cycle at near constant rate (marked C), while a further five, although synthesized in greater amounts at later times were also made in appreciable amounts at early times (marked LC for late-continuous). These 31 to 32 bands account for a total protein mol. wt. of about t'3 x lO8 or about 8o of the assumed coding capacity of the TI genome. The significance of the 2o or so bands present in trace amounts is not clear at present. (The particle protein P8 migrates to a position which almost overlaps with an early band, this gives the impression of a continuous band but is really an early band overlapping with a late band.) The infected cell samples shown in Fig. 2 show all the particle protein bands with the exception of P1 and P3. While PI appears on other gels and is clearly a bonafide intracellular protein, P3 has never been observed in cell extracts. This makes it very unlikely that P3 as such is synthesized as a TI-specified polypeptide. Amber mutants from several different complementation groups failed to synthesize particle protein P7 (mol. wt. 33 ooo). Some of these mutants accumulated a band of mol. wt. 4oooo instead, while others made neither, suggesting that the heavier band may be a precursor of P7. This was confirmed by showing that after a short period of labelling, infected cell samples contained large amounts of the mol. wt. 4o ooo band but very little P7 whereas after a chase with unlahelled amino acids this pattern was reversed (Fig. 3). P7 thus appears to be derived from the 4o ooo precursor protein by post-translational cleavage. The residual fragment(s) of total mol. wt. 7o0o could not be resolved on this gel. A comparison of the 2I-2
0
l
2
3
4
5
6
7
8
9
l0
11
12
13
14
15
16
17
f
TI+~
15
20
30
50
100
Fig. 2. Autoradiogram of electrophoresed polypeptides from u.v.-irradiated E. coli strain B infected with TI + phage. Polypeptides were labelled with 14C-amino acids in a z min pulse followed by a 3 rain chase with unlabelled amino acids. The time after infection at which pulses were started is given under each track. Bands are labelled: E (early), EC (early-continuous), C (continuous), LC (late-continuous) and L (late). The left-hand track (U) shows the uninfected cell pattern (labelled for 2 min immediately before infection) with the dots indicating bands which co-migrate with bands synthesized after infection. The right-hand track shows the Tt + particle proteins with lines indicating the corresponding intracellular polypeptide. Some of the minor bands which appear after infection are marked with an X. Migration is from top to bottom.
U
C-
E-
EC. EC"
EC"
EC
C.
EC' E'
E"
150
o
×
7
"i3
Z
-q
4~
L,O
Phage Tl-specified polypeptides (a)
315
(b)
P2
P4 P5 P6
P7
PIO
PI1
Fig. 3. Post-translational cleavage of TI particle proteins. Autoradiogram of electrophoresed polypeptides from TI + infected E. coli B cultures labelled with 14C-amino acids. (a) Polypeptides labelled during a I min pulse given between io and I I min after infection. (b) Polypeptides labelled during a x min pulse from 9 to Io rain after infection followed by a chase with unlabelled amino acids from IO to I I min after infection. Bands labelled P7P and Plop refer to the intracellular precursors of particle polypeptides P7 and PIo respectively. Some additional particle polypeptides are labelled for reference. Migration is from top to bottom.
pre- and post-chased samples in Fig. 3 reveals other differences, suggesting that posttranslational cleavage may well affect other polypeptides, PIo for example. The cases of mutants a m 22r and a m 28o are worth noting. A m 22I is unable to synthesize Ti DNA in the non-permissive host and corresponds to the DO gene z described by Figurski & Christensen (~974)- The gel of a m 22I pulsed early (I to 3 min) after infection showed that only one band, an E band of about 65 ooo mol. wt. was missing which suggests that this band is the protein product ofgene z (Fig. 4A). More interestingly the polypeptides made late during a m 2zi infection contain all late proteins in roughly normal amounts, including P7. Apparently, normal levels of phage DNA synthesis are not required for late protein synthesis although the a m 22I function is required for normal lysis (Figurski & Christensen, I974). With a m 280 the polypeptide pattern obtained late in infection of a non-permissive host was very similar to the normal early pattern (Fig. 4B; Fig. 2 can also be used for comparison). The defect may therefore affect a function required to control the early to late switch in gene expression.
D. T. M. MARTIN AND OTHERS
316
A (a) (b)
B (a) (b)
O
Fig. 4. Autoradiograms of electrophoresed polypeptides from u.v.-irradiated E. coli B cells infected with TI + and TI am mutant phage. (A) Early samples pulsed from i to 3 min after infection with 14C-amino acids; (a) am z z I , (b) TI +. The band marked with a filled circle in (b) is the band missing in (a) and the band marked with an open circle in (a) is possibly an extra band representing the prematurely terminated polypeptide fragment. (B) Late samples pulsed from IZ to I5 min after infection with a4C-amino acids; (a) am a8o, (b) TI +. All samples were chased for 3 min with unlabelled amino acids at the end of the radioactive pulse. Migration is from top to bottom. DISCUSSION T I phage particles released about I4 distinct polypeptide components when disrupted by boiling with SDS and mercaptoethanol and then electrophoresed on polyacrylamide gels. Three major polypeptides, P7, P I o and P I I with mol. wt. of 33ooo, 26000 and 16o0o respectively, contribute over 85 ~ of the particle mass. P7, by virtue of its largest contribution (about 5° ~), is the most likely candidate for the major head subunit. Moreover, P7 and probably Pro are the products of post-translational cleavage, a modification also known to occur during the morphogenesis of phages T4 (Laemmli, I97o),/l (Murialdo & Siminovitch, I972), P2 (Lengyel e t al. I973) and T5 (Zweig & Cummings, 1973). Of the I4 polypeptides all but P3 have been identified as being synthesized during T I infection. No band at the position of P3 has been observed in gels of Ti-infected cell extracts and the origin of this particle component is thus, for the moment, unclear. Possibly P3 represents an encapsidated host protein or a degradation product of P I and Pz; an aggregate of lower mol. wt. polypeptides appears unlikely in view of the extraction procedure. Comparison of our analysis with that reported recently by Toni e t al. (I976) shows a large measure of agreement, particularly for the more prominent bands. For example, the
Phage TI-specified polypeptides
317
tool. wt. of their three major bands, V8 (3oooo), VIo (26000) and VI2 (I6ooo) compare well with our bands P7 (33 ooo), P Io (26 ooo) and P~ I (I 6 ooo) respectively. The relative proportions of VIo and PIo (I5"5 % and I6"3 %) and of VIz and PII (23.2 ~ooand 2o.I %) are also in agreement. Polypeptide V8, however, is estimated by Toni et aL 0976) to be present in the particle in only half the quantity that we calculated for P7. If P7 and V8 each represent the major head protein then they are likely in reality to be present in similar amounts. The discrepancy is probably due to the relative amounts of the minor bands which tend to be much lower in our analysis. Moreover, at present, we cannot account for the differences in the two estimates for the tool. wt. of the three largest polypeptides (P~, P2 and P3 in our terminology). Our analysis of the phage genes responsible for the synthesis of each of the particle proteins is incomplete. However, preliminary studies with lysates of TI amber mutants grown under non-permissive conditions indicate that Pz is missing from gene Io mutant infections (tail synthesis); P4 is not synthesized by mutants in two further tail genes (3 and 9); P5 is absent from gene 15 mutant lysates (head formation); P8 is not made by am 216, a mutant in a recently discovered gene of unknown function. In addition, mutations in genes IO (tail) and t3, I4 and I6 (head) all lead to absence ofP 7 and appear unable to convert the precursor protein, P7P, to P7. Am 246, another unmapped and uncharacterized mutant, makes neither P7P nor P7 and may therefore identify the structural gene for P7. As with other phage systems post infection polypeptide synthesis can only be analysed when host protein synthesis has been largely eliminated by u.v. irradiation (Levinthal, Hosoda & Shub, 1967; Murialdo & Siminovitch, ~972; Studier, ~972). Under these conditions some 40 to 5o bands were seen of which about 3o were quite distinct from the residual host polypeptides. Summation of the mol. wt. of these 3o bands indicates that the amount of protein product corresponds to an estimated 8o % of the coding potential of the TI genome, assuming that all DNA codes for polypeptides and accounting for the terminally repetitious duplication of about 6"5 % (Thomas & MacHattie, r967; MacHattie, Rhoades & Thomas i972). Extension of this calculation also suggests that the total number of TI-specified polypeptides is about 35 or even higher if the remaining proteins are of lower than average mol. wt., as is likely since the low mol. wt. regions of these gels are the least well defined. Classification of the 3I post infection bands according to their time of synthesis points to five distinguishable classes of polypeptide: Class I, synthesized only during the early stages of infection; Class II, synthesized continuously at constant rate; Class III, synthesized only during the later stages of infection; Class IV, synthesized continuously but at decreasing rate; Class V, synthesized continuously but at increasing rate. While Classes I, II and III are readily distinguishable in terms of their period of synthesis, the differences between Classes II, IV and V are quantitative and therefore less clear cut. The I6 to ~7 Class III late polypeptides, as expected, include many of the particle proteins (PI, P2, P4, P8, P9, PIo and PI I). More interestingly, the particle proteins P5 and P6 and P7P, the precursor protein of P7, are made continuously (P6 and P7P being of the late-continuous class) and must presumably respond to a regulatory stimulus different from that which turns on the late proteins. However, the function which cleaves the P7 precursor is clearly of the late class since the cleavage reaction does not start until about 6 rain after infection. The difference in gel systems used by Toni et al. 0976) and in the present work make precise comparison of the post-infection polypeptide patterns difficult. However, at the general level there are several points of agreement. Both estimates of the total number of TI-specified polypeptides give a figure of about 30. Only three early proteins have been
318
D . T . M . MARTIN AND OTHERS
detected. The band of 65 ooo mol. wt., identified tentatively as the product of gene 2 (am 22I; Fig. 2 and 4A), corresponds to the band 3 protein of Toni et al. (I976), and our early polypeptide of tool. wt. about I7OOOis probably the same as their band t5. Post translational cleavage of the major capsid protein was also observed by Toni et al. (I976). In their case polypeptide 7, a late band of 36 50o mol. wt., was converted to the 30 ooo mol. wt. V8. In the present study the precursor P7P is a late-continuous product estimated to be about 40000 mol. wt. and the product is the 3300o tool. wt. P7. By way of contrast, Toni et al. (I976) contended that there were only three classes, corresponding to Classes I, II and III of our classification. In our opinion however, proteins which are being synthesized continuously at constant, decreasing and increasing rates are likely to represent different classes, both in functional terms and in terms of possible mechanisms for the regulation of Tr-directed protein synthesis. (Of course, transcription products of different stability could provide a single control for the early, continuous and early-continuous classes.) The data so far point to a control system for TI protein synthesis broadly similar to that observed for other large DNA-containing phages in which different classes of proteins are synthesized during fixed and specific periods of the growth cycle, e.g. T4 (Levinthal et al. I967), T5 (McCorquodale, Oleson & Buchanan, I967) and T7 (Studier, I972). Infection by TI leads to the rapid inhibition of host D N A synthesis (Figurski & Christensen, I974) and within i min of infection to the appearance of the early Class I proteins and the continuous proteins (Classes II, IV and V). The time of switch-off for early proteins, which more or less coincides with the turn-on of late protein synthesis may well involve the action of the a m 280 product in which both controls appear to be affected in non-permissive cells. Unlike the situation with phage T4 (Wiberg, et al. I962), late protein synthesis is not strongly coupled to T i D N A synthesis as judged from the results with the gene 2 mutant a m 22I. Furthermore, our unpublished results from studies with rifampicin, which show a continuing requirement for the host cell RNA polymerase throughout infection, would suggest that transcription is not controlled by switching transcription from the host RNA polymerase to a completely new transcriptase as with phage T7 (Chamberlin, McGrath & Waskell, I97O), but rather that the host enzyme is modified, as apparently occurs for T4 (Wu et al. I973). We are indebted to Dr H. S. Marsden for providing the molecular weight marker polypeptides and for general advice on the techniques of polyacrylamide gel electrophoresis, and to Professor J. H. Subak-Sharpe for his comments on the manuscript.
REFERENCES ALBERTSSON, V. A. (1967). T w o phase separation o f viruses. In Methods" in Virology, vol. II, chapter Io, pp. 303-322. Edited by K . M a r a m o r o s c h , a n d H. Koprowski. N e w Y o r k : A c a d e m i c Press.
ANDERSON,E. H. (I946). Growth requirements of virus-resistant mutants of Escherichia eoli strain B. Proceedings of the National Academy of Sciences of the United States of America 32, 12o--128. CHAMBERLIN, M., McGRATH, J. & WASKELL, L. (I970). Isolation and characterisation of a n e w R N A p o l y m e r a s e f r o m E. eo/i infected with bacteriophage T7. Nature, London 228, 227-232. EAIRBANKS, ~., JUN., LEVl~qTHAL, C. & REEDER, R. H. 0965). Analyses of l~C-labelled protein by disc electrophoresis. Biochemical and Biophysical Research Communications 20, 393-399. FIGURSKI, D. H. & CI-IRlSTENSEN, J. R. (I974). Functional characterisation o f the genes of bacteriophage T I . Virology 59, 397-4o7. HERSHEY,A. O. & CHASE,M. (I952). Independent functions of viral protein and nucleic acid in growth of bacteriophage. Journal of General Physiology 36 , 39-56.
Phage T i-specified polypeptides
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LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature, London 227, 68o-685. LENGYEL, J. A., GOLDSTEIN, R. N., MARSH, M., SUNSHINE, M. G. & CALENDAR, R. (I973). Bacteriophage P2 head morphogenesis: cleavage of the major capsid protein. Virology 53, 1-23. LEVINTHAL, C., HOSODA, J. & SHUB, D. (I967). Control of protein synthesis after phage infection. In The Molecular Biology of Viruses, pp. 71-87. Edited by J. S. CoRer & W. Paranchych. New York: Academic Press. MACHATTIE, L. A., RHOADES, M. & THOMAS, C. A., JUN. (I972). Large repetition in the non-permuted nucleotide sequence of bacteriophage TI DNA. Journal of Molecular Biology 72, 645-656. MAIZEL, S. V., JUN. (1969)- Acrylamide gel electrophoresis of proteins and nucleic acids. In Fundamental Techniques in Virology, chapter 32, pp. 334-362. Edited by K. Habel and N. P. Salzman. New York: Academic Press. MARSDEN, H. S., CROMBIE, I. K. & SUBAK-SHARPE, J. H. (1976)- Control of protein synthesis in herpesvirusinfected cells: analysis of the polypeptides induced by wild type and. sixteen temperature sensitive mutants of HSV strain 17. Journal o f General Virology 31, 347-372. MATTHEWS, R. E. F. (I960). Properties of nucleoprotein fractions isolated from turnip yellow mosaic virus preparations. Virology x2, 52t-539. McCORQUODALE, D. J., OLESON, A. E. & BUCHANAN, J. M. (I967). Control of virus-induced enzyme synthesis in bacteria. In The Molecular Biology of Viruses, pp. 31-54. Edited by J. S. Colter and W. Paranchych. New York: Academic Press. MICHALKE, W. (1967). Erhohte Rekombinationshaufigeit an den Enden des TI-Chromosoms. Molecular and General Genetics 99, I2-23. MURIALDO, H. & SIMINOVlTCH,L. (1972). Morphogenesis of bacteriophage lambda. IV. Identification of gene products and control of the expression of the morphogenetic information. Virology 48, 785-823. STENT, G. S. (1963). In Molecular Biology of Bacterial Viruses, chapter 5, P. IOO. San Francisco and London: W. H. Freeman and Company. STUDIER, ~. W. (1972). Bacteriophage T7. Science, New York I76, 367-376. THOMAS, C. A., JUN. & MACHATTIE,L. A. (I967). The anatomy of viral DNA molecules. In Annual Review of Biochemistry, pp. 485-518. Edited by P. D. Boyer. Palo Alto: Annual Reviews Inc. TONI, M., CONT1, ~. & SCHITO, O. C. (1976). Viral protein synthesis during replication of bacteriophage TI. Biochemical and Biophysical Research Communications 68, 545-552. W'IBERG, J. S., DIRKSEN, M.-L., EPSTEIN, R. H., LURIA, S. E. & BUCHANAN, J. M. (1962). Early enzyme synthesis and its control in E. eoli infected with some amber mutants of bacteriophage T4. Proceedings of the National Academy of Sciences of the United States of America 48, 293-3o2. WU, R., GEIDUSCHEK, E. V., RABUSSAY, D. & CASCINO, A. (I973). Regulation of transcription in bacteriophage T4-infected 17. coB- A brief review of some recent results. In Virus Research, pp. 181-2o4. Edited by C. R. Fox and W. S. Robinson. New York: Academic Press. ZWEI6, M. & CUMMENGS,19. J. (I973). Cleavage of head and tail proteins during bacteriophage T5 assembly: selective host involvement in the cleavage of a tail protein. Journal of Molecular Biology 80, 5o5-518.
(Received IZ April I976)
