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The process of infection with coliphage T7
J. Mol. Biol. (1970) 49, 116-123
The Process of Infection with Coliphage T7 III.? Control of Phagespecific RNA Synthesis in viva by an Early Phage Gene RUTH B. SIEQEL AND WILUAM C. SUMMERS Radiobiology Laboratories Yale University School of Medicine New Haven, Corm. 06510, U.S.A.
(Received 20 August 1969) After infection of Escher-ichia coli with bacteriophage T7, 12 or 13 new phagespecific RNA species are synthesized. These are resolvable as discrete bands by polyacrylamide-agarose gel electrophoresis. Initiation of all T7 RNA species occurs to some extent before the onset of phage DNA synthesis. Only four of the normal T7 RNA’s are made if phage-specific protein synthesis is blocked with chloramphenicol or kanamycin and one of these RNA’s (mol. wt 1.1 x 108) is probably the messenger RNA from gene 1. RNA made after infection with T7 carrying amber mutations in gene 1 has only three discrete species of T7 messenger RNA’s. The RNA from cells infected with T7 + in the presence of 400 pg chloramphenicol/ ml. is very similar to T7 ana (gene 1) RNA as judged by electrophoretic analysis and by RNA-DNA hybridization competition experiments. On the other hand, RNA made after infection with T7 carrying amber mutations in other genes is very similar to T7 + RNA. This result parallels that reported on the control of T7 protein synthesis which showed that the gene 1 protein controlled the production of all or nearly all other T7-induced proteins. These results suggest that while gene 1 is transoribed in the absence of phage protein synthesis, the gene 1 protein is required to transcribe the remainder of the T7 genome. The control of T7 protein synthesis is therefore mediated at the level of transcription by means of positive regulation.
1. Introduction The detailed analysis of the course of bacteriophage development has shown that there is a fairly precise control of the sequence of events after infection (e.g. Levinthal, Hosoda t Shub, 1967). In several phage systems these analyses have revealed that
phage genes are responsible for this control. The Q gene in bacteriophage X seems to be required for the expression of a whole class of genes concerned with phage coat and tail production (Joyner, Isaacs, Echols & Sly, 1966; Dove, 1966). In T4 phage, the genes responsible for controlling phage DNA synthesis also control, in some way, the expression of ‘late’ phage genes (Hosoda & Levinthal, 1968; Bolle, Epstein, Salser & Geiduschek, 196&z; Mathews, 1968). Recently evidence has been reported for SPOl phage-induced proteins which act in vitro to turn off transcription of ‘early ’ phage genes (Wilson & Geiduschek, 1969) and to turn on transcription of ‘late’ genes (Geiduschek, Wilson & Gage, 1969). t Paper II in this series is Summers & Siegel, 1969. 115
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The mechanism by which a phage pre-empte host cell metabolism and sequentially activates its own genome, should give insight into the nature of positive control of genes. In phage T7 the control of gene expression seems particularly clear and amenable to experimental analysis. Recent work by Studier & Maize1 (1969) has shown that amber mutations in the first gene (gene 1) at the left end (amino-terminus) of the map seem to exert a pleiotropic effect on protein synthesis, abolishing the labeling of nearly all phage-induced protein speoies. None of the mutations in other genes caused this pleiotropic effect. All the other mutants tested showed an almost normal labeling of phage-induced proteins, with an alteration only in the protein which was coded for by the gene in which the amber lesion was located. It thus appeared that mutants in gene 1 can synthesize almost no phage-specific proteins, whereas mutants in all other genes can synthesize almost all phage-specific proteins. In the study reported here, we present evidence that the pleiotropic effect on protein synthesis of a mutation in gene 1 of T7 is exerted at the level of transcription of RNA.
2. Materials and Methods (8) Bacteria and bctdri~hage T7L was grown and purified aa previously described (Summers, 1969). The host bacteria used in this study was E. c& B, -JTl. K medium (M9 medium of Adams (1969) supplemented with 1% glucose, 1% Caeamin oecids, and 0.2% thiamin) was used in all experiments. Cultures were grown at 30% with aeration. Amber mutents of T7L and the permissive host, E. c&i Oil’, were obtained from the collection of F. W. Studier of the Brookheven National Laboratory. A stock of T7 u~niV71 WBB from R. Hauranann of the University of Freiburg. (b) RNA;extracW RNA for hybridization experiments was extracted from protoplaets made from T7infeotecl oells (Summers, 1969) &ng the hot-phenol method previously described (Summers & Szybalski, 1968). Pulse-labeled RNA for gel eleotrophoresia was prepared (Summers, 1909) from aulturea (10 ml.) to which [14C]edenine end [14C]ur&l (O-24 &ml. of each) had been added 3 min before harvesting the oells. Thiz was done by rapid chilling on ice chips in the presence of 1 m-KCN followed by centrifugetion et 0 to 2’C et 8000 g for 10 min. The cells were next converted to protoplasts and lyzed in the presence of sodium dodecyl sulfate and then extrected using e mixture of 800 g phenol, 70 g m-creaol, 0.6 g 8-hydroxyquinoline and 56 g weter (Kirby, 1965). (c) Amdytkal polyaaylamide-gel ekxtroplunv&a The method described by Summ ere (1969) wes used in this study. [14C]RNA samples were electrophoreeed on gels containing 2.6% acrylamide and O-6o/o agerose. The gels then were sliced longitudinally (F&&u&s, Levinthal & Reeder, 1965). dried under suction made. and contact r8diO8U~Ojp8phs (d) RNA-DNA Aybridizuhon competdion eapkmenta These experiments were carried out following a modification of the methods of Bolle, Epstein, Selser & Geidueohek (ISSSa). Only the r-strandt of the T7 DNA wae used in the hybridization mixture. This prevented renaturation of DNA during the hybrid&&ion reection. The complementary DNA strands were preparatively separated as previously described (Summers & Szybalski, 1968). The reaction mixture contained 1 a DNA, [‘%]RNA in excees, and various emounts of unlebeled competitor RNA, in 0.6 ml. 2 x SSC (SSC is 0.15 M-N&, 0.016 M-tri-sodium t The r-strand of T7 DNA is defined aa the DNA strand whioh is transcribed to the right with the molecule in the standard orientation having the B’pT end on the left. The r-strand binds
Q-riah polyribonuoleotides sayhelski, 19681.
and is the “heavy”
fraction in
8
C&l density-gradient
(Summere &
THE
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T7
citrate, pH 7.6). Hybridization was carried out for 5 hr at 60°C. The samples were cooled, pancreatic RNase (20 pg/ml.) was added, and the samples were incubated for 30 min at room temperature. They were diluted to 15 ml. in 3 x SSC and filtered through washed nitrocellulose filters (Schleicher & Schuell, B6, 24 mm). The alters were washed with 100 ml. 3 x SSC, dried and counted by the liquid-scintillation method.
3. Results (a) Hybridization-competition
ana$y&
T7-specific RNA made after infection with T7+ in the presence of chloramphenicol, T7 am23 (gene l), T7 amN71 (gene 5), or T7 am9 (gene 16) was compared with T7 + RNA by RNA-DNA hybridization competition experiments and by polyacrylamideTABLE 1
Characteristics of T7 mutants Phage
Phenotype
T7+ T7 cam23 T7 amiVY
wild DO DO
T7 am9
non-infectious DNA-containing particle
Cistron
Phage-directed
1 (DO-A) 6 (DO-B)
25 to 30 ape&s 3 species 24 to 29 species, 80,000 mol. wt species absent
16
proteins
Reference Studier, 1069 Studier, 1969. Hausmann & Gomez, 1967, Studier, 1909. Studier, 1909.
24 to 29 species, 170,000 mol. wt species absent
DO indicates DNA negative phenotype in which no phage-directed DNA synthesis is observed. DO-A and DO-B are designations for two different DO cistrons (Hausmann & Gomez, 1967). Patterns of phsge-directed protein synthesis as analyzed by gel electrophoresis (Studier & Maizel, 1969) are indicated.
/-strand
S’pTpC
DO DA DA DO DO DA
N
5 Fs
3 ss4
7
8
I
2
5
6
TC Fs 9
S
Tail NC NC S
N
IO
II
13
Coat
I2
14
S
S
NC S
15
16
17 I8
Fs Fs 19 Gp Ap 5’
r-stmnd 5zc----
__-------
-------
-m RNA
NH2--+
--*--*
--* Protein
Fm. 1. Genetic and molecular map of coliphage T7. The order of the 19 amber complement&ion groups is according to Studier (1969) and Studier & Hausmann (1960). DO indicates absolute blocks to DNA synthesis and DA indicates a DNA “arrested” phenotype (Studier, 1969; Hausmann & Gomez, 1967). N indicates the production of a non$nfectious particle containing DNA; NC is such a particle which can be complemented in extracts (Studier & Maizel, 1969). Fs indicates that a fast-sedimenting form of phage DNA accumulates in the infected cell (Hausmann 8ELaRue, 1960). Genes 8,10,12,15,16 and 17 code for structural proteins (8) of the mature phage particle (Studier & Maizel, 1969). “Top component” (TC) is a protein which is recovered in CsCl gradients at a density of about 1.3 g/cm3. 8s is a locus responsible for autorestriction in certain 6’higeZZa strains (Hausmann, Gomez & Moody, 1968). The orientation of mRNA transcription was shown by Summers & Szybalski (1968) and was corroborated by the study of the direction of translation of genes 1, 12, 16 and 16 by Studier & Maize1 (1969). The 6’ te rminal dinucleotides were determined by Weiss & Richardson (1967).
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gel electrophoresis. The characteristics of these amber mutants are summarized in Table 1. A schematic map of T7 is shown in Figure 1. The RNA-DNA hybridization competition experiments (Fig. 2) showed that the T7 RNA made after infection of non-permissive cells with T7 am23 (gene 1) was only a sub-class of the T7 RNA made after infection with T7 + , since unlabeled T7 am23 RNA could only dilute about 20 to 25% of the labeled, hybridizable RNA from T7 +-infected cells (Fig. 2(a)), whereas unlabeled T7 + RNA could dilute all the labeled T7-RNA species in the T7 am23-infeoted cell (Fig. 2(b)). RNA from cells infected with T7 + in the presence of chloramphenicol(4.00 pg/ml.) was very similar to T7 RNA from the T7 am23 (gene l)-infected cells, since unlabeled T7 + CM RNA? competed poorly with labeled T7 + RNA (Fig. 2(c)) whereas labeled T7 + CM RNA could be almost completely diluted by T7 am23 RNA (Fig. 2(d)). On the other hand, the RNA from T7 amN714nfected cells was similar to T7 + RNA rather than to T7 am23 RNA (Fig. 2(e) and (f)).
T7CM
A
[k), , , 0.3
0.1 I
I
I
--x-+-T--l-
T7am.?3
9
T7am23 “\
A--&
cd)
x
\ A’A :)
T7+ \A
mg Unlabeled
competitor
RNA added/ml.
FIG. 2. RNA-DNA hybridization competition analysis of T7 RNA. The amount of the [‘*C]RNA which hybridized with a limiting amount of the r-strand of T7 DNA (1 to 3 pg) ia plotted wraua the concentration (mg/ml.) of added unlabeled “competitor” RNA in the hybridization mixture. Hybridization efficiency without added unlabeled RNA was between 20 and 30%. The results were normalized using this value ae 100%. The labeled RNA species is indioated along the vertical axis; the “oompetitor” species is indicated adjacent to the experimental points.
t Abbreviations used: T7 + CM RNA, RNA from cells infeoted with T7 wild type in the presence of 400 pg chlorampheniool/ml. ; mRNA, messenger RNA.
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+
FIG. 3. Polyacrylemide-gel electrophoresis of T7 [rV]RNA’s. Microdensitometer tracings are of contact radioautographs of longitudinally shoed, dried gels. RNA from T7+-infected cells labeled 6 to 9 min after infection (30°C) is shown in top left trace. RNA’s from cells infected and labeled under the same oonditions 8re 81~0 shown: center left trace, cells tre8ted with ohloramphenicol (400 pg/ml.) 8nd infected with T7+; bottom left trace, cells infected with T7 am23 (gene 1) ; top right trece, cells infected with T7 amN71 (gene 6) ; bottom right trace, cells infected with T7 am9 (gene 16). Tracings were sligned by position of 23 s ribosomal RNA marker run in 8 parallel gel in which the markers and the T7 RNA were mixed before eleotrophoresis. The position of the precursor (~16 s) to the 16 s ribosomd RNA is also indicated. Distortions of the gel slsbs can occur during the slioing and drying process which accounts for the slight variability in distanoes between the markers on different gels. Some radioactivity ocetLsionelly is trapped at the top of the gel and gives the origin srtifsct labeled “0” on some of the tracings. Estimated mol. wts ( x 10-s) of some phage RNA% are also included.
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(b) Gel ekctrophoresis of T7 RNA’s Analysis of the various T7 RNA’s by polyecrylamide-gel eledrophoresis (Summers, 1969) corroborated the results of the RNA-DNA hybridization experiments. Only three of the 12 to 13 normal T7 messenger RNA species were present in T7 am23 (gene 1) RNA and only four in T7 + CM RNA (Fig. 3). These two samples completely lacked the T7 RNA’s of molecular weight greater than about 1 x 106. However, T7 amN71 (gene 5) RNA and T7 am9 (gene 16) RNA were similar to the wild-type RNA. In cells infected with T7 am23 (gene l), T7 umN72 (gene 5) or T7 + CM, there was labeling of host, i.e. ribosomal RNA. In the T7 + CM RNA there was also an additional new T7 RNA species, which was not seen under normal conditions (molecular weight about 0.35 x 106). This band, and three others in T7 + CM RNA, were apparently phage-induced because they were labeled in proportion to the amount of T7-specific RNA in the sample, which could be increased from 5 to 40% by changing the multiplicity of infection of the chloramphenicol-treated cells from 3 to 40. In addition, this same new RNA species appeared after infection of cells in which protein synthesis was inhibited by 490 pg kanamycin/ml. (Weisblum & Davies, 1968). 4. Discussion In their elegant study of the control of phage-protein synthesis in T7-infected cells, Studier & Maize1 (1969) demonstrated that only mutations in gene 1 affect the synthesis of the protein products of the other T7 genes. By analysis of T7-induced proteins on sodium dodecyl sulfate polyacrylsmide gels, they showed that mutations in genes 2 through 19 either had no observable effect, or else deleted only one of the 25 to 30 protein species made in T7 +-infected cells, On the other hand, an amber mutation in gene 1 had a pleiotropic effect on T7 protein synthesis since only two of the usual T7 proteins were made in the absence of a functional gene 1 protein. The results of the present study show that the control of T7-protein synthesis observed by Studier & Maize1 (1969) is in concert with the control of T7 mRNA synthesis and occurs, therefore, at the level of transcription. The RNA made in the absence of phage-induced protein synthesis (i.e. in chloramphenicol) or after infection with T7 am 23 (gene 1) contains only a few of the usual T7 mRNA’s. On the other hand, mutations in gene 5 (amN71) and gene 16 (um9) are not pleiotropic in their effect on T7 RNA synthesis. The RNA species made in T7 am23-infected cells are most easily interpreted. Host RNA synthesis is not turned off completely after infection with this mutant, so the 16 s and 23 s host-ribosomal RNA’s are labeled as well as three phage-specific RNA’s. The approximate size of these RNA’s can be estimated to be 0.45 x 106, O-29 x 106, and O-20 x 106, from log molecular weight versus migration plots with the host ribosomal RNA’s as intern&l markers. Two of the RNA’s may correspond to the two phage proteins of the approximate molecular weights 37,000, and 10,000 to 15,000 seen in T7 am23-infected cells by Studier & Maize1 (1969). One of the smaller RNA’s could code for a small protein which could have been overlooked in their gel system, which was designed to resolve larger proteins. There was no T7 RNA species observed that was large enough (about 1-O x lo6 molecular weight) to be a completed mRNA for the gene 1 protein (100,000 molecular weight). The amber mutation may result in a truncated mRNA molecule as in the case of the mRNA for the tryptophan operon (Imamoto & Yanofsky, 1967).
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The am23 mutation maps very close to the amino-terminal end of gene 1 (Studier, 1969) and may produce an mRNA fragment too small to be observed in the usual electrophoretic analysis. Therefore, the mutations am94 and am342, which map in the middle and near the carboxyl-terminus, respectively, of gene 1 (Studier, 1969), were also examined for discrete classes of truncated mRNA. No short mRNA’s of a uniform size (‘bands’ on gel) were observed except the 0*45,0*29, and 0.20 x lo6 molecular weight species. The absence of a band may be the result of non-specific termination of mRNA synthesis in the region of an amber mutation which would generate a heterogeneous population of mRNA’s which would not form a discrete zone during electrophoresis. Alternatively, the incomplete mRNA may undergo an abnormally rapid degradation in the cell as a result of premature termination of translation. The fact that discrete species of T7 RNA are seen on the gels, means that completed RNA molecules can accumulate to high levels in the cell before even one-strand cleavage. In the presence of 400 pg of chloramphenicol/ml., protein synthesis ( [3H]leucine incorporation) was inhibited by about 95% in T7 +-infected cells. Phage-specific RNA synthesis was also greatly depressed, while host RNA synthesis was relatively unaffected. In addition to the three T7 RNA’s present in T7 am23 (gene 1) RNA, the T7 + CM RNA contains a new RNA species of approximately 0.35 x log molecular weight (Fig. 2). This species is not normally present in T7 + RNA and its appearance in chloramphenicol-treated, T7 +-infected cells is not understood. This new species may be the result of either premature termination, or initiation at a new site induced by inhibitors of protein synthesis such as chloramphenicol or kanamycin. Chloramphenicol treatment has been reported to relieve polar blocks in the transcription of the luc operon (Contease & Gros, 1968). This observation suggests the hypothesis that chloramphenicol somehow gives rise to a new initiation site for mRNA synthesis. The T7 + CM RNA also contains a T7 mRNA of about 1-l x lo6 molecular weight. This species is the same size as one of those found in T7 +-infected cells and most probably represents the mRNA for the gene 1 protein. The RNA synthesized in the presence of chloramphenicol should resemble that made by a gene 1 mutant except that the gene 1 messenger should also be present. Since protein synthesis is blocked by chloramphenicol and no gene 1 protein can be synthesized, the rest of the normal T7 RNA’s will not be made. In the absence of a functional gene 1 product, only three T7 polypeptides were found by Studier & Maize1 (1969) (10,000 mol. wt 37,000 mol. wt and amber peptides of length dependent on the location of the amber mutation). They estimate that the molecular weight of the gene 1 protein is about 100,000 which requires an mRNA of at least 1 x lo6 molecular weight. It is likely that the 1.1 x lo6 molecular weight RNA species is the mRNA for gene 1, since it is the only mRNA that is large enough in T7 + CM RNA. This implies that gene 1 is transcribed monooistronically, since the messenger is of minimum size. The possibility that mutations in gene 1 control transcription by a polar effect on an adjacent gene is rendered unlikely by this observation. The T7 RNA species made in T7 amN71 (gene 5)- and T7 am9 (gene 16)~infected cells are similar to the T7 + RNA’s. This is reasonable since these mutant phages direct the synthesis of all but one of the normal T7-directed proteins, i.e. the protein coded by the gene in which the amber codon is located (Studier t Maize& 1969). Labeling of host RNA was occasionally present following infection by T7 amN71 and was probably the result of some uninfected cells in the culture. This mutant has been shown to break down host DNA as does T7 + (Hausmann & Gomez, 1967), so the host template should
R. B. SIEGEL
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no longer be available in infected cells five to seven minutes after infection. However, the time required for complete breakdown of host DNA may be longer for this mutant. It is interesting to note that T7 amN71 (gene 5) is a DO phenotype (defective in phage-induced DNA synthesis), yet it is capable of directing nearly normal T7 RNA production. This independence of late phage functions from DNA replication in T7 has been noted previously (Hausmann & Comez, 1967; Studier & Maizel, 1969) and is in contrast to late transcription in T4 (Boll8 et aE., 1968b), in h (Joyner et al., 1966) and in &&%Ls s&i& phage 2C (Pene & Marnun, 1967). Studier & Maize1 (1969) suggested that the gene 1 protein might function to alter the host RNA polymerase in such a way as to permit transcription of the remaining T7 genes. Since gene 1 has an approximate molecular weight of 100,000 (Studier Bc Maizel, 1969), they suggested that it might function in a manner analogous to the sigma specificity subunit (96,000 molecular weight) of E. coli RNA polymerase (Burgess, Travers, Dunn & Bautz, 1969) and change the initiation speci6city of the catalytic portion of the enzyme. Recent evidence supports this model for positive control by the gene 1 product of T7 transcription in vitro (Summers & Siegel, 1969). After infection with T7 + , both host protein synthesis (Studier & Maizel, 1969) and host RNA synthesis (Summers & Szybalski, 1968; Summers, 1969) gradually cease. This has been attributed to the degradation of host DNA by a T7-induced DNase (Hausmann & Gomez, 1968). An alternative explanation might be that when the host RNA polymer&se is modified by the substitution of the gene 1 protein for the normal host specificity factor, the host DNA can no longer be transcribed. The fact that hoat RNA synthesis can continue after infection by T7 carrying amber mutants in gene 1 is not unexpected because the gene 1 amber peptides may not be able to displace the sigma factor from host polymerase. Indeed, recent experiments (Summers, unpublished results) with T7 harboring a thermosensitive mutation in gene 1 suggest that host fl;NA synthesis is inhibited at the non-permissive temperature despite absence of a functional gene 1 protein. Perhaps the complete, thermosensitive-gene 1 protein can displace the normal host sigma factor and thereby prevent transcription of the host DNA while also rendering the modified enzyme thermosensitive in its ability to transcribe T7 RNA (Summers & Siegel, 1969). If this hypothesis is valid, it would be an interesting example of linkage of positive and negative regulation of gene function. The experiments presented here show that gene 1 of T7 exerts a positive control over the transcription of the phage genome in vivo. Phage-directed protein synthesis is required for shut-off of host RNA synthesis. Whether or not this shut-off is also mediated by the gene 1 product is not yet known. We thank Dr Studier and Dr Hausmann for giving us strains of bacteriophage. Dr Studier also has generously communicated bitr results before public&ion. This work was e~ppoti by U.S. Public Health Service grant no. CAO6619. REFERENCES p. 446. New York: Interscience. Adams, M. H., (1969). Bacteriophagea, Belle, A., Epstein, R. H., Salser, W. & Geiduschek, E. P. (1963u). J. Mol. Biol. 33, 339. Belle, A., Epstein, R. H., Salser, W. & Geiduschek, E. P. (19683). J. Mol. Biol. 31, 326. Burgess, R. R., !&avers, A. A., Dunn, J. J. & Bautz, E. K. F. (1969). Natare, 221, 43. Cm&we, G. L%Gros, F. (1968). C. R. Acad. Sci. Pa&, 266D, 263. Dove, W. F. (1966). J. Mol. Biol. IQ, 187. Fairbanks, G., Jr., Levintbel, C. & Reeder, R. H. (1966). Biochem. B&o&p. Rec. Cm. 20, 393.
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Geiduschek, E. P., Wilson, D. L. & Gage, L. P. (1969). J. CeZZ Physiol. (Suppl. 1) 74, 81. Hausmann, R. & Gomez, B. (1967). J. Viral. 1, 779. Hausmann, R. & Gomez, B. (1968). J. VifoZ. 2, 266. Hausmann, R., Gomez, B. t Moody, B. (1968). J. Viral. 2, 335. Hausmtum, R., t LaRue, K. (1969). J. VGoZ. 3, 278. Hosoda, J. & Levinthal, C. (1968). Virology, 34, 709. Imamoto, F. & Yanofsky, C. (1967). J. Mol. Biol. 28, 25. Joyner, A., Isaacs, L. N., Echols, H. &z Sly, W. S. (1966). J. Mol. BioZ. 19, 174. Kirby, K. S. (1966). Biochem. J. 96, 266. Levinthal, C., Hosoda, J. & Shub, D. (1967). In The MoZecuZar Biology of Viruses, ed. by J. S. Colter & W. Paranchych, p. 71. New York: Academic Press. Mathews, C. K. (1968). J. BioZ. Chem. 243,561O. Pene, J. J. & Marmur, J. (1967). J. VG-OZ. 1, 86. Studier, F. W. (1969). Virology, 39, 562. Studier, F. W. t Hausmann, R. (1969). Virology, 39, 587. Studier, F. W. & Maize& J. V., Jr.. (1969). Virology, 39, 575. Summers, W. C. (1969). V
The Process of Infection with Coliphage T7 III.? Control of Phagespecific RNA Synthesis in viva by an Early Phage Gene RUTH B. SIEQEL AND WILUAM C. SUMMERS Radiobiology Laboratories Yale University School of Medicine New Haven, Corm. 06510, U.S.A.
(Received 20 August 1969) After infection of Escher-ichia coli with bacteriophage T7, 12 or 13 new phagespecific RNA species are synthesized. These are resolvable as discrete bands by polyacrylamide-agarose gel electrophoresis. Initiation of all T7 RNA species occurs to some extent before the onset of phage DNA synthesis. Only four of the normal T7 RNA’s are made if phage-specific protein synthesis is blocked with chloramphenicol or kanamycin and one of these RNA’s (mol. wt 1.1 x 108) is probably the messenger RNA from gene 1. RNA made after infection with T7 carrying amber mutations in gene 1 has only three discrete species of T7 messenger RNA’s. The RNA from cells infected with T7 + in the presence of 400 pg chloramphenicol/ ml. is very similar to T7 ana (gene 1) RNA as judged by electrophoretic analysis and by RNA-DNA hybridization competition experiments. On the other hand, RNA made after infection with T7 carrying amber mutations in other genes is very similar to T7 + RNA. This result parallels that reported on the control of T7 protein synthesis which showed that the gene 1 protein controlled the production of all or nearly all other T7-induced proteins. These results suggest that while gene 1 is transoribed in the absence of phage protein synthesis, the gene 1 protein is required to transcribe the remainder of the T7 genome. The control of T7 protein synthesis is therefore mediated at the level of transcription by means of positive regulation.
1. Introduction The detailed analysis of the course of bacteriophage development has shown that there is a fairly precise control of the sequence of events after infection (e.g. Levinthal, Hosoda t Shub, 1967). In several phage systems these analyses have revealed that
phage genes are responsible for this control. The Q gene in bacteriophage X seems to be required for the expression of a whole class of genes concerned with phage coat and tail production (Joyner, Isaacs, Echols & Sly, 1966; Dove, 1966). In T4 phage, the genes responsible for controlling phage DNA synthesis also control, in some way, the expression of ‘late’ phage genes (Hosoda & Levinthal, 1968; Bolle, Epstein, Salser & Geiduschek, 196&z; Mathews, 1968). Recently evidence has been reported for SPOl phage-induced proteins which act in vitro to turn off transcription of ‘early ’ phage genes (Wilson & Geiduschek, 1969) and to turn on transcription of ‘late’ genes (Geiduschek, Wilson & Gage, 1969). t Paper II in this series is Summers & Siegel, 1969. 115
116
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W. C. SUMMERS
The mechanism by which a phage pre-empte host cell metabolism and sequentially activates its own genome, should give insight into the nature of positive control of genes. In phage T7 the control of gene expression seems particularly clear and amenable to experimental analysis. Recent work by Studier & Maize1 (1969) has shown that amber mutations in the first gene (gene 1) at the left end (amino-terminus) of the map seem to exert a pleiotropic effect on protein synthesis, abolishing the labeling of nearly all phage-induced protein speoies. None of the mutations in other genes caused this pleiotropic effect. All the other mutants tested showed an almost normal labeling of phage-induced proteins, with an alteration only in the protein which was coded for by the gene in which the amber lesion was located. It thus appeared that mutants in gene 1 can synthesize almost no phage-specific proteins, whereas mutants in all other genes can synthesize almost all phage-specific proteins. In the study reported here, we present evidence that the pleiotropic effect on protein synthesis of a mutation in gene 1 of T7 is exerted at the level of transcription of RNA.
2. Materials and Methods (8) Bacteria and bctdri~hage T7L was grown and purified aa previously described (Summers, 1969). The host bacteria used in this study was E. c& B, -JTl. K medium (M9 medium of Adams (1969) supplemented with 1% glucose, 1% Caeamin oecids, and 0.2% thiamin) was used in all experiments. Cultures were grown at 30% with aeration. Amber mutents of T7L and the permissive host, E. c&i Oil’, were obtained from the collection of F. W. Studier of the Brookheven National Laboratory. A stock of T7 u~niV71 WBB from R. Hauranann of the University of Freiburg. (b) RNA;extracW RNA for hybridization experiments was extracted from protoplaets made from T7infeotecl oells (Summers, 1969) &ng the hot-phenol method previously described (Summers & Szybalski, 1968). Pulse-labeled RNA for gel eleotrophoresia was prepared (Summers, 1909) from aulturea (10 ml.) to which [14C]edenine end [14C]ur&l (O-24 &ml. of each) had been added 3 min before harvesting the oells. Thiz was done by rapid chilling on ice chips in the presence of 1 m-KCN followed by centrifugetion et 0 to 2’C et 8000 g for 10 min. The cells were next converted to protoplasts and lyzed in the presence of sodium dodecyl sulfate and then extrected using e mixture of 800 g phenol, 70 g m-creaol, 0.6 g 8-hydroxyquinoline and 56 g weter (Kirby, 1965). (c) Amdytkal polyaaylamide-gel ekxtroplunv&a The method described by Summ ere (1969) wes used in this study. [14C]RNA samples were electrophoreeed on gels containing 2.6% acrylamide and O-6o/o agerose. The gels then were sliced longitudinally (F&&u&s, Levinthal & Reeder, 1965). dried under suction made. and contact r8diO8U~Ojp8phs (d) RNA-DNA Aybridizuhon competdion eapkmenta These experiments were carried out following a modification of the methods of Bolle, Epstein, Selser & Geidueohek (ISSSa). Only the r-strandt of the T7 DNA wae used in the hybridization mixture. This prevented renaturation of DNA during the hybrid&&ion reection. The complementary DNA strands were preparatively separated as previously described (Summers & Szybalski, 1968). The reaction mixture contained 1 a DNA, [‘%]RNA in excees, and various emounts of unlebeled competitor RNA, in 0.6 ml. 2 x SSC (SSC is 0.15 M-N&, 0.016 M-tri-sodium t The r-strand of T7 DNA is defined aa the DNA strand whioh is transcribed to the right with the molecule in the standard orientation having the B’pT end on the left. The r-strand binds
Q-riah polyribonuoleotides sayhelski, 19681.
and is the “heavy”
fraction in
8
C&l density-gradient
(Summere &
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T7
citrate, pH 7.6). Hybridization was carried out for 5 hr at 60°C. The samples were cooled, pancreatic RNase (20 pg/ml.) was added, and the samples were incubated for 30 min at room temperature. They were diluted to 15 ml. in 3 x SSC and filtered through washed nitrocellulose filters (Schleicher & Schuell, B6, 24 mm). The alters were washed with 100 ml. 3 x SSC, dried and counted by the liquid-scintillation method.
3. Results (a) Hybridization-competition
ana$y&
T7-specific RNA made after infection with T7+ in the presence of chloramphenicol, T7 am23 (gene l), T7 amN71 (gene 5), or T7 am9 (gene 16) was compared with T7 + RNA by RNA-DNA hybridization competition experiments and by polyacrylamideTABLE 1
Characteristics of T7 mutants Phage
Phenotype
T7+ T7 cam23 T7 amiVY
wild DO DO
T7 am9
non-infectious DNA-containing particle
Cistron
Phage-directed
1 (DO-A) 6 (DO-B)
25 to 30 ape&s 3 species 24 to 29 species, 80,000 mol. wt species absent
16
proteins
Reference Studier, 1069 Studier, 1969. Hausmann & Gomez, 1967, Studier, 1909. Studier, 1909.
24 to 29 species, 170,000 mol. wt species absent
DO indicates DNA negative phenotype in which no phage-directed DNA synthesis is observed. DO-A and DO-B are designations for two different DO cistrons (Hausmann & Gomez, 1967). Patterns of phsge-directed protein synthesis as analyzed by gel electrophoresis (Studier & Maizel, 1969) are indicated.
/-strand
S’pTpC
DO DA DA DO DO DA
N
5 Fs
3 ss4
7
8
I
2
5
6
TC Fs 9
S
Tail NC NC S
N
IO
II
13
Coat
I2
14
S
S
NC S
15
16
17 I8
Fs Fs 19 Gp Ap 5’
r-stmnd 5zc----
__-------
-------
-m RNA
NH2--+
--*--*
--* Protein
Fm. 1. Genetic and molecular map of coliphage T7. The order of the 19 amber complement&ion groups is according to Studier (1969) and Studier & Hausmann (1960). DO indicates absolute blocks to DNA synthesis and DA indicates a DNA “arrested” phenotype (Studier, 1969; Hausmann & Gomez, 1967). N indicates the production of a non$nfectious particle containing DNA; NC is such a particle which can be complemented in extracts (Studier & Maizel, 1969). Fs indicates that a fast-sedimenting form of phage DNA accumulates in the infected cell (Hausmann 8ELaRue, 1960). Genes 8,10,12,15,16 and 17 code for structural proteins (8) of the mature phage particle (Studier & Maizel, 1969). “Top component” (TC) is a protein which is recovered in CsCl gradients at a density of about 1.3 g/cm3. 8s is a locus responsible for autorestriction in certain 6’higeZZa strains (Hausmann, Gomez & Moody, 1968). The orientation of mRNA transcription was shown by Summers & Szybalski (1968) and was corroborated by the study of the direction of translation of genes 1, 12, 16 and 16 by Studier & Maize1 (1969). The 6’ te rminal dinucleotides were determined by Weiss & Richardson (1967).
118
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C. SUMMERS
gel electrophoresis. The characteristics of these amber mutants are summarized in Table 1. A schematic map of T7 is shown in Figure 1. The RNA-DNA hybridization competition experiments (Fig. 2) showed that the T7 RNA made after infection of non-permissive cells with T7 am23 (gene 1) was only a sub-class of the T7 RNA made after infection with T7 + , since unlabeled T7 am23 RNA could only dilute about 20 to 25% of the labeled, hybridizable RNA from T7 +-infected cells (Fig. 2(a)), whereas unlabeled T7 + RNA could dilute all the labeled T7-RNA species in the T7 am23-infeoted cell (Fig. 2(b)). RNA from cells infected with T7 + in the presence of chloramphenicol(4.00 pg/ml.) was very similar to T7 RNA from the T7 am23 (gene l)-infected cells, since unlabeled T7 + CM RNA? competed poorly with labeled T7 + RNA (Fig. 2(c)) whereas labeled T7 + CM RNA could be almost completely diluted by T7 am23 RNA (Fig. 2(d)). On the other hand, the RNA from T7 amN714nfected cells was similar to T7 + RNA rather than to T7 am23 RNA (Fig. 2(e) and (f)).
T7CM
A
[k), , , 0.3
0.1 I
I
I
--x-+-T--l-
T7am.?3
9
T7am23 “\
A--&
cd)
x
\ A’A :)
T7+ \A
mg Unlabeled
competitor
RNA added/ml.
FIG. 2. RNA-DNA hybridization competition analysis of T7 RNA. The amount of the [‘*C]RNA which hybridized with a limiting amount of the r-strand of T7 DNA (1 to 3 pg) ia plotted wraua the concentration (mg/ml.) of added unlabeled “competitor” RNA in the hybridization mixture. Hybridization efficiency without added unlabeled RNA was between 20 and 30%. The results were normalized using this value ae 100%. The labeled RNA species is indioated along the vertical axis; the “oompetitor” species is indicated adjacent to the experimental points.
t Abbreviations used: T7 + CM RNA, RNA from cells infeoted with T7 wild type in the presence of 400 pg chlorampheniool/ml. ; mRNA, messenger RNA.
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+
FIG. 3. Polyacrylemide-gel electrophoresis of T7 [rV]RNA’s. Microdensitometer tracings are of contact radioautographs of longitudinally shoed, dried gels. RNA from T7+-infected cells labeled 6 to 9 min after infection (30°C) is shown in top left trace. RNA’s from cells infected and labeled under the same oonditions 8re 81~0 shown: center left trace, cells tre8ted with ohloramphenicol (400 pg/ml.) 8nd infected with T7+; bottom left trace, cells infected with T7 am23 (gene 1) ; top right trece, cells infected with T7 amN71 (gene 6) ; bottom right trace, cells infected with T7 am9 (gene 16). Tracings were sligned by position of 23 s ribosomal RNA marker run in 8 parallel gel in which the markers and the T7 RNA were mixed before eleotrophoresis. The position of the precursor (~16 s) to the 16 s ribosomd RNA is also indicated. Distortions of the gel slsbs can occur during the slioing and drying process which accounts for the slight variability in distanoes between the markers on different gels. Some radioactivity ocetLsionelly is trapped at the top of the gel and gives the origin srtifsct labeled “0” on some of the tracings. Estimated mol. wts ( x 10-s) of some phage RNA% are also included.
120
R. B. SIEGEL
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(b) Gel ekctrophoresis of T7 RNA’s Analysis of the various T7 RNA’s by polyecrylamide-gel eledrophoresis (Summers, 1969) corroborated the results of the RNA-DNA hybridization experiments. Only three of the 12 to 13 normal T7 messenger RNA species were present in T7 am23 (gene 1) RNA and only four in T7 + CM RNA (Fig. 3). These two samples completely lacked the T7 RNA’s of molecular weight greater than about 1 x 106. However, T7 amN71 (gene 5) RNA and T7 am9 (gene 16) RNA were similar to the wild-type RNA. In cells infected with T7 am23 (gene l), T7 umN72 (gene 5) or T7 + CM, there was labeling of host, i.e. ribosomal RNA. In the T7 + CM RNA there was also an additional new T7 RNA species, which was not seen under normal conditions (molecular weight about 0.35 x 106). This band, and three others in T7 + CM RNA, were apparently phage-induced because they were labeled in proportion to the amount of T7-specific RNA in the sample, which could be increased from 5 to 40% by changing the multiplicity of infection of the chloramphenicol-treated cells from 3 to 40. In addition, this same new RNA species appeared after infection of cells in which protein synthesis was inhibited by 490 pg kanamycin/ml. (Weisblum & Davies, 1968). 4. Discussion In their elegant study of the control of phage-protein synthesis in T7-infected cells, Studier & Maize1 (1969) demonstrated that only mutations in gene 1 affect the synthesis of the protein products of the other T7 genes. By analysis of T7-induced proteins on sodium dodecyl sulfate polyacrylsmide gels, they showed that mutations in genes 2 through 19 either had no observable effect, or else deleted only one of the 25 to 30 protein species made in T7 +-infected cells, On the other hand, an amber mutation in gene 1 had a pleiotropic effect on T7 protein synthesis since only two of the usual T7 proteins were made in the absence of a functional gene 1 protein. The results of the present study show that the control of T7-protein synthesis observed by Studier & Maize1 (1969) is in concert with the control of T7 mRNA synthesis and occurs, therefore, at the level of transcription. The RNA made in the absence of phage-induced protein synthesis (i.e. in chloramphenicol) or after infection with T7 am 23 (gene 1) contains only a few of the usual T7 mRNA’s. On the other hand, mutations in gene 5 (amN71) and gene 16 (um9) are not pleiotropic in their effect on T7 RNA synthesis. The RNA species made in T7 am23-infected cells are most easily interpreted. Host RNA synthesis is not turned off completely after infection with this mutant, so the 16 s and 23 s host-ribosomal RNA’s are labeled as well as three phage-specific RNA’s. The approximate size of these RNA’s can be estimated to be 0.45 x 106, O-29 x 106, and O-20 x 106, from log molecular weight versus migration plots with the host ribosomal RNA’s as intern&l markers. Two of the RNA’s may correspond to the two phage proteins of the approximate molecular weights 37,000, and 10,000 to 15,000 seen in T7 am23-infected cells by Studier & Maize1 (1969). One of the smaller RNA’s could code for a small protein which could have been overlooked in their gel system, which was designed to resolve larger proteins. There was no T7 RNA species observed that was large enough (about 1-O x lo6 molecular weight) to be a completed mRNA for the gene 1 protein (100,000 molecular weight). The amber mutation may result in a truncated mRNA molecule as in the case of the mRNA for the tryptophan operon (Imamoto & Yanofsky, 1967).
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The am23 mutation maps very close to the amino-terminal end of gene 1 (Studier, 1969) and may produce an mRNA fragment too small to be observed in the usual electrophoretic analysis. Therefore, the mutations am94 and am342, which map in the middle and near the carboxyl-terminus, respectively, of gene 1 (Studier, 1969), were also examined for discrete classes of truncated mRNA. No short mRNA’s of a uniform size (‘bands’ on gel) were observed except the 0*45,0*29, and 0.20 x lo6 molecular weight species. The absence of a band may be the result of non-specific termination of mRNA synthesis in the region of an amber mutation which would generate a heterogeneous population of mRNA’s which would not form a discrete zone during electrophoresis. Alternatively, the incomplete mRNA may undergo an abnormally rapid degradation in the cell as a result of premature termination of translation. The fact that discrete species of T7 RNA are seen on the gels, means that completed RNA molecules can accumulate to high levels in the cell before even one-strand cleavage. In the presence of 400 pg of chloramphenicol/ml., protein synthesis ( [3H]leucine incorporation) was inhibited by about 95% in T7 +-infected cells. Phage-specific RNA synthesis was also greatly depressed, while host RNA synthesis was relatively unaffected. In addition to the three T7 RNA’s present in T7 am23 (gene 1) RNA, the T7 + CM RNA contains a new RNA species of approximately 0.35 x log molecular weight (Fig. 2). This species is not normally present in T7 + RNA and its appearance in chloramphenicol-treated, T7 +-infected cells is not understood. This new species may be the result of either premature termination, or initiation at a new site induced by inhibitors of protein synthesis such as chloramphenicol or kanamycin. Chloramphenicol treatment has been reported to relieve polar blocks in the transcription of the luc operon (Contease & Gros, 1968). This observation suggests the hypothesis that chloramphenicol somehow gives rise to a new initiation site for mRNA synthesis. The T7 + CM RNA also contains a T7 mRNA of about 1-l x lo6 molecular weight. This species is the same size as one of those found in T7 +-infected cells and most probably represents the mRNA for the gene 1 protein. The RNA synthesized in the presence of chloramphenicol should resemble that made by a gene 1 mutant except that the gene 1 messenger should also be present. Since protein synthesis is blocked by chloramphenicol and no gene 1 protein can be synthesized, the rest of the normal T7 RNA’s will not be made. In the absence of a functional gene 1 product, only three T7 polypeptides were found by Studier & Maize1 (1969) (10,000 mol. wt 37,000 mol. wt and amber peptides of length dependent on the location of the amber mutation). They estimate that the molecular weight of the gene 1 protein is about 100,000 which requires an mRNA of at least 1 x lo6 molecular weight. It is likely that the 1.1 x lo6 molecular weight RNA species is the mRNA for gene 1, since it is the only mRNA that is large enough in T7 + CM RNA. This implies that gene 1 is transcribed monooistronically, since the messenger is of minimum size. The possibility that mutations in gene 1 control transcription by a polar effect on an adjacent gene is rendered unlikely by this observation. The T7 RNA species made in T7 amN71 (gene 5)- and T7 am9 (gene 16)~infected cells are similar to the T7 + RNA’s. This is reasonable since these mutant phages direct the synthesis of all but one of the normal T7-directed proteins, i.e. the protein coded by the gene in which the amber codon is located (Studier t Maize& 1969). Labeling of host RNA was occasionally present following infection by T7 amN71 and was probably the result of some uninfected cells in the culture. This mutant has been shown to break down host DNA as does T7 + (Hausmann & Gomez, 1967), so the host template should
R. B. SIEGEL
122
AND
W. C. SUMMERS
no longer be available in infected cells five to seven minutes after infection. However, the time required for complete breakdown of host DNA may be longer for this mutant. It is interesting to note that T7 amN71 (gene 5) is a DO phenotype (defective in phage-induced DNA synthesis), yet it is capable of directing nearly normal T7 RNA production. This independence of late phage functions from DNA replication in T7 has been noted previously (Hausmann & Comez, 1967; Studier & Maizel, 1969) and is in contrast to late transcription in T4 (Boll8 et aE., 1968b), in h (Joyner et al., 1966) and in &&%Ls s&i& phage 2C (Pene & Marnun, 1967). Studier & Maize1 (1969) suggested that the gene 1 protein might function to alter the host RNA polymerase in such a way as to permit transcription of the remaining T7 genes. Since gene 1 has an approximate molecular weight of 100,000 (Studier Bc Maizel, 1969), they suggested that it might function in a manner analogous to the sigma specificity subunit (96,000 molecular weight) of E. coli RNA polymerase (Burgess, Travers, Dunn & Bautz, 1969) and change the initiation speci6city of the catalytic portion of the enzyme. Recent evidence supports this model for positive control by the gene 1 product of T7 transcription in vitro (Summers & Siegel, 1969). After infection with T7 + , both host protein synthesis (Studier & Maizel, 1969) and host RNA synthesis (Summers & Szybalski, 1968; Summers, 1969) gradually cease. This has been attributed to the degradation of host DNA by a T7-induced DNase (Hausmann & Gomez, 1968). An alternative explanation might be that when the host RNA polymer&se is modified by the substitution of the gene 1 protein for the normal host specificity factor, the host DNA can no longer be transcribed. The fact that hoat RNA synthesis can continue after infection by T7 carrying amber mutants in gene 1 is not unexpected because the gene 1 amber peptides may not be able to displace the sigma factor from host polymerase. Indeed, recent experiments (Summers, unpublished results) with T7 harboring a thermosensitive mutation in gene 1 suggest that host fl;NA synthesis is inhibited at the non-permissive temperature despite absence of a functional gene 1 protein. Perhaps the complete, thermosensitive-gene 1 protein can displace the normal host sigma factor and thereby prevent transcription of the host DNA while also rendering the modified enzyme thermosensitive in its ability to transcribe T7 RNA (Summers & Siegel, 1969). If this hypothesis is valid, it would be an interesting example of linkage of positive and negative regulation of gene function. The experiments presented here show that gene 1 of T7 exerts a positive control over the transcription of the phage genome in vivo. Phage-directed protein synthesis is required for shut-off of host RNA synthesis. Whether or not this shut-off is also mediated by the gene 1 product is not yet known. We thank Dr Studier and Dr Hausmann for giving us strains of bacteriophage. Dr Studier also has generously communicated bitr results before public&ion. This work was e~ppoti by U.S. Public Health Service grant no. CAO6619. REFERENCES p. 446. New York: Interscience. Adams, M. H., (1969). Bacteriophagea, Belle, A., Epstein, R. H., Salser, W. & Geiduschek, E. P. (1963u). J. Mol. Biol. 33, 339. Belle, A., Epstein, R. H., Salser, W. & Geiduschek, E. P. (19683). J. Mol. Biol. 31, 326. Burgess, R. R., !&avers, A. A., Dunn, J. J. & Bautz, E. K. F. (1969). Natare, 221, 43. Cm&we, G. L%Gros, F. (1968). C. R. Acad. Sci. Pa&, 266D, 263. Dove, W. F. (1966). J. Mol. Biol. IQ, 187. Fairbanks, G., Jr., Levintbel, C. & Reeder, R. H. (1966). Biochem. B&o&p. Rec. Cm. 20, 393.
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Geiduschek, E. P., Wilson, D. L. & Gage, L. P. (1969). J. CeZZ Physiol. (Suppl. 1) 74, 81. Hausmann, R. & Gomez, B. (1967). J. Viral. 1, 779. Hausmann, R. & Gomez, B. (1968). J. VifoZ. 2, 266. Hausmann, R., Gomez, B. t Moody, B. (1968). J. Viral. 2, 335. Hausmtum, R., t LaRue, K. (1969). J. VGoZ. 3, 278. Hosoda, J. & Levinthal, C. (1968). Virology, 34, 709. Imamoto, F. & Yanofsky, C. (1967). J. Mol. Biol. 28, 25. Joyner, A., Isaacs, L. N., Echols, H. &z Sly, W. S. (1966). J. Mol. BioZ. 19, 174. Kirby, K. S. (1966). Biochem. J. 96, 266. Levinthal, C., Hosoda, J. & Shub, D. (1967). In The MoZecuZar Biology of Viruses, ed. by J. S. Colter & W. Paranchych, p. 71. New York: Academic Press. Mathews, C. K. (1968). J. BioZ. Chem. 243,561O. Pene, J. J. & Marmur, J. (1967). J. VG-OZ. 1, 86. Studier, F. W. (1969). Virology, 39, 562. Studier, F. W. t Hausmann, R. (1969). Virology, 39, 587. Studier, F. W. & Maize& J. V., Jr.. (1969). Virology, 39, 575. Summers, W. C. (1969). V