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A test of a hypothesis concerning ultraviolet irradiation damage and phage functions
VIROLOGY 26, 390--393
(1965)
A Test of A Hypothesis Concerning Ultraviolet Irradiation Damage and Phage Functions' K. EBISUZAKI Department of Biological Chemistry, Harvard Medical School, and the Biochemical Research Laboratory, The Massachusetts General Hospital, Boston 14, Massachusetts Accepted March 22, 1965
The formation of the T4 phage-induced lysozymeis highly sensitive to ultraviolet (UV) irradiation. The formation of this "late" protein, in cells infected with nonirradiated, lysozymelessphage and UVdrradiated, lysozyme-producingphage shows an undiminished, high sensitivity to UV irradiation. These results are discussed in the context of current ideas on the control of phage development and of irradiation damage. INTRODUCTION
able hypothesis whereby this UV effect can be explained on the basis of a "clock" model of regulation. This model, which was originally proposed by Jacob (1960) and by Jacob and Wollman (1961), assumes that phage development occurs by a series of consecutive reactions in which inhibition of any one of the reactions prevents a terminal reaction. Stent proposes that UV irradiation inactivates an "early" cistron and this interrupts the specific induction sequence. This communication is concerned with a direct test of the Stent hypothesis. Experiments were done to study the ability of UVirradiated bacteriophage T4 to form the "late" protein, lysozyme, in cells mixedly infected with an unirradiated nonlysozymeproducing mutant. The results suggest that the Stent hypothesis does not apply and that the very high UV sensitivity of late phage genes must be explained by other means. MATERIAL AND METHODS Bacteria. E. coli B, E. coli B-3 (thymineless), and E. coli CR63 were used. Phage. T4D (wild type) was grown on E. coli B. "Amber" mutants, T4e am (lysozymeless) and T4 amA453 (defective in an 1 This work was supported by grants from the early function, cistron 32) were grown on E. National Institutes of Health and from the coli CR63 (Epstein et al., 1963). ("Amber" mutants do not grow on E. coli B.) These National Science Foundation. 390 Infection of Escherichia coli with the Teven bacteriophages results in the formation of two primary classes of proteins. These proteins are simply classified on the basis of their temporal appearance as "early" and "late." The "early" proteins are concerned primarily with DNA metabolism, whereas the "late" proteins are predominantly components of the phage structure. The mechanism by which the phage genome regulates the synthesis of "early" and "late" proteins is largely unknown. Somehow the synthesis of "early" proteins is mandatory for the synthesis of "late" proteins (Epstein et al., 1963; Ebisuzaki, 1963), and the synthesis of "late" proteins is accompanied by a repression of the formation of "early" proteins (Watanabe, 1957a; Dirksen et al., 1960). However, this regulatory balance is disrupted if the phage are given small doses of UV irradiation; the "early" proteins are continuously synthesized (Dirksen et al., 1960; Delihas, 1961) and the synthesis of "late" proteins is repressed (Watanabe, 1957b; K. Mark, personal communication; Sekiguchi and Cohen, 1964). Stent (1963) has suggested a very reason-
TEST OF COMPLEMENTATION OF UV DAMAGE two m u t a n t s will simply be referred to as T 4 e - and T4 32-, respectively. Lysozyme assay. Lysozyme assays were performed with supernatants obtained from cells disrupted with a French pressure cell and centrifuged at 6000 g for 20 minutes. Lyophilized E. cell B cells were used as substrate as suggested b y K. h~[ark, Dep a r t m e n t of Molecular Biology, University of Oregon. T h e enzyme was assayed with 0.6 m g of cells, 50 ~moles T r i s ( h y d r o x y methyl)aminomethane (Tris), p H 7.5, 4 ~,moles e t h y l e n e d i a m i n e t e t r a a c e t i c acid (neutralized), in a total volume of 1 ml. The enzyme was assayed at room temperature for a 2-minute period at 600 rag, under
z.o
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o
:
o
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~
o.,
~ o.os ~.
o.ol
]
391
I
r
I
I
I0
20
30
40
0.9
SECONDS OF IRRADIATION
FIG. 2. Survival of the capacity of the lysozyme gene with varying doses of UV irradiation. Experimental conditions were identical to those described for Fig. 1. Lysozyme was assayed on extracts of cells incubated for 20 minutes after infection. Phage formation is equated with the ability of the phage to form a plaque. • • phage formation; O O ]ysozyme formation.
0.8
0.7
o.~
o.5
conditions where enzyme activity is proportional to enzyme concentration. 0.4
RESULTS AND DISCUSSION
"M
0.3
/
d
/i
0.2
•
II I
0.1
/ i0
0
I0
I
I
20
MINUTES
FIG. 1. Formation of lysozyme with phage T4D and UV-irradiated T4D. E. cell B-3 was infected with phage at a multiplicity of infection 0.25 and incubated at 37° in a modified Herriot and Barlow medium (Ebisuzaki, 1963), containing 5 vg thymine per milliliter. Phage, which were suspended in cold 0.05 M phosphate buffer, pH 6.8, were irradiated with a General Electric germicidal lamp GST5 at a distance of 64 cm. Lysozyme activity is expressed as change in optical density per minute per milligram protein. • • no UV; O - - - O 20 seconds UV.
The kinetics of lysozyme formation in E. cell cells infected with bacteriophage T 4 D and UV-irradiated T 4 D is presented in Fig. 1. To avoid complications resulting from multiple infection, the experiments were carried out with low multiplicities of phage; and to minimize the possibility of reinfection, ]ysozyme synthesis was followed only for the first 20 minutes after the addition of phage. The effect of the UV dose on lysozyme formation (lysozyme formed at 20 minutes after infection) and on phage survival can be compared as a ratio of the slopes of two inactivation curves (Fig. 2). In Fig. 2, a comparison was made of the residual survival of phage and the ability to synthesize lysozyme. The average value for this ratio in two separate experiments was 0.35. As a comparison, in a similar experiment involving the formation of phage structural proteins, the ratio of the two slopes was
392
EBISUZAKI
1.0
0.5
% _.
O.I
_. 0 . 0 5
o.ol 0
I
]
I
I
I0
20
50
40
SECONDS OF IRRI~DIATION FIG. 3. Survival of the capacity of the lysozyme gene with varying doses of irradiation under conditions of complementation with lysozymeless phage. E. coli B was infected with T4e- and UV-irradiated T4e + in the following medium: 75 ml of modified tterriot and Barlow medium and 25 ml 20% sucrose containing 0.02 M MgC12. This medium was used to decrease the lysis that occurred with high multiplicities of phage. Other conditions were similar to those described for Fig. I, and the notations
are the same
as in Fig. 2.
0.47 (Ebisuzaki, unpublished; see also Watanabe, 1957b). As mentioned earlier, the high sensitivity of "late" functioning genes to UV irradiation could be due to a cumulative effect where the inactivation of one or more of the preceding steps indirectly results in the inactivation of a terminal step. To test this possibility, E. coli B was infected with T4e- (lysozymeless) at a multiplicity of infection (moi) of 4 and UV-irradiated T4e + at a moi of 0.3. T4e- is able to perform "early" and "late" functions except for the production of lysozyme (Epstein et al., 1963); thus the ability of UV-irradiated T4e + to produce lysozyme would be measured by the survival of the functional capacity of the eistron. Although the "early" functions are presumably carried out by the nonirradiated phage, (eomplementation), UV irradiation destroyed the functional capacity of the lysozynm gene at nearly
the same rate as i n the singly infected cells (Fig. 3). There is a remote possibility that the formation of lysozyme occurred primarily in cells singly infected with T4e+. In order to force eomplementation, E. coli B was infected with T4e-32+(moi = 4) and UVirradiated T4e+a2-(moi = 0.3). Under these conditions, eomplementation would be a prerequisite for the formation of lysozyme. The ratio, lys0ayme survival: phage survival, was 0.33 and 0.27 in two separate experiments, indicating again that eomplementation does not greatly alter the UV sensitivity of lysozyme formation. Some wild-type reeombinants were observed (a few per cent of the total yield), but they did not appreciably change the survival curve for the function of the lysozyme gene. If the last experiment is reversed, in the sense that the infection is carried out with T4e+32-(moi = 0.5) and irradiated T4e-32 + (moi = 4), lysozyme formation is virtually unaffected by UV irradiation (with UV doses up to 120 minutes). Cistron 32, like other early functions tested, is relatively resistant to UV irradiation (Ebisuzaki, unpublished) and, under the conditions of the experiment, apparently complements the T4e+32 - phage. This result means that the effect of UV irradiation is noneomplementing in that it influences only the genome of the irradiated phage. These eomplementation experiments appear to rule out Stent's hypothesis on the mechanism by which UV irradiation damages the capacity of T4 phage to produce "late" proteins. Since small doses of UV irradiation are involved, a specific gene such as that of lysozyme is probably not the direct target of the irradiation. Instead, it seems that UV irradiation must invoke some sort of damage involving a critical step, which in turn prevents the expression of the lysozyme gene. It has been suggested that the formation of "late" proteins or the cessation of the synthesis of "early" proteins is regulated by DNA synthesis (Wiberg et al., 1962; Luria, 1962; Epstein et al., 1963) and that UV irradiation may inhibit "late" functions by inhibiting DNA synthesis (Sekiguchi and Cohen, 1964). If these theories are correct, it follows that the
TEST OF COMPLEMENTATION OF UV DAMAGE failure to form lysozyme must be due to a failure to replicate the D N A of the irradiated phage, even though, presumably, the milieu for D N A synthesis is provided. Regardless of the correctness of the theory, the failure of complementation to appreciably alter the inactivation curves for lysozyme synthesis suggests that a large portion of the phage genome cannot express itself. I t is interesting to note that "early" functions such as d C M P hydroxymethylase and d C M P hydroxymethylkinase are inactivated by UV irradiation with a ratio of enzyme survival:phage survival of about 0.08 for phage T4D (Ebisuzaki, unpublished), while their high sensitivity to pa2 decay suggests that a large number of genes are somehow coordinated (Ebisuzaki, 1962). The sensitivity of the r I I eistrons to UV irradiation (Krieg, 1959) and to p~2 decay (unpublished experiments of Steinberg, reviewed by Stahl, 1959) also resembles the results obtained with the "early" enzymes. Viewed with this background, it seems especially pertinent to question why the UV damage appears to be so specific in inactivating late functions. Is it the inactivation of the D N A template as a replication unit (for instance, through thymine dimers), so that the formation of "late" proteins is inhibited? Or could there be loci of high UV sensitivity in the phage genome which specifically affect "late" functions? ACKNOWLEDGMENTS The author is particularly indebted to Dr. It. M. Kalckar for many helpful discussions and criticism. He also wishes to thank Drs. S. E. Luria, R. S. Edgar, Henry Wu, and Stanley Hattman for their constructive criticism and help in the writing of this manuscript. Dr. R. S. Edgar kindly provided the T4 amber mutants used in these studies, and Dr. K. Mark provided helpful advice with the lysozyme assay. REFERENCES DEI~IHAS, N. (1961). The ability of irradiated bacteriophage T2 to initiate the synthesis of
393
deoxycytidylate hydroxymethylase in Escherichia coll. Virology 13,242-248.
DIRKSEN, M. L., WIBERG,J. S., KOERNER,J. F., and BUCHANAN,J. M. (1960). Effect of ultraviolet irradiation of bacteriophage T2 on enzyme synthesis in host cells. Proc. Natl. Acad. Sci. U.S. 46, 1425-1430. EBISUZAKI,I{. (1962). Functional interdependence of genes in bacteriophage T2. J. Mol. Biol. 5, 506-510. EBISLTZAKI,K. (1963). On the regulation of the morphogenesis of bacteriophage T4. J. Mol. Biol. 7, 379-387. EPSTEIN~ R. H., BOLLIX,A., STEINBERG, C. M., KELLENBERGER, E., BoY DE LA TOVR, E.,
CItEVALLEY, R., EDGAR, R. S., SUSMAN, M., DENHARDT, G. H., and LIELAUSIS, A. (1963). Cold Spring Harbor Syrup. Quant. Biol. 28, 375-392. JACOB, F. (1960). Genetic control of viral functions. Harvey Lectures Ser. 54, 1-39. JAcoB, F., and WOLL]~,IAN,E. L. (1961). "Sexuality and the Genetics of Bacteria," p. 300. Academic Press, New York. KRIEO, D. R. (1959). A study of gene action in ultraviolet-irradiated bacteriophage T4. Virology 8, 80-98. LucIA, S. E. (1962). Genetics of bacteriophage. Ann. Rev. Microbiol. 16, 205-240. SE~:IGUCm,M., and COHEN,S. S. (1964). The synthesis of messenger RNA without protein synthesis. J. Mol. Biol. 8, 638-659. STAHL, F. W. (1959). Radiobiology of bacteriophage. In "The Viruses" (F. M. Burner and W. M. Stanley, eds.), Vol. 2. Academic Press, New York. STENT, G. S. (1963). In "Molecular Biology of Bacterial Viruses," pp. 424-425. Freeman, San Francisco, California. WATANABE, I. (1957a). Formation of non phageantigenic protein in E. coli infected with T2 phage. Biochim. Biophys. Acta 25, 665-666. W.~T.aNABE, I. (1957b). The effect of ultraviolet light on the production of bacterial virus prorein. J. Gen. Physiol. 40, 521-531.
WIBERG, J. F., DIRKSEN,hi[. L., EPSTEIN, R. H., LL-RIA, S. E., and BUCHANAN, J. M. (1962). Early enzyme synthesis and its control in E. coli infected with some amber mutants of bacteriophage T4. Proc. Natl. Acad. Sci. U.S. 48, 293-302.
(1965)
A Test of A Hypothesis Concerning Ultraviolet Irradiation Damage and Phage Functions' K. EBISUZAKI Department of Biological Chemistry, Harvard Medical School, and the Biochemical Research Laboratory, The Massachusetts General Hospital, Boston 14, Massachusetts Accepted March 22, 1965
The formation of the T4 phage-induced lysozymeis highly sensitive to ultraviolet (UV) irradiation. The formation of this "late" protein, in cells infected with nonirradiated, lysozymelessphage and UVdrradiated, lysozyme-producingphage shows an undiminished, high sensitivity to UV irradiation. These results are discussed in the context of current ideas on the control of phage development and of irradiation damage. INTRODUCTION
able hypothesis whereby this UV effect can be explained on the basis of a "clock" model of regulation. This model, which was originally proposed by Jacob (1960) and by Jacob and Wollman (1961), assumes that phage development occurs by a series of consecutive reactions in which inhibition of any one of the reactions prevents a terminal reaction. Stent proposes that UV irradiation inactivates an "early" cistron and this interrupts the specific induction sequence. This communication is concerned with a direct test of the Stent hypothesis. Experiments were done to study the ability of UVirradiated bacteriophage T4 to form the "late" protein, lysozyme, in cells mixedly infected with an unirradiated nonlysozymeproducing mutant. The results suggest that the Stent hypothesis does not apply and that the very high UV sensitivity of late phage genes must be explained by other means. MATERIAL AND METHODS Bacteria. E. coli B, E. coli B-3 (thymineless), and E. coli CR63 were used. Phage. T4D (wild type) was grown on E. coli B. "Amber" mutants, T4e am (lysozymeless) and T4 amA453 (defective in an 1 This work was supported by grants from the early function, cistron 32) were grown on E. National Institutes of Health and from the coli CR63 (Epstein et al., 1963). ("Amber" mutants do not grow on E. coli B.) These National Science Foundation. 390 Infection of Escherichia coli with the Teven bacteriophages results in the formation of two primary classes of proteins. These proteins are simply classified on the basis of their temporal appearance as "early" and "late." The "early" proteins are concerned primarily with DNA metabolism, whereas the "late" proteins are predominantly components of the phage structure. The mechanism by which the phage genome regulates the synthesis of "early" and "late" proteins is largely unknown. Somehow the synthesis of "early" proteins is mandatory for the synthesis of "late" proteins (Epstein et al., 1963; Ebisuzaki, 1963), and the synthesis of "late" proteins is accompanied by a repression of the formation of "early" proteins (Watanabe, 1957a; Dirksen et al., 1960). However, this regulatory balance is disrupted if the phage are given small doses of UV irradiation; the "early" proteins are continuously synthesized (Dirksen et al., 1960; Delihas, 1961) and the synthesis of "late" proteins is repressed (Watanabe, 1957b; K. Mark, personal communication; Sekiguchi and Cohen, 1964). Stent (1963) has suggested a very reason-
TEST OF COMPLEMENTATION OF UV DAMAGE two m u t a n t s will simply be referred to as T 4 e - and T4 32-, respectively. Lysozyme assay. Lysozyme assays were performed with supernatants obtained from cells disrupted with a French pressure cell and centrifuged at 6000 g for 20 minutes. Lyophilized E. cell B cells were used as substrate as suggested b y K. h~[ark, Dep a r t m e n t of Molecular Biology, University of Oregon. T h e enzyme was assayed with 0.6 m g of cells, 50 ~moles T r i s ( h y d r o x y methyl)aminomethane (Tris), p H 7.5, 4 ~,moles e t h y l e n e d i a m i n e t e t r a a c e t i c acid (neutralized), in a total volume of 1 ml. The enzyme was assayed at room temperature for a 2-minute period at 600 rag, under
z.o
0.5 i
o
:
o
_~
~
o.,
~ o.os ~.
o.ol
]
391
I
r
I
I
I0
20
30
40
0.9
SECONDS OF IRRADIATION
FIG. 2. Survival of the capacity of the lysozyme gene with varying doses of UV irradiation. Experimental conditions were identical to those described for Fig. 1. Lysozyme was assayed on extracts of cells incubated for 20 minutes after infection. Phage formation is equated with the ability of the phage to form a plaque. • • phage formation; O O ]ysozyme formation.
0.8
0.7
o.~
o.5
conditions where enzyme activity is proportional to enzyme concentration. 0.4
RESULTS AND DISCUSSION
"M
0.3
/
d
/i
0.2
•
II I
0.1
/ i0
0
I0
I
I
20
MINUTES
FIG. 1. Formation of lysozyme with phage T4D and UV-irradiated T4D. E. cell B-3 was infected with phage at a multiplicity of infection 0.25 and incubated at 37° in a modified Herriot and Barlow medium (Ebisuzaki, 1963), containing 5 vg thymine per milliliter. Phage, which were suspended in cold 0.05 M phosphate buffer, pH 6.8, were irradiated with a General Electric germicidal lamp GST5 at a distance of 64 cm. Lysozyme activity is expressed as change in optical density per minute per milligram protein. • • no UV; O - - - O 20 seconds UV.
The kinetics of lysozyme formation in E. cell cells infected with bacteriophage T 4 D and UV-irradiated T 4 D is presented in Fig. 1. To avoid complications resulting from multiple infection, the experiments were carried out with low multiplicities of phage; and to minimize the possibility of reinfection, ]ysozyme synthesis was followed only for the first 20 minutes after the addition of phage. The effect of the UV dose on lysozyme formation (lysozyme formed at 20 minutes after infection) and on phage survival can be compared as a ratio of the slopes of two inactivation curves (Fig. 2). In Fig. 2, a comparison was made of the residual survival of phage and the ability to synthesize lysozyme. The average value for this ratio in two separate experiments was 0.35. As a comparison, in a similar experiment involving the formation of phage structural proteins, the ratio of the two slopes was
392
EBISUZAKI
1.0
0.5
% _.
O.I
_. 0 . 0 5
o.ol 0
I
]
I
I
I0
20
50
40
SECONDS OF IRRI~DIATION FIG. 3. Survival of the capacity of the lysozyme gene with varying doses of irradiation under conditions of complementation with lysozymeless phage. E. coli B was infected with T4e- and UV-irradiated T4e + in the following medium: 75 ml of modified tterriot and Barlow medium and 25 ml 20% sucrose containing 0.02 M MgC12. This medium was used to decrease the lysis that occurred with high multiplicities of phage. Other conditions were similar to those described for Fig. I, and the notations
are the same
as in Fig. 2.
0.47 (Ebisuzaki, unpublished; see also Watanabe, 1957b). As mentioned earlier, the high sensitivity of "late" functioning genes to UV irradiation could be due to a cumulative effect where the inactivation of one or more of the preceding steps indirectly results in the inactivation of a terminal step. To test this possibility, E. coli B was infected with T4e- (lysozymeless) at a multiplicity of infection (moi) of 4 and UV-irradiated T4e + at a moi of 0.3. T4e- is able to perform "early" and "late" functions except for the production of lysozyme (Epstein et al., 1963); thus the ability of UV-irradiated T4e + to produce lysozyme would be measured by the survival of the functional capacity of the eistron. Although the "early" functions are presumably carried out by the nonirradiated phage, (eomplementation), UV irradiation destroyed the functional capacity of the lysozynm gene at nearly
the same rate as i n the singly infected cells (Fig. 3). There is a remote possibility that the formation of lysozyme occurred primarily in cells singly infected with T4e+. In order to force eomplementation, E. coli B was infected with T4e-32+(moi = 4) and UVirradiated T4e+a2-(moi = 0.3). Under these conditions, eomplementation would be a prerequisite for the formation of lysozyme. The ratio, lys0ayme survival: phage survival, was 0.33 and 0.27 in two separate experiments, indicating again that eomplementation does not greatly alter the UV sensitivity of lysozyme formation. Some wild-type reeombinants were observed (a few per cent of the total yield), but they did not appreciably change the survival curve for the function of the lysozyme gene. If the last experiment is reversed, in the sense that the infection is carried out with T4e+32-(moi = 0.5) and irradiated T4e-32 + (moi = 4), lysozyme formation is virtually unaffected by UV irradiation (with UV doses up to 120 minutes). Cistron 32, like other early functions tested, is relatively resistant to UV irradiation (Ebisuzaki, unpublished) and, under the conditions of the experiment, apparently complements the T4e+32 - phage. This result means that the effect of UV irradiation is noneomplementing in that it influences only the genome of the irradiated phage. These eomplementation experiments appear to rule out Stent's hypothesis on the mechanism by which UV irradiation damages the capacity of T4 phage to produce "late" proteins. Since small doses of UV irradiation are involved, a specific gene such as that of lysozyme is probably not the direct target of the irradiation. Instead, it seems that UV irradiation must invoke some sort of damage involving a critical step, which in turn prevents the expression of the lysozyme gene. It has been suggested that the formation of "late" proteins or the cessation of the synthesis of "early" proteins is regulated by DNA synthesis (Wiberg et al., 1962; Luria, 1962; Epstein et al., 1963) and that UV irradiation may inhibit "late" functions by inhibiting DNA synthesis (Sekiguchi and Cohen, 1964). If these theories are correct, it follows that the
TEST OF COMPLEMENTATION OF UV DAMAGE failure to form lysozyme must be due to a failure to replicate the D N A of the irradiated phage, even though, presumably, the milieu for D N A synthesis is provided. Regardless of the correctness of the theory, the failure of complementation to appreciably alter the inactivation curves for lysozyme synthesis suggests that a large portion of the phage genome cannot express itself. I t is interesting to note that "early" functions such as d C M P hydroxymethylase and d C M P hydroxymethylkinase are inactivated by UV irradiation with a ratio of enzyme survival:phage survival of about 0.08 for phage T4D (Ebisuzaki, unpublished), while their high sensitivity to pa2 decay suggests that a large number of genes are somehow coordinated (Ebisuzaki, 1962). The sensitivity of the r I I eistrons to UV irradiation (Krieg, 1959) and to p~2 decay (unpublished experiments of Steinberg, reviewed by Stahl, 1959) also resembles the results obtained with the "early" enzymes. Viewed with this background, it seems especially pertinent to question why the UV damage appears to be so specific in inactivating late functions. Is it the inactivation of the D N A template as a replication unit (for instance, through thymine dimers), so that the formation of "late" proteins is inhibited? Or could there be loci of high UV sensitivity in the phage genome which specifically affect "late" functions? ACKNOWLEDGMENTS The author is particularly indebted to Dr. It. M. Kalckar for many helpful discussions and criticism. He also wishes to thank Drs. S. E. Luria, R. S. Edgar, Henry Wu, and Stanley Hattman for their constructive criticism and help in the writing of this manuscript. Dr. R. S. Edgar kindly provided the T4 amber mutants used in these studies, and Dr. K. Mark provided helpful advice with the lysozyme assay. REFERENCES DEI~IHAS, N. (1961). The ability of irradiated bacteriophage T2 to initiate the synthesis of
393
deoxycytidylate hydroxymethylase in Escherichia coll. Virology 13,242-248.
DIRKSEN, M. L., WIBERG,J. S., KOERNER,J. F., and BUCHANAN,J. M. (1960). Effect of ultraviolet irradiation of bacteriophage T2 on enzyme synthesis in host cells. Proc. Natl. Acad. Sci. U.S. 46, 1425-1430. EBISUZAKI,I{. (1962). Functional interdependence of genes in bacteriophage T2. J. Mol. Biol. 5, 506-510. EBISLTZAKI,K. (1963). On the regulation of the morphogenesis of bacteriophage T4. J. Mol. Biol. 7, 379-387. EPSTEIN~ R. H., BOLLIX,A., STEINBERG, C. M., KELLENBERGER, E., BoY DE LA TOVR, E.,
CItEVALLEY, R., EDGAR, R. S., SUSMAN, M., DENHARDT, G. H., and LIELAUSIS, A. (1963). Cold Spring Harbor Syrup. Quant. Biol. 28, 375-392. JACOB, F. (1960). Genetic control of viral functions. Harvey Lectures Ser. 54, 1-39. JAcoB, F., and WOLL]~,IAN,E. L. (1961). "Sexuality and the Genetics of Bacteria," p. 300. Academic Press, New York. KRIEO, D. R. (1959). A study of gene action in ultraviolet-irradiated bacteriophage T4. Virology 8, 80-98. LucIA, S. E. (1962). Genetics of bacteriophage. Ann. Rev. Microbiol. 16, 205-240. SE~:IGUCm,M., and COHEN,S. S. (1964). The synthesis of messenger RNA without protein synthesis. J. Mol. Biol. 8, 638-659. STAHL, F. W. (1959). Radiobiology of bacteriophage. In "The Viruses" (F. M. Burner and W. M. Stanley, eds.), Vol. 2. Academic Press, New York. STENT, G. S. (1963). In "Molecular Biology of Bacterial Viruses," pp. 424-425. Freeman, San Francisco, California. WATANABE, I. (1957a). Formation of non phageantigenic protein in E. coli infected with T2 phage. Biochim. Biophys. Acta 25, 665-666. W.~T.aNABE, I. (1957b). The effect of ultraviolet light on the production of bacterial virus prorein. J. Gen. Physiol. 40, 521-531.
WIBERG, J. F., DIRKSEN,hi[. L., EPSTEIN, R. H., LL-RIA, S. E., and BUCHANAN, J. M. (1962). Early enzyme synthesis and its control in E. coli infected with some amber mutants of bacteriophage T4. Proc. Natl. Acad. Sci. U.S. 48, 293-302.