23 Jerne, N. K., A. A. Nordin, and C. Henry, in Cell Bound Antibodies, ed. B. Amos aid H .... H., N. J. Rosebrough, A. J. Farr, and R. J. Randall, J. Biol. Chem., 193 ...
832
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PROC. N. A. S.
14Three human X L-chains were blocked in the N-terminal position; hence in the N-terminal analysis of pooled human -yG L-chains one is probably looking only at K chain residues. 15 Dray, S., G. 0. Young, and L. Gerald, J. Immunol., 91, 403 (1963). 16 Pauling, L., J. Am. Chem. Soc., 62, 2643 (1940). 17 Karush, F., Trans. N. Y. Acad. Sci. (Series II), 20, 581 (1958). 18 Lederberg, J., Science, 129, 1649 (1959). 19 Smithies, O., Nature, 199, 1231 (1963). 10 Burnet, M., Nature, 203, 451 (1964). 21 Smithies, O., Science, 149, 151 (1965). 22 Potter, M., E. Apella, and S. Geisser, J. Mol. Biol., 14, 361 (1965). 23 Jerne, N. K., A. A. Nordin, and C. Henry, in Cell Bound Antibodies, ed. B. Amos aid H. Koprowski (Philadelphia: Wistar Inst. Press, 1963), p. 109. f 24Mach, B., and E. L. Tatum, these PROCEEDINGS, 52, 876 (1964). 25 Campbell, A. M., Advan. Genet., 11, 90 (1962). 26Nirenberg, M., P. Leder, M. Bernfield, R. Brimacombe, J. Trupin, F. Rottman, and C. O'Neal, these PROCEEDINGS, 53, 1161 (1965). 27 Dayhoff, M. O., R. V. Eck, M. A. Chang, and M. R. Sochard, Atlas of Protein Sequence and Structure (National Biomedical Research Foundation, 1965).
INHIBITION OF LYSOZYME SYNTHESIS BY ACTINOMYCIN D IN BACTERIOPHAGE T4-INFECTED CELLS OF ESCHERICHIA COLI BY JAY J. PROTASS AND DAVID KORN LABORATORY OF BIOCHEMICAL PHARMACOLOGY, NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES, NATIONAL INSTITUTES OF HEALTH
Communicated by C. B. Anfinsen, February 16, 1966
There is considerable evidence that in T-even bacteriophage infection of cells of E. coli there is a temporal sequence of phage-directed protein synthesis.'-4 However, the mechanisms controlling "early" and "late" protein formation are not well understood. Two opposing theories are as follows: (a) The input phage genome transcribes only "early" messenger RNA (mRNA), while "late" mRNA is transcribed from newly synthesized genome replicas.5'6 This theory thus proposes that regulation operates at the level of DNA transcription. (b) Both "early" and "late" mRNA species can be transcribed from the input phage genome, but "late" mRNA is not expressed during the early phase of the infectious cycle. This theory, then, assigns a major role in regulation to mRNA translation. Indirect evidence supporting this latter hypothesis has recently been published.7 In this paper we present some initial results of our study of factors controlling phage-directed protein synthesis. Using T4-infected, EDTA-treated8 cells of E. coli, we have investigated the effect of actinomycin D on the production of the "late" phage-directed enzyme, lysozyme. The data indicate that the formation of mRNA capable of directing lysozyme synthesis does not significantly precede the time at which lysozyme activity becomes demonstrable in the infected cells. Materials and Methods.-The experiments were performed with bacteriophage T4 and E. coli CR34. Conditions of infection and the method of sensitization of E. coli to actinomycin D by EDTA treatment were as previously described.9 Crude extracts were prepared from infected cells by rapidly chilling aliquots of the culture
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and collecting the cells by centrifugation. Approximately 6 X 109 cells were suspended in 1 ml of cold 0.01 M Tris-chloride buffer, pH 7.5, and were disrupted by sonicating. for 15 sec at 0C with a Branson sonifier, model S75. The sonicate was centrifuged at low speed to remove debris and the resulting supernatant comprised the crude extract. Protein concentration was determined by the method of Lowry et al.'0 Lysozyme activity was assayed by a modification of the method of Sekiguchi and Cohen.6 The incubation mixture contained in a final volume of 1 ml about 1 X 108 chloroform-treated cells of E. coli CR34, 50 Mmoles Tris-chloride buffer, pH 7.5, and crude extract. The reaction was carried out at room temperature in cuvettes with a 10-mm light path. Decrease in optical density was followed at 0.-min intervals for 2 min in a Beckman DU spectrophotometer at 650 my. The unit of enzyme activity was defined as a decrease of 0.100 optical density unit per min. The assay was linear over a tenfold range of extract protein and a 5-min time period. Results.-Under our experimental conditions (latent period about 35 min), lysozyme activity in T4-infected cultures is first demonstrable between the eighth and twelfth minutes of the latent period (Fig. 1A) and increases steadily thereafter. Thus the time of appearance of lysozyme corresponds closely to the time of onset of phage DNA synthesis (Fig. 1C). If actinomycin D (25 Mg/inl) is added to sensitized, infected cells at time 0, no
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FIG. 1.-Actinomycin D (25 ,ug/ml) effect on lysozyme synthesis (A), amino acid incorporation (B), and DNA synthesis (C), in T4-infected, EDTA-treated E. coli CR34. (A) Lysozyme activity was assayed as described in the text from 0 to 30 min of the latent period (am------ 0). Actinomycin D was added to half the culture at 12 min ( *). (B) C14-leucine, specific activity 0.45 mc/mM, was added at 12 min and its incorporation into acid-insoluble product measured as previously described9 (- ------ -). At 12 min, actinomycin D was added to half the culture (e-e). (C) H3-thymine, specific activity 5 mc/mM, was added at 0 min of infection and its incorporation into acid-insoluble product followed (0-- --- -). At 12 min, actinomycin D was added to half the culture (* *).
834
BIOCHEMISTRY: PROTASS AND KORN
PROC. N. A. S.
lysozyme activity can be detected over the subsequent 40 min. Addition of the drug up to the twelfth minute of the latent period leads to almost total inhibition of lysozyme formation (Fig. 1A). DNA synthesis, on the other hand, is unaffected by actinomycin D added at 12 min (Fig. 1C). In contrast to the dramatic effect of the antibiotic on lysozyme production, actinomycin D, added at the twelfth minute of infection, results in a more gradual curtailment of total protein synthesis (Fig. 1B), similar to that produced in B. subtilis" and in uninfected E. coli.8 This gradual cessation of protein synthesis is believed to reflect the rate of degradation of functional mRNA present at the time of actinomycin treatment.'1 Comparison of Figures 1A and B indicates that while the infected cells at the twelfth minute do indeed contain mRNA that can be translated into protein, an extremely small fraction of this RNA represents message for lysozyme. Studies on the effect of actinomycin D on lysozyme formation at later times during infection have been somewhat more difficult. Although phage production in these cells remains exquisitely sensitive to the antibiotic throughout the latent period,9 the ability of the drug to inhibit mRNA synthesis rapidly decreases beyond the twelfth minute. Thus actinomycin inhibits uridine incorporation in the infected cells by 94 per cent at the eleventh minute (Table 1) but by only 62 per TABLE 1 EFFECT OF ADDITION OF ACTINOMYCIN D AT DIFFERENT TIMES DURING THE LATENT PERIOD (A) HL-Uridine Incorporation Rate (cpm/min) Actinomycin D (25 g/ml) H'-uridine Per cent Actinomycin added at: Minus Plus inhibition added at:
1 min 11m 19m
2 min 12 min 20 min
780 438 300
0 25 113
100 94.3 62.3
(B) Lysozyme Specific Activity Actinomycin D (25 jg/ml) Per cent Actinomycin added at: Minus Plus inhibition
0 min 12 min 15 min
15 14.1 12.5
0 1
5.4
100 92.9 56.8
(A) Hs-uridine, specific activity 10 mc/mM, was added at the times indicated to T4-infected, sensitized cells of E. coli CR34. Incorporation into acid-insoluble material was assayed' during the ensuing 9 min in the presence or absence of actinomycin D. (B) Aliquots of infected cultures were removed at 0, 12, and 15 min for determination of lysozyme activity. Immediately thereafter, actinomycin D was added to half of each culture and incubation continued until the 30th minute of the latent period. At this time aliquots of each culture were again removed. The numbers in the table show the increase in specific activity of lysozyme during the intervals 0-30, 12-30, and 15-30 min, respectively, in the presence or absence of actinomycin D.
cent at the nineteenth minute. It is clear, however, that the effect of actinomycin D on lysozyme synthesis corresponds with its effect on mRNA formation throughout that portion of the latent period we have investigated. At no time studied did lysozyme synthesis become insensitive to the antibiotic. Discussion and Conclusions.-If we assume that the effect of actinomycin D on protein synthesis is primarily due to the inhibition of DNA-directed RNA (mRNA) synthesis,1" 12 we may suggest on the basis of the data presented that virtually no functional lysozyme mRNA is present in T4-infected CR34 during the first 12 min of the latent period. The data also indicate that the continuing synthesis of lysozyme during later portions of the latent period is dependent upon the continuing synthesis of mRNA. These results support the hypothesis that in T4-infected cells the mechanism of regulation of "early" and "late" protein synthesis operates at the level of DNA transcription. Although mRNA competent to direct the formation of lysozyme first appears at about the time that phage DNA synthesis begins, the question of whether
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this "late" message is copied from the original infecting genome or from replicas remains unanswered. 1 Cohen, S. S., Federation Proc., 29, 641 (1961). 2Kornberg, A., S. Zimmerman, S. R. Kornberg, and J. Josse, these PROCEEDINGS, 45, 772
(1959). 3DeMars, R. I., Virology, 1, 83 (1955). 4 Wiberg, J. S., M. L. Dirksen, R. H. Epstein, S. E. Luria, and J. M. Buchanan, these PROCEED-
INGS, 48, 293 (1962). 5 Hall, B. D., A. P. Nygaard, and M. H. Green, J. Mol. Biol., 9, 143 (1964). 6 Sekiguchi, M., and S. S. Cohen, J. Mol. Biol., 8, 638 (1964). 7 Edlin, G., J. Mol. Biol., 12, 363 (1965). 8 Leive, L., Biochem. Biophys. Res. Commun., 18, 13 (1965). 9 Korn, D., J. J. Protass, and L. Leive, Biochem. Biophys. Res. Commun., 19, 473 (1965). 10 Lowry, 0. H., N. J. Rosebrough, A. J. Farr, and R. J. Randall, J. Biol. Chem., 193, 265 (1951). 11 Levinthal, C., D. P. Fan, A. Higa, and R. A. Zimmerman, in Cold Spring Harbor Symposia on Quantitative Biology, vol. 28 (1963), p. 183. 12Hurwitz, J., J. J. Furth, M. Malamy, and M. Alexander, these PROCEEDINGS, 48, 1222 (1962).
SIMILARITY OF EFFECTS OF OXYGEN, SULFUR, AND SELENIUM ISOLOGS ON THE ACETYLCHOLINE RECEPTOR IN EXCITABLE MEMBRANES OF JUNCTIONS AND AXONS* BY PHILIP ROSENBERG, HENRY G. MAUTNER, AND DAVID NACHMANSOHN DEPARTMENTS OF NEUROLOGY AND BIOCHEMISTRY, COLLEGE OF PHYSICIANS AND SURGEONS, COLUMBIA UNIVERSITY, AND DEPARTMENT OF PHARMACOLOGY, YALE UNIVERSITY
Communicated February 18, 1966
Excitable membranes have, during electrical activity, the ability of changing their permeability to ions in a specific, rapid, and reversible way. The assumption of a purely physical nature of these changes, as proposed by Hodgkin,' has become untenable in the light of several recent developments. The strong initial heat production coinciding with electrical activity2' 3 provides evidence for the assumption that chemical reactions control the permeability cycle. Failure to affect electrical parameters significantly by drastic modifications of the ion composition in the internal and external fluid of the axon4 is hard to reconcile with the views referred to as the "ionic theory" of conduction. On the other hand, a large amount of evidence has accumulated over the last few decades in favor of the assumption that the action of acetylcholine (ACh) is essential for controlling the permeability cycle in all excitable membranes during electrical activity.5-8 The original hypothesis that ACh is a chemical mediator between two cells was based on results obtained with classical methods of pharmacology. The assumption proved unacceptable in the light of biochemical investigations on cellular, subcellular, and molecular levels, an approach more adequate for the analysis of an event taking place in a membrane of less than 100 A thickness in a few millionths of a second. Experimental data indicate that ACh apparently induces a conformational change in the ACh-receptor protein with a shift of charge, thereby initiating a chain
