ANMICROBIAL AGENTS AND CHEMOTHERAPY, May 1975, P. 487-493
Copyright i 1975 American Society for Microbiology
Vol 7, No. 5 Printed in U.S.A.
Nalidixic Acid and Macromolecular Metabolism in Tetrahymena pyriformis: Effects on Protein Synthesis J. F. DE CASTRO,* J. F. 0. CARVALHO, N. MOUSSATCH1I, AND F. T. DE CASTRO Laboratbrio de Metabolismo Macromolecular, Instituto de Bioffsica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil
Received for publication 25 November 1974
A study on the effect of nalidixic acid on macromolecular metabolism, particularly of protein, in Tetrahymena pyriformis was performed. It was shown that the compound is a potent inhibitor of deoxyribonucleic acid, ribonucleic acid, and protein synthesis for this organism. A conspicuous breakdown of polysomes, accompanied by the accumulation of 80S ribosomes, occurred in cells incubated for 10 min with the drug; polysome formation was prevented. The accumulating 80S particles were shown to be run-off ribosomal units. The incorporation of amino acids by a cell-free system is not affected by nalidixic acid. In nonproliferating cells the incorporation was also not prevented, unless the cells were previously incubated with the drug. These results are discussed in terms of the possible mechanism of action of nalidixic acid in T. pyriformis.
Since the synthesis by Lesher et al. in 1962 (11) of a series of naphthyridine derivatives, one of them, 1-ethyl-7-methyl-4-oxo-1,8-naphthyridine 3-carboxylic qcid, named nalidixic acid (NX), has called the attention of a number of workers (3, 4, 7, 9, 16, 20, 21). Accumulated evidence indicates that the drug specifically blocks deoxyribonucleic acid (DNA) synthesis in bacteria; ribonucleic acid (RNA) and protein synthesis are not directly affected (8). A few reports have appeared bearing on the effect of NX on eucaryotic systems. The restults obtained so far are not particularly elucidative of the mechanism of action of the drug. Growth of the yeast Kluyveromyces lactis is arrested and a conspicuous and preferential repression of mitochondriajl DNA synthesis is observed (12). In Saccharomyces cerevisiae no selective inhibition of DNA could be demonstrated. Instead a transient block of total DNA, RNA, and protein synthesis was found (14). In Euglena gracilis NX blocks chloroplast replication (13). In this paper work is reported which shows that NX blocks the synthesis of DNA, RNA, and protein in cells of Tetrahymena pyriformis. The inhibition of protein synthesis is not transient and seems to be independent of the repression of RNA synthesis. Furthermore, some of the results here presented are compatible with the occurence of inhibition, caused by the antibiotic, of one or more steps in the process of initiation of new polypeptide chains.
cells were grown in the following medium: 0.2% peptone, 0.2% yeast extract, and 0.5% glucose. The medium was sterilized in the autoclave at 121 C for 30 min. Cultures were grown in 1-liter Erlenmeyer flasks containing 500 ml of medium with shaking at 29 C in a New Brunswick gyratory shaker set at 125 turns per min. The generation time under these conditions was 3.8 h. Growth was measured by the absorption at 660 nm using a standard curve as reference. In vivo isotope incorporation. DNA, RNA, and protein synthesis in whole cells were estimated by the incorporation, respectively, of radioactive thymidine, uracil, and leucine into the cold trichloroacetic acidprecipitable cell fraction. For measurements of radioactivity 2-ml samples were withdrawn from the cell suspension and mixed with 2 ml of cold 10% trichloroacetic acid. The precipitates were collected on glass fiber filter disks and washed three times with cold 5% trichloroacetic acid. The filters were glued on aluminum planchets and dried, and the radioactivity was counted in a gas flow counter at 35% efficiency. Cell-free system. A detailed description of the procedure used for the preparation of the system is given elsewhere (J. F. 0. Carvalho, J. F. de Castro, N. Moussatche, and F. T. de Castro, manuscript in preparation). Briefly, a crude homogenate was obtained by treating the cells with 1% Nonidet P-40 in TMKG buffer [10 mM tris(hydroxymethyl)aminomethane-hydrochloride, pH 7.8; 5 mM KCl; 10 mM magnesium acetate; 6 mM reduced glutathione]. After removal of nuclei, mitochondria, and cell debris by centrifugation at 12,000 x g for 10 min, polysomes were collected at 33,000 rpm in the Spinco rotor 40 for 30 min through a discontinuous gradient made of 2 ml each of two sucrose solutions in TMKG buffer, one 20% and the other 5%. As a source of soluble components, a 0 to 0.7 saturated ammonium sulfate fraction MATERIALS AND METHODS prepared from the high speed supernatant was used. Incubation conditions for amino acid Organisms and conditions of cultivation. T. pyriformis GL was used throughout this work. The incorporation. The standard incubation mixture of 487
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0.25 ml contained: 0.5 pmol of adenosine triphosphate; 0.015 Amol of guanosine triphosphate; 1.5 Mmol of phosphoenolpyruvate; 3.2 U of phosphoenolpyruvate kinase; 12.5 umol of KCl; 2.5 gmol of magnesium acetate; 3 Mmol of glutathione; 25 umol of tris(hydroxymethyl)aminomethane-hydrochloride, pH 7.8; 0.05 Amol each of methionine, tryptophane, and cysteine; 0.1 tiCi of "4C-labeled amino acid mixture; 6 absorption units at 260 nm of polysome fraction; ammonium sulfate fraction, 60 ug of protein. Incubation was carried out at 28 C for 30 min. The reaction was stopped by addition of 0.25 ml of 10% trichloroacetic acid. The mixture was centrifuged and the residue was dissolved in 0.5 ml of 0.2 N NaOH and reprecipitated by adding 1 ml of 10% trichloroacetic acid. The samples were collected on Whatman GF/A glass fiber filters and washed three times with 5% trichloroacetic acid. The radioactivity was determined as indicated before. Preparation and zone centrifugation analysis of the ribosomes. Cell samples were chilled and washed twice by centrifugation at 1,000 x g for 1 min in TMK buffer [10 mM tris(hydroxymethyDEaminomethane-hydrochloride, pH 7.6; 7.5 mM magnesium acetate; 120 mM KCl] containing 200 Mg of cycloheximide per ml. All the subsequent steps were carried at 2 to 4 C. The washed cells were suspended in a volume of the same buffer equivalexnt to 1/50 of the volume of the original sample. Toluene was added for a final concentration of 65 gVml, and the preparation was vigorously shaken in a vortex mixer for two 30-s periods, being chilled in the interval. The crude cell lysate was centrifuged at 12,000 x g for 10 min and a portion of the supernatant was transferred to the top of a 10 to 30% sucrose gradient in TMK buffer constructed over a cushion of 0.9 ml of a 50% sucrose solution. The gradients were centrifuged at 2 C for 120 min at 25,000 rpm in the SW50.1 Spinco rotor. After centrifugation the solution was taken from the top of the tubes through an ISCO gradient analyser. The S values assigned to the components of the gradient are only approximate. Chemicals. Commercial sources of chemicals were as follows: adenosine triphosphate, disodium salt; guanosine triphosphate, sodium salt; phosphoenolpyruvate, trisodium salt; phosphoenolpyruvate kinase; glutathione, reduced form; cycloheximide (Sigma Chemical Co.); L-tryptophan; L-cysteine; Lmethionine (California Corporation for Biochemical Research); "4C-labeled L-amino acid mixture (uniformly labeled); ['C]leucine, 305 mCi/mmol (New England Nuclear); [14C]uracil, 62 mCi/mmol (Radiochemical Centre, Amersham); [methyl-14C]thymidine, 33 mCi/mmol (Schwartz/Mann); NX and Nonidet P-40 were generously supplied by Winthrop Products Co. and Shell Chemical Co., respectively.
RESULTS Effect of NX on population growth. The addition of NX to a culture of T. pyriformis developing exponentially resulted in growth suppression. As is shown in Fig. 1, within 1 h of exposure of the cells to the drug, growth had stopped completely. Examined under the mi-
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5 3 TIME-HOURS FIG. 1. Effect of nalidixic acid on the growth of T. pyriformis. At zero time the culture was divided into two portions and nalidixic acid, at a concentration of 50 gg/ml, was added to one portion. Samples were taken, at the indicated time intervals, for the determination of absorbance at 660 nm. Symbols: 0, nalidixic acid-treated cultures; *, control.
croscope during the time of incubation the cells were seen to move freely. Effect of NX on DNA, RNA, and protein synthesis. Figure 2 shows the effect of NX, at different concentrations, on the incorporations of thymidine (A), uracil (B), and leucine (C and D). For the first three experiments the cells were incubated for 30 min with the respective radioactive precursor before NX was added. In the experiment summarized in Fig. 2D, the precursor and the inhibitor were added at zero time. At the chosen time intervals portions were taken and treated for the measurement of radioactivity as indicated above. As can be seen (Fig. 2A-C), the kinetics of incorporation of all three precursors in the presence of the inhibitor were very similar. In each case, within 30 min of incubation with NX inhibition was already apparent for each one of the three levels of the drug. As shown in Fig. 2D, the inhibition was not reversible. Even when the concentration of NX was as low as 1 ,g/ml there was no indication of a significant recovery within 24 h of treatment. Effect of NX on amino acid incorporation in a cell-free system. In contrast with its effect on protein synthesis in whole cells of exponen-
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In a nonproliferating medium, Tetrahymena cells rapidly lose polyribosomes, accumulating 80S ribosomes (C. Samel and F. T. de Castro,
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manuscript in preparation). Under these circumstances the frequency of initiation of new polypeptide chains is expected to be relatively low. According to this reasoning we could infer that NX does not block peptide chain elongation. One possibility is that in Tetrahymena NX prevents the initiation of new polypeptide chains. If this were the case, there should be a decrease in amino acid incorporation if nongrowing cells were previously incubated with NX to allow for the completion of growing pep-
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possibility the fol-
lowing experiment was performed. Cells from an exponentially growing culture were collected by centrifugation, washed three times, and suspended in NPM buffer (10 mM disodium io so
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nesium acetate; 5 mM glucose). After 60 min the suspension was divided, and NX was added to one of the portions and the other was kept as a control, without addition. After 120
FIG. 2. Effect of nalidixic acid on the incorporation of thymidine, uracil, and leucine by cells of T. pyriformis. All cultures weere developing exponentilly. In experiments A, B, and C, the isotope was added at zero time; at 15 a?nd 30 min one sample of min, radioactive leucine was added to both suseach culture was taken for ra dioactivity measurement. pensions and, at the indicated time intervals, The cultures were then dit)ided into four portions: samples were taken for the determination of the one was kept as an untreated control; to the others radioactivity incorporated into the cold trinalidixic acid was added at different concentrations. chloroacetic acid fraction. The of Samples were taken at the chosen time intervals for the previous incubation with NX was a conthe determination of radio zctivity as described. In .. experiment D, the general procedure followed was spicuous inhibition of leucine incorporation the same except that leucin e and nalidixic acid were (Fig. 4). When uracil was used instead of leucine, under the same experimental conditions, added at zero time. (A) ["14 C]thymidine, 0.1 (B) [14C]uracil, 0.05 uCi/ml; (C) [V4C]eucine, 0.05 MgCi/ml; (A, B, C) 0, Control. Nalidixic acid addiTABLE 1. Effect of nalidixic acid on amino acid tions were as follows (1sg/ml): 0, 10; A, 20; *, 50. incorporation in a cell-free systema (D) 0, Control. Nalidixic acid additions (gg/ml): 0, 1; A, 2; *, 5. Counts/min per Mixture mg of protein consequence
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tially growing cultures, NX was not inhibitory Complete 3,271 to amino acid incorporation in a cell-free system Complete + nalidixic acid 3,129 (100 Ag/ml) (Table 1). Complete - ATP, GTP, 185 Effect of NX on protein synthesis in nonPEP, and PEPK proliferating cells. The antibiotic did not prevent protein synthesis in whole cells maintained a The standard incubation mixture of 0.25 ml in nonproliferating conditions. Under these cir- contained: 0.5 gmol of adenosine triphosphate (ATP); cumstances, the level of leucine incorporated 0.015 ;&mol of guanosine triphosphate (GTP); 1.5 was almost identical either in the presence or in Mmol of phosphoenolpyruvate (PEP); 3.2 U of phosphoenolpyruvate kinase (PEPK); 12.5 umol of KCl; the absence of the inhibitor (Fig. 3). In the cell-free experiment, the amino acid 2.5 umol of magnesium acetate; 3 Mmol of glutathione incorporated gave a measure of the amount of (reduced); 25 jmol of tris(hydroxymethyl)aminopH 7.8; 0.05 ,mol each of chain elongation performed by the system, since methane-hydrochloride, tryptophane, and cysteine; 0.1 MCi of under the conditions adopted there is no notice- methionine, amino acid mixture; 6 absorbancy units able recycling of the ribosomes, that is to say no at4C-labeled 260 nm of polysome fraction; ammonium sulfate manifest initiation of polypeptide chains occurs fraction, 60 ug of protein. Incubation was carried out (unpublished data). The same is most probably at 28 C for 30 min. Further treatment of the samples true for the experiment with nongrowing cells. and measurement of radioactivity were as described.
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transfer RNA: that is to say, they do not result from fragmentation of polysomal mRNA. It thus appears that the 80S particles accumulated in cells treated with NX are run-off ribosomes. This conclusion is in aggreement with the antagonistic effect exerted by cycloheximide on the degradation of polysomes in cells treated with NX. Cycloheximide is known to freeze polysomes of eucaryotic cells by blocking the translocation step in the ribosome cycle (18). This property makes the drug suitable to counteract the effect of disaggregating agents (2). In cells that had previously been incubated with cycloheximide, the polyribosome degradation in the presence of NX was much less conspicuous (Fig. 6). The effect of NX is also manifested in the formation of polysomes, as can be shown by the next experiment (Fig. 7). Cells of T. pyriformis rapidly lose their polysomes when they are washed and suspended, at a high population density, in NPM buffer. Transfer of the cells thus treated back into a fresh peptone medium resulted in almost complete polyribosome recovery within 45 min (Fig. 7A, B). When NX was present polyribosome formation was repressed (Fig. 7C).
FIG. 3. Effect of nalidixic acid on the incorporation of leucine in nonproliferating cells of T. pyriformis. Cells from an exponentially growing culture were collected by centrifugation at 1,000 x g for 1 min, washed three times, and suspended in 10 mM disodium hydrogen phosphate (pH 7.0), 3 mM magnesium acetate, and 5 mM glucose; the cell density was adusted to 0.3 absorbance units at 660 nm. The suspension was allowed to stand for 1 h and was dlivided into two portions: to one was added [14CJleuDISCUSSION cane (0.05 uCi/mb) and to the other ['4C]leucine, at The here indicate that the results presented the same concentration, plus nalidixic acid to a final concentration of 20 gg/ml. At the indicated time effect exerted by NX on Tetrahymena is more intervals samples were taken for the determination of general than that reported for Escherichia coli radioactivity as described. Symbols: 0, control; 0, nalidixic acid treated.
the level of radioactivity incorporated in the alkali-labile fraction was about the same either in the presence or in the absence of NX (not shown). Effect of NX on polyribosome stability and formation. The addition of NX to exponentially growing cultures was followed, in a relatively short time, by extensive degradation of cell polysomes with the accumulation of 80S particles. Figure 5 shows the level of the polysomes in cells treated for 10 min with NX at different concentrations. The nature of the 80S particles formed was investigated by probing their stability on high salt buffer. It has been shown (10, 22) that this treatment causes the dissociation of run-off ribosomes but not of the ribosomal units still attached to fragmented polysomal messenger (m)RNA. Figure 5E shows that the 80S peak formed after NX treatment was completely dissociated in the presence of 0.5 M KCl. This behavior indicates that such particles are not associated with mRNA fragments and peptidyl-
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FIG. 4. Effect of preincubation with nalidixic acid on leucine incorporation in nonproliferating cells. The general procedure followed was as described in the legend of Fig. 3, except that nalidixic acid was added to one of the suspensions 60 min before the addition of leucine. Symbols: *, control; 0, nalidixic acid treated.
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(7). In this organism, although the inhibition of DNA synthesis is demonstrable within a few minutes after the addition of the drug, RNA and protein production continue without modification for about one generation time. In r \ It Tetrahymena, DNA, RNA, and protein synthesis are inhibited simultaneously and to about -l the same extent. The kinetics of the incorpora3 l tion of the precursor in the presence of the inhibitor suggests that the inhibition of protein synthesis is not only a consequence of the block in RNA production. For if such were the case the decrease in the amino acid incorporation 03 l l - should correlate with the decay of mRNA as has been shown to happen in cells treated with actinomycin D (19). In S. cerevisiae, NX prevents DNA, RNA, 0o1 and protein synthesis coordinately and quickly _ \ l___\ after its addition to the culture medium; the inhibition lasts about 1 h and then total macror___ l l ll l l |molecular synthesis is resumed at a rate compaQ9 -i rable to that in the untreated cells (14). SimiD | 1t 1B I l l ll l | larly, in Tetrahymena the repression of macromolecular synthesis is an early event and, as stressed above, the synthesis of DNA, RNA, and Q7 protein are also coordinated. The salient character of the inhibition in Tetrahymena, in ll llt g 1 1 contrast with that observed in Saccharomyces, E l lil l l is that it is not transitory, but persists for at Osh least 24 h. NX has no lethal effect on Tetrahymena; cells continue to move freely for hours, even H1 S \ 1 when the drug concentration in the medium is 0.3 F || | 0.3 ~~~~~~~~~~relatively high. The protection exerted by cycloheximide against the disaggregating effect of NX on Tetrahymena polysomes indicates that the disI JL V \ j ruption 0.1 of these structures by the drug requires protein synthesis. On the other hand the fact X Top Bottom that amino acid incorporation in a cell-free system is not inhibited by NX speaks against a 0l9 El ^ possible puromycin-like mechanism of this antibiotic through a premature termination of r 11 1 ggrowing polypeptide chains (1, 15). || 2The general picture emerging from the experiOy A
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FIG. 5. State of the ribosomes in cells incubated with nalidixic acid. Five different cultures were treated as follows: (A) Control without addition. To the other cultures nalidixic acid was added at different concentrations (#g/ml): (B) 10; (C) 20; (D) and (E) 50. After 10 min the cells of all cultures were collected for the preparation and analysis of ribosomes: (A-D) as described in Materials and Methods; (E) KCI, at the final concentration of 500 mM, was added to the postmitochondrial fraction. The preparations were layered on a 5 to 20% linear sucrose gradient with a bottom layer of 50% sucrose solution, and centrifuged in an SW50.1 Spinco rotor for 3 h at 35,000 rpm.
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Bottom
FIG. 7. Effect of nalidixic acid on polysome reformation. Cells from an exponentially growing culture were collected and suspended in NPM buffer as FIG. 6. Cycloheximide-mediated prevention of described for Fig. 3, except that the cell density was polysome disaggregation in cells treated with nali- adjusted to 1.2 absorbance units at 660 nm. After dixic acid. Three cultures growing exponentially were resting for 10 min the suspension was divided into treated as follows: (A) a control without addition; (B) three portions: (A) immediately taken for the prepaincubated for 10 min with nalidixic acid (50 ,g/ml); ration of ribosomes; (B) diluted with fresh peptone (C) incubated for 15 min with cycloheximide (200 and (C) diluted with peptone medium 1.g/ml), followed bS nalidixic acid (50 1.g/ml) for 30 medium; containing nalidixic acid (20 ug/ml). After 45 min the min. The cells of each culture were collected by cells of both (B) and (C) were collected for the centrifugation and treated for the preparation of preparation of ribosomes. The ribosomes of all three ribosomes as indicated in Materials and Methods. samples were analyzed as indicated in Materials and Methods.
ments described here suggests that the effect of A complex of 40S/met-transfer RNA has now NX on protein synthesis in Tetrahymena is likely to be the result of a specific inhibition of been demonstrated in eucaryotic cells (6, 17). one or more steps required for the initiation of The formation of this complex is thought to constitute the first of three events that comprise polypeptide chains.
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the initiation of protein synthesis in eucaryotes; the other two complexes are formed by the successive addition of two new components: mRNA and 60S subunits (6). Work now in progress in our laboratory is aimed at examining the possible interference of NX with the formation of one or more of the above mentioned complexes.
10.
11.
12.
ACKNOWLEDGMENTS This work was supported in part by funds from Conselho Nacional de Pesquisas, Banco Nacional do Desenvolvimento Economico, contract FUNTEC-74, and by Conselho de Pesquisas da Universidade Federal do Rio de Janeiro.
LITERATURE CITED 1. Allen, D. W., and P. C. Zamecnik. 1962. The effect of puromycin on rabbit reticulocyte ribosomes. Biochim. Biophys. Acta 55:865-874. 2. Baliga, B. S., S. A. Cohen, and H. N. Munro, 1970. Effects of cycloheximide on the reaction of puromycin with polysome bound peptidyl t-RNA. FEBS Lett. 8:249-252. 3. Barbour, S. D. 1967. Effect of nalidixic acid on conjugational transfer and expression of episomal lac genes in E. coli K-12. J. Mol. Biol. 28:373-376. 4. Boyle, J. V., W. A. Goss, and T. M. Cook. 1967. Induction of excessive deoxyribonucleic acid synthesis in Escherichia coli by nalidixic acid. J. Bacteriol. 94:1664-1671. 5. Bourguignon, G. J., M. Levitt, and R. Sternglahz. 1973. Studies on the mechanism of action of nalidixic acid. Antimicrob. Agents Chemother. 4:479-486. 6. Darnbrough, C., S. Legon, T. Hunt, and R. J. Jackson. 1973. Initiation of protein synthesis: evidence for messenger RNA-independent binding of methionyltransfer RNA to the 40 S ribosomal subunit. J. Mol. Biol. 76:379-403. 7. Goss, W. A., W. H. Deitz, and T. M. Cook. 1964. Mechanism of action of nalidixic acid on Escherichia coli. J. Bacteriol. 88:1112-1118. 8. Goss, W. A., W. H. Deitz, and T. M. Cook. 1965. Mechanism of action of nalidixic acid on E. coli. H. Inhibition of DNA synthesis. J. Bacteriol. 89:10681074. 9. Hane, M. W., and T. W. Wood. 1969. Escherichia coli
13. 14.
15.
16.
17.
18.
19. 20. 21. 22.
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K-12 mutants resistant to nalidixic acid-genetic mapping and dominance studies. J. Bacteriol. 99:238-241. Lawford, G. R. 1969. The effect of incubation with puromycin on the dissociation of rat liver ribosomes into active subunits. Biochem. Biophys. Res. Commun. 37:143-150. Lescher, G. Y., E. J. Froelich, M. D. Gruett, J. H. Bailey, and R. P. Brundage. 1962. 1,8-Naphthyridine derivates. A new class of chemotherapeutic agents. J. Med. Pharm. Chem. 5:1063-1065. Luha, A. A., L. E. Sarcoe, and P. A. Whittaker. 1971. Biosynthesis of yeast mitochondrial drug effects on the petite negative yeast Kluyveromyces lactis. Biochem. Biophys. Res. Commun. 44:396-402. Lyman, H. 1967. Specific inhibition of chloroplast replication in Euglena gracilis by nalidixic acid. J. Cell. Biol. 35:726-733. Michels, C. A., J. Blamire, B. Goldfinger, and J. Marmur. 1973. Studies on the action of nalidixic acid in the yeast Saccharomyces cerevisiae. Antimicrob. Agents Chemother. 3:562-567. Nathans, D. 1964. Puromycin inhibition of protein synthesis: incorporation of puromycin into peptide chains. Proc. Natl. Acad. Sci. U.S.A. 51:585-592. Ramareddy, G., and H. Reiter. 1969. Specific loss of newly replicated deoxyribonucleic acid in nalidixic acid-treated Bacillus subtilis 168. J. Bacteriol. 100:724-729. Schreier, H., and T. Staehelin. 1974. Initiation of eukaryotic protein synthesis: (met-tRNA, .40 S ribosome) initiation complex catalyzed by purified initiation factors in the absence of mRNA. Nature (London) New Biol. 242:35-38. Siegel, M. R., and H. D. Sisler. 1964. Site of action of cycloheximide in cells of Saccharomyces pastorianus. II. The nature of inhibition of protein synthesis in a cell-free system. Biochim. Biophys. Acta 87:83-89. Singer, R. H., and S. Penman. 1972. Stability of HeLa cell mRNA in actinomycin. Nature (London) 240:100-102. Taketo, A., and H. Watanabe. 1967. Effect of nalidixic acid on growth of bacterial viruses. J. Biochem. Jpn. 61:520-522. Winshell, E. B., and H. S. Rosenkranz. 1970. Nalidixic acid and the metabolism of E. coli. J. Bacteriol. 104:1168-1175. Zilber, E. A., and S. Penman. 1970. The effect of high ionic strength on monomers, polyribosomes, and puromycin-treated polyribosomes. Biochim. Biophys. Acta 204:221-229.
Copyright i 1975 American Society for Microbiology
Vol 7, No. 5 Printed in U.S.A.
Nalidixic Acid and Macromolecular Metabolism in Tetrahymena pyriformis: Effects on Protein Synthesis J. F. DE CASTRO,* J. F. 0. CARVALHO, N. MOUSSATCH1I, AND F. T. DE CASTRO Laboratbrio de Metabolismo Macromolecular, Instituto de Bioffsica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil
Received for publication 25 November 1974
A study on the effect of nalidixic acid on macromolecular metabolism, particularly of protein, in Tetrahymena pyriformis was performed. It was shown that the compound is a potent inhibitor of deoxyribonucleic acid, ribonucleic acid, and protein synthesis for this organism. A conspicuous breakdown of polysomes, accompanied by the accumulation of 80S ribosomes, occurred in cells incubated for 10 min with the drug; polysome formation was prevented. The accumulating 80S particles were shown to be run-off ribosomal units. The incorporation of amino acids by a cell-free system is not affected by nalidixic acid. In nonproliferating cells the incorporation was also not prevented, unless the cells were previously incubated with the drug. These results are discussed in terms of the possible mechanism of action of nalidixic acid in T. pyriformis.
Since the synthesis by Lesher et al. in 1962 (11) of a series of naphthyridine derivatives, one of them, 1-ethyl-7-methyl-4-oxo-1,8-naphthyridine 3-carboxylic qcid, named nalidixic acid (NX), has called the attention of a number of workers (3, 4, 7, 9, 16, 20, 21). Accumulated evidence indicates that the drug specifically blocks deoxyribonucleic acid (DNA) synthesis in bacteria; ribonucleic acid (RNA) and protein synthesis are not directly affected (8). A few reports have appeared bearing on the effect of NX on eucaryotic systems. The restults obtained so far are not particularly elucidative of the mechanism of action of the drug. Growth of the yeast Kluyveromyces lactis is arrested and a conspicuous and preferential repression of mitochondriajl DNA synthesis is observed (12). In Saccharomyces cerevisiae no selective inhibition of DNA could be demonstrated. Instead a transient block of total DNA, RNA, and protein synthesis was found (14). In Euglena gracilis NX blocks chloroplast replication (13). In this paper work is reported which shows that NX blocks the synthesis of DNA, RNA, and protein in cells of Tetrahymena pyriformis. The inhibition of protein synthesis is not transient and seems to be independent of the repression of RNA synthesis. Furthermore, some of the results here presented are compatible with the occurence of inhibition, caused by the antibiotic, of one or more steps in the process of initiation of new polypeptide chains.
cells were grown in the following medium: 0.2% peptone, 0.2% yeast extract, and 0.5% glucose. The medium was sterilized in the autoclave at 121 C for 30 min. Cultures were grown in 1-liter Erlenmeyer flasks containing 500 ml of medium with shaking at 29 C in a New Brunswick gyratory shaker set at 125 turns per min. The generation time under these conditions was 3.8 h. Growth was measured by the absorption at 660 nm using a standard curve as reference. In vivo isotope incorporation. DNA, RNA, and protein synthesis in whole cells were estimated by the incorporation, respectively, of radioactive thymidine, uracil, and leucine into the cold trichloroacetic acidprecipitable cell fraction. For measurements of radioactivity 2-ml samples were withdrawn from the cell suspension and mixed with 2 ml of cold 10% trichloroacetic acid. The precipitates were collected on glass fiber filter disks and washed three times with cold 5% trichloroacetic acid. The filters were glued on aluminum planchets and dried, and the radioactivity was counted in a gas flow counter at 35% efficiency. Cell-free system. A detailed description of the procedure used for the preparation of the system is given elsewhere (J. F. 0. Carvalho, J. F. de Castro, N. Moussatche, and F. T. de Castro, manuscript in preparation). Briefly, a crude homogenate was obtained by treating the cells with 1% Nonidet P-40 in TMKG buffer [10 mM tris(hydroxymethyl)aminomethane-hydrochloride, pH 7.8; 5 mM KCl; 10 mM magnesium acetate; 6 mM reduced glutathione]. After removal of nuclei, mitochondria, and cell debris by centrifugation at 12,000 x g for 10 min, polysomes were collected at 33,000 rpm in the Spinco rotor 40 for 30 min through a discontinuous gradient made of 2 ml each of two sucrose solutions in TMKG buffer, one 20% and the other 5%. As a source of soluble components, a 0 to 0.7 saturated ammonium sulfate fraction MATERIALS AND METHODS prepared from the high speed supernatant was used. Incubation conditions for amino acid Organisms and conditions of cultivation. T. pyriformis GL was used throughout this work. The incorporation. The standard incubation mixture of 487
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0.25 ml contained: 0.5 pmol of adenosine triphosphate; 0.015 Amol of guanosine triphosphate; 1.5 Mmol of phosphoenolpyruvate; 3.2 U of phosphoenolpyruvate kinase; 12.5 umol of KCl; 2.5 gmol of magnesium acetate; 3 Mmol of glutathione; 25 umol of tris(hydroxymethyl)aminomethane-hydrochloride, pH 7.8; 0.05 Amol each of methionine, tryptophane, and cysteine; 0.1 tiCi of "4C-labeled amino acid mixture; 6 absorption units at 260 nm of polysome fraction; ammonium sulfate fraction, 60 ug of protein. Incubation was carried out at 28 C for 30 min. The reaction was stopped by addition of 0.25 ml of 10% trichloroacetic acid. The mixture was centrifuged and the residue was dissolved in 0.5 ml of 0.2 N NaOH and reprecipitated by adding 1 ml of 10% trichloroacetic acid. The samples were collected on Whatman GF/A glass fiber filters and washed three times with 5% trichloroacetic acid. The radioactivity was determined as indicated before. Preparation and zone centrifugation analysis of the ribosomes. Cell samples were chilled and washed twice by centrifugation at 1,000 x g for 1 min in TMK buffer [10 mM tris(hydroxymethyDEaminomethane-hydrochloride, pH 7.6; 7.5 mM magnesium acetate; 120 mM KCl] containing 200 Mg of cycloheximide per ml. All the subsequent steps were carried at 2 to 4 C. The washed cells were suspended in a volume of the same buffer equivalexnt to 1/50 of the volume of the original sample. Toluene was added for a final concentration of 65 gVml, and the preparation was vigorously shaken in a vortex mixer for two 30-s periods, being chilled in the interval. The crude cell lysate was centrifuged at 12,000 x g for 10 min and a portion of the supernatant was transferred to the top of a 10 to 30% sucrose gradient in TMK buffer constructed over a cushion of 0.9 ml of a 50% sucrose solution. The gradients were centrifuged at 2 C for 120 min at 25,000 rpm in the SW50.1 Spinco rotor. After centrifugation the solution was taken from the top of the tubes through an ISCO gradient analyser. The S values assigned to the components of the gradient are only approximate. Chemicals. Commercial sources of chemicals were as follows: adenosine triphosphate, disodium salt; guanosine triphosphate, sodium salt; phosphoenolpyruvate, trisodium salt; phosphoenolpyruvate kinase; glutathione, reduced form; cycloheximide (Sigma Chemical Co.); L-tryptophan; L-cysteine; Lmethionine (California Corporation for Biochemical Research); "4C-labeled L-amino acid mixture (uniformly labeled); ['C]leucine, 305 mCi/mmol (New England Nuclear); [14C]uracil, 62 mCi/mmol (Radiochemical Centre, Amersham); [methyl-14C]thymidine, 33 mCi/mmol (Schwartz/Mann); NX and Nonidet P-40 were generously supplied by Winthrop Products Co. and Shell Chemical Co., respectively.
RESULTS Effect of NX on population growth. The addition of NX to a culture of T. pyriformis developing exponentially resulted in growth suppression. As is shown in Fig. 1, within 1 h of exposure of the cells to the drug, growth had stopped completely. Examined under the mi-
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5 3 TIME-HOURS FIG. 1. Effect of nalidixic acid on the growth of T. pyriformis. At zero time the culture was divided into two portions and nalidixic acid, at a concentration of 50 gg/ml, was added to one portion. Samples were taken, at the indicated time intervals, for the determination of absorbance at 660 nm. Symbols: 0, nalidixic acid-treated cultures; *, control.
croscope during the time of incubation the cells were seen to move freely. Effect of NX on DNA, RNA, and protein synthesis. Figure 2 shows the effect of NX, at different concentrations, on the incorporations of thymidine (A), uracil (B), and leucine (C and D). For the first three experiments the cells were incubated for 30 min with the respective radioactive precursor before NX was added. In the experiment summarized in Fig. 2D, the precursor and the inhibitor were added at zero time. At the chosen time intervals portions were taken and treated for the measurement of radioactivity as indicated above. As can be seen (Fig. 2A-C), the kinetics of incorporation of all three precursors in the presence of the inhibitor were very similar. In each case, within 30 min of incubation with NX inhibition was already apparent for each one of the three levels of the drug. As shown in Fig. 2D, the inhibition was not reversible. Even when the concentration of NX was as low as 1 ,g/ml there was no indication of a significant recovery within 24 h of treatment. Effect of NX on amino acid incorporation in a cell-free system. In contrast with its effect on protein synthesis in whole cells of exponen-
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NALIDIXIC ACID AND PROTEIN SYNTHESIS
/
In a nonproliferating medium, Tetrahymena cells rapidly lose polyribosomes, accumulating 80S ribosomes (C. Samel and F. T. de Castro,
/-t~
manuscript in preparation). Under these circumstances the frequency of initiation of new polypeptide chains is expected to be relatively low. According to this reasoning we could infer that NX does not block peptide chain elongation. One possibility is that in Tetrahymena NX prevents the initiation of new polypeptide chains. If this were the case, there should be a decrease in amino acid incorporation if nongrowing cells were previously incubated with NX to allow for the completion of growing pep-
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10
489
00
TM-mum
o
tide chains. To check this
possibility the fol-
lowing experiment was performed. Cells from an exponentially growing culture were collected by centrifugation, washed three times, and suspended in NPM buffer (10 mM disodium io so
so To-MUTES
D
soi
5
10
\/-14
hydrogen phosphate, pH 7.0; 3 mM
mag-
nesium acetate; 5 mM glucose). After 60 min the suspension was divided, and NX was added to one of the portions and the other was kept as a control, without addition. After 120
FIG. 2. Effect of nalidixic acid on the incorporation of thymidine, uracil, and leucine by cells of T. pyriformis. All cultures weere developing exponentilly. In experiments A, B, and C, the isotope was added at zero time; at 15 a?nd 30 min one sample of min, radioactive leucine was added to both suseach culture was taken for ra dioactivity measurement. pensions and, at the indicated time intervals, The cultures were then dit)ided into four portions: samples were taken for the determination of the one was kept as an untreated control; to the others radioactivity incorporated into the cold trinalidixic acid was added at different concentrations. chloroacetic acid fraction. The of Samples were taken at the chosen time intervals for the previous incubation with NX was a conthe determination of radio zctivity as described. In .. experiment D, the general procedure followed was spicuous inhibition of leucine incorporation the same except that leucin e and nalidixic acid were (Fig. 4). When uracil was used instead of leucine, under the same experimental conditions, added at zero time. (A) ["14 C]thymidine, 0.1 (B) [14C]uracil, 0.05 uCi/ml; (C) [V4C]eucine, 0.05 MgCi/ml; (A, B, C) 0, Control. Nalidixic acid addiTABLE 1. Effect of nalidixic acid on amino acid tions were as follows (1sg/ml): 0, 10; A, 20; *, 50. incorporation in a cell-free systema (D) 0, Control. Nalidixic acid additions (gg/ml): 0, 1; A, 2; *, 5. Counts/min per Mixture mg of protein consequence
aCi/mld
tially growing cultures, NX was not inhibitory Complete 3,271 to amino acid incorporation in a cell-free system Complete + nalidixic acid 3,129 (100 Ag/ml) (Table 1). Complete - ATP, GTP, 185 Effect of NX on protein synthesis in nonPEP, and PEPK proliferating cells. The antibiotic did not prevent protein synthesis in whole cells maintained a The standard incubation mixture of 0.25 ml in nonproliferating conditions. Under these cir- contained: 0.5 gmol of adenosine triphosphate (ATP); cumstances, the level of leucine incorporated 0.015 ;&mol of guanosine triphosphate (GTP); 1.5 was almost identical either in the presence or in Mmol of phosphoenolpyruvate (PEP); 3.2 U of phosphoenolpyruvate kinase (PEPK); 12.5 umol of KCl; the absence of the inhibitor (Fig. 3). In the cell-free experiment, the amino acid 2.5 umol of magnesium acetate; 3 Mmol of glutathione incorporated gave a measure of the amount of (reduced); 25 jmol of tris(hydroxymethyl)aminopH 7.8; 0.05 ,mol each of chain elongation performed by the system, since methane-hydrochloride, tryptophane, and cysteine; 0.1 MCi of under the conditions adopted there is no notice- methionine, amino acid mixture; 6 absorbancy units able recycling of the ribosomes, that is to say no at4C-labeled 260 nm of polysome fraction; ammonium sulfate manifest initiation of polypeptide chains occurs fraction, 60 ug of protein. Incubation was carried out (unpublished data). The same is most probably at 28 C for 30 min. Further treatment of the samples true for the experiment with nongrowing cells. and measurement of radioactivity were as described.
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TIME-MINUTES
transfer RNA: that is to say, they do not result from fragmentation of polysomal mRNA. It thus appears that the 80S particles accumulated in cells treated with NX are run-off ribosomes. This conclusion is in aggreement with the antagonistic effect exerted by cycloheximide on the degradation of polysomes in cells treated with NX. Cycloheximide is known to freeze polysomes of eucaryotic cells by blocking the translocation step in the ribosome cycle (18). This property makes the drug suitable to counteract the effect of disaggregating agents (2). In cells that had previously been incubated with cycloheximide, the polyribosome degradation in the presence of NX was much less conspicuous (Fig. 6). The effect of NX is also manifested in the formation of polysomes, as can be shown by the next experiment (Fig. 7). Cells of T. pyriformis rapidly lose their polysomes when they are washed and suspended, at a high population density, in NPM buffer. Transfer of the cells thus treated back into a fresh peptone medium resulted in almost complete polyribosome recovery within 45 min (Fig. 7A, B). When NX was present polyribosome formation was repressed (Fig. 7C).
FIG. 3. Effect of nalidixic acid on the incorporation of leucine in nonproliferating cells of T. pyriformis. Cells from an exponentially growing culture were collected by centrifugation at 1,000 x g for 1 min, washed three times, and suspended in 10 mM disodium hydrogen phosphate (pH 7.0), 3 mM magnesium acetate, and 5 mM glucose; the cell density was adusted to 0.3 absorbance units at 660 nm. The suspension was allowed to stand for 1 h and was dlivided into two portions: to one was added [14CJleuDISCUSSION cane (0.05 uCi/mb) and to the other ['4C]leucine, at The here indicate that the results presented the same concentration, plus nalidixic acid to a final concentration of 20 gg/ml. At the indicated time effect exerted by NX on Tetrahymena is more intervals samples were taken for the determination of general than that reported for Escherichia coli radioactivity as described. Symbols: 0, control; 0, nalidixic acid treated.
the level of radioactivity incorporated in the alkali-labile fraction was about the same either in the presence or in the absence of NX (not shown). Effect of NX on polyribosome stability and formation. The addition of NX to exponentially growing cultures was followed, in a relatively short time, by extensive degradation of cell polysomes with the accumulation of 80S particles. Figure 5 shows the level of the polysomes in cells treated for 10 min with NX at different concentrations. The nature of the 80S particles formed was investigated by probing their stability on high salt buffer. It has been shown (10, 22) that this treatment causes the dissociation of run-off ribosomes but not of the ribosomal units still attached to fragmented polysomal messenger (m)RNA. Figure 5E shows that the 80S peak formed after NX treatment was completely dissociated in the presence of 0.5 M KCl. This behavior indicates that such particles are not associated with mRNA fragments and peptidyl-
15
10 a.
X )
TIME-MINUTES
FIG. 4. Effect of preincubation with nalidixic acid on leucine incorporation in nonproliferating cells. The general procedure followed was as described in the legend of Fig. 3, except that nalidixic acid was added to one of the suspensions 60 min before the addition of leucine. Symbols: *, control; 0, nalidixic acid treated.
NALIDIXIC ACID AND PROTEIN SYNTHESIS
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491
(7). In this organism, although the inhibition of DNA synthesis is demonstrable within a few minutes after the addition of the drug, RNA and protein production continue without modification for about one generation time. In r \ It Tetrahymena, DNA, RNA, and protein synthesis are inhibited simultaneously and to about -l the same extent. The kinetics of the incorpora3 l tion of the precursor in the presence of the inhibitor suggests that the inhibition of protein synthesis is not only a consequence of the block in RNA production. For if such were the case the decrease in the amino acid incorporation 03 l l - should correlate with the decay of mRNA as has been shown to happen in cells treated with actinomycin D (19). In S. cerevisiae, NX prevents DNA, RNA, 0o1 and protein synthesis coordinately and quickly _ \ l___\ after its addition to the culture medium; the inhibition lasts about 1 h and then total macror___ l l ll l l |molecular synthesis is resumed at a rate compaQ9 -i rable to that in the untreated cells (14). SimiD | 1t 1B I l l ll l | larly, in Tetrahymena the repression of macromolecular synthesis is an early event and, as stressed above, the synthesis of DNA, RNA, and Q7 protein are also coordinated. The salient character of the inhibition in Tetrahymena, in ll llt g 1 1 contrast with that observed in Saccharomyces, E l lil l l is that it is not transitory, but persists for at Osh least 24 h. NX has no lethal effect on Tetrahymena; cells continue to move freely for hours, even H1 S \ 1 when the drug concentration in the medium is 0.3 F || | 0.3 ~~~~~~~~~~relatively high. The protection exerted by cycloheximide against the disaggregating effect of NX on Tetrahymena polysomes indicates that the disI JL V \ j ruption 0.1 of these structures by the drug requires protein synthesis. On the other hand the fact X Top Bottom that amino acid incorporation in a cell-free system is not inhibited by NX speaks against a 0l9 El ^ possible puromycin-like mechanism of this antibiotic through a premature termination of r 11 1 ggrowing polypeptide chains (1, 15). || 2The general picture emerging from the experiOy A
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FIG. 5. State of the ribosomes in cells incubated with nalidixic acid. Five different cultures were treated as follows: (A) Control without addition. To the other cultures nalidixic acid was added at different concentrations (#g/ml): (B) 10; (C) 20; (D) and (E) 50. After 10 min the cells of all cultures were collected for the preparation and analysis of ribosomes: (A-D) as described in Materials and Methods; (E) KCI, at the final concentration of 500 mM, was added to the postmitochondrial fraction. The preparations were layered on a 5 to 20% linear sucrose gradient with a bottom layer of 50% sucrose solution, and centrifuged in an SW50.1 Spinco rotor for 3 h at 35,000 rpm.
492
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ANTiMICROB. AGENTS CHEMOTHER.
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Ec
EC
Top
Bottom
FIG. 7. Effect of nalidixic acid on polysome reformation. Cells from an exponentially growing culture were collected and suspended in NPM buffer as FIG. 6. Cycloheximide-mediated prevention of described for Fig. 3, except that the cell density was polysome disaggregation in cells treated with nali- adjusted to 1.2 absorbance units at 660 nm. After dixic acid. Three cultures growing exponentially were resting for 10 min the suspension was divided into treated as follows: (A) a control without addition; (B) three portions: (A) immediately taken for the prepaincubated for 10 min with nalidixic acid (50 ,g/ml); ration of ribosomes; (B) diluted with fresh peptone (C) incubated for 15 min with cycloheximide (200 and (C) diluted with peptone medium 1.g/ml), followed bS nalidixic acid (50 1.g/ml) for 30 medium; containing nalidixic acid (20 ug/ml). After 45 min the min. The cells of each culture were collected by cells of both (B) and (C) were collected for the centrifugation and treated for the preparation of preparation of ribosomes. The ribosomes of all three ribosomes as indicated in Materials and Methods. samples were analyzed as indicated in Materials and Methods.
ments described here suggests that the effect of A complex of 40S/met-transfer RNA has now NX on protein synthesis in Tetrahymena is likely to be the result of a specific inhibition of been demonstrated in eucaryotic cells (6, 17). one or more steps required for the initiation of The formation of this complex is thought to constitute the first of three events that comprise polypeptide chains.
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NALIDIXIC ACID AND PROTEIN SYNTHESIS
the initiation of protein synthesis in eucaryotes; the other two complexes are formed by the successive addition of two new components: mRNA and 60S subunits (6). Work now in progress in our laboratory is aimed at examining the possible interference of NX with the formation of one or more of the above mentioned complexes.
10.
11.
12.
ACKNOWLEDGMENTS This work was supported in part by funds from Conselho Nacional de Pesquisas, Banco Nacional do Desenvolvimento Economico, contract FUNTEC-74, and by Conselho de Pesquisas da Universidade Federal do Rio de Janeiro.
LITERATURE CITED 1. Allen, D. W., and P. C. Zamecnik. 1962. The effect of puromycin on rabbit reticulocyte ribosomes. Biochim. Biophys. Acta 55:865-874. 2. Baliga, B. S., S. A. Cohen, and H. N. Munro, 1970. Effects of cycloheximide on the reaction of puromycin with polysome bound peptidyl t-RNA. FEBS Lett. 8:249-252. 3. Barbour, S. D. 1967. Effect of nalidixic acid on conjugational transfer and expression of episomal lac genes in E. coli K-12. J. Mol. Biol. 28:373-376. 4. Boyle, J. V., W. A. Goss, and T. M. Cook. 1967. Induction of excessive deoxyribonucleic acid synthesis in Escherichia coli by nalidixic acid. J. Bacteriol. 94:1664-1671. 5. Bourguignon, G. J., M. Levitt, and R. Sternglahz. 1973. Studies on the mechanism of action of nalidixic acid. Antimicrob. Agents Chemother. 4:479-486. 6. Darnbrough, C., S. Legon, T. Hunt, and R. J. Jackson. 1973. Initiation of protein synthesis: evidence for messenger RNA-independent binding of methionyltransfer RNA to the 40 S ribosomal subunit. J. Mol. Biol. 76:379-403. 7. Goss, W. A., W. H. Deitz, and T. M. Cook. 1964. Mechanism of action of nalidixic acid on Escherichia coli. J. Bacteriol. 88:1112-1118. 8. Goss, W. A., W. H. Deitz, and T. M. Cook. 1965. Mechanism of action of nalidixic acid on E. coli. H. Inhibition of DNA synthesis. J. Bacteriol. 89:10681074. 9. Hane, M. W., and T. W. Wood. 1969. Escherichia coli
13. 14.
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K-12 mutants resistant to nalidixic acid-genetic mapping and dominance studies. J. Bacteriol. 99:238-241. Lawford, G. R. 1969. The effect of incubation with puromycin on the dissociation of rat liver ribosomes into active subunits. Biochem. Biophys. Res. Commun. 37:143-150. Lescher, G. Y., E. J. Froelich, M. D. Gruett, J. H. Bailey, and R. P. Brundage. 1962. 1,8-Naphthyridine derivates. A new class of chemotherapeutic agents. J. Med. Pharm. Chem. 5:1063-1065. Luha, A. A., L. E. Sarcoe, and P. A. Whittaker. 1971. Biosynthesis of yeast mitochondrial drug effects on the petite negative yeast Kluyveromyces lactis. Biochem. Biophys. Res. Commun. 44:396-402. Lyman, H. 1967. Specific inhibition of chloroplast replication in Euglena gracilis by nalidixic acid. J. Cell. Biol. 35:726-733. Michels, C. A., J. Blamire, B. Goldfinger, and J. Marmur. 1973. Studies on the action of nalidixic acid in the yeast Saccharomyces cerevisiae. Antimicrob. Agents Chemother. 3:562-567. Nathans, D. 1964. Puromycin inhibition of protein synthesis: incorporation of puromycin into peptide chains. Proc. Natl. Acad. Sci. U.S.A. 51:585-592. Ramareddy, G., and H. Reiter. 1969. Specific loss of newly replicated deoxyribonucleic acid in nalidixic acid-treated Bacillus subtilis 168. J. Bacteriol. 100:724-729. Schreier, H., and T. Staehelin. 1974. Initiation of eukaryotic protein synthesis: (met-tRNA, .40 S ribosome) initiation complex catalyzed by purified initiation factors in the absence of mRNA. Nature (London) New Biol. 242:35-38. Siegel, M. R., and H. D. Sisler. 1964. Site of action of cycloheximide in cells of Saccharomyces pastorianus. II. The nature of inhibition of protein synthesis in a cell-free system. Biochim. Biophys. Acta 87:83-89. Singer, R. H., and S. Penman. 1972. Stability of HeLa cell mRNA in actinomycin. Nature (London) 240:100-102. Taketo, A., and H. Watanabe. 1967. Effect of nalidixic acid on growth of bacterial viruses. J. Biochem. Jpn. 61:520-522. Winshell, E. B., and H. S. Rosenkranz. 1970. Nalidixic acid and the metabolism of E. coli. J. Bacteriol. 104:1168-1175. Zilber, E. A., and S. Penman. 1970. The effect of high ionic strength on monomers, polyribosomes, and puromycin-treated polyribosomes. Biochim. Biophys. Acta 204:221-229.
