Cell, Vol. 13. 329-334.
February
Mistranslation
1978, Copyright
Q 1978 by MIT
in a Eucaryotic
Edward Palmer and James M. Wilhelm Department of Microbiology University of Rochester School of Medicine and Dentistry Rochester. New York 14642
Summary Previous work from our laboratory has demonstrated that a subclass of the aminoglycoside antibiotics, those containing the drug fragment paromamine, stimulates mistranslatlon in cellfree protein-synthesizing systems derived from eucaryotlc cells. We report here experiments which show that the ciliate Tetrahymena thermophila (formerly T. pyriformis, syngen 1) is sensltlve to the paromamlne-containing aminoglycoside antibiotics. The drugs are active with respect to growth inhibition, inhibition of protein synthesis In the whole organism, inhibition of protein synthesis in vitro and the stimulation of mistranslation in cell-free protein-synthesizing systems. Because of their misreading properties, these drugs may be useful in isolating and prop agating strains carrying mutatfons which can be translationally suppressed (that Is, missense and nonsense mutations). Introduction Gorini and his colleagues have demonstrated that streptomycin can suppress ‘a number of auxotrophic mutations in E. coli (for review, see Gorini, 1974). This phenomenon has been called phenotypic suppression and most probably results from the mistranslation of RNA, which occurs in the presence of this antibiotic (Davies, Gilbert and Gorini, 1964; Friedman and Weinstein, 1964). Previous work has shown that streptomycin is inactive with cytoplasmic ribosomes derived from eucaryotic organisms (Weinstein, Ochoa and Friedman, 1966; Friedman, Berezney and Weinstein, 1968). There are a large number of other aminoglycoside antibiotics whose structures are detailed elsewhere (for example, Benveniste and Davies, 1973), and Wilhelm, Pettit and Jessop (1978) have recently identified a specific subclass of the aminoglycosides which can stimulate misreading in cellfree protein-synthesizing systems derived from eucaryotic cells. These particular aminoglycosides are distinguished by the fact that they all contain the drug fragment paromamine (or 3’-deoxyparomamine). Paromamaine is a rather simple two-ring molecule, consisting of D-glucosamine linked to an aminocycylitol, 2-deoxystreptamine.
Organism
It would be extremely useful to distinguish a genetically manipulable eucaryotic organism which is sensitive to these agents. Utilizing these drugs, one could potentially isolate and propagate strains carrying mutations which can be translationally suppressed (nonsense and missense mutations). We have found that the ciliate Tetrahymena thermophila is sensitive to the paromamine-containing aminoglycoside antibiotics, and report experiments designed to examine the action of these antibiotics on protein synthesis in this organism.
Results We were interested in distinguishing a eucaryotic organism which is sensitive in vivo to aminoglycoside antibiotics known to stimulate eucaryotic ribosomes to mistranslate RNA in cell-free systems (Wilhelm et al., 1978).
Growth Is Sensitive to Aminoglycosides Of the drugs which are active in eucaryotic systems, paromomycin is the most readily available. The data of Figure 1 show that the growth of Tetrahymena thermophila is extremely sensitive to paromomycin. Increasing amounts of the drug increasingly inhibit growth until it is completely eliminated in 100 PM paromomycin. We were able to determine the effects of paromomycin on cell viability by measuring the number of cells which formed clones when singly dispersed in medium without drug after various lengths of exposure to paromomycin. The data in Table 1 indicate that while 50 PM paromomycin virtually eliminates growth, it does not decrease cell viability even after 25 hr. On the other hand, 500 PM paromomycin drastically reduces cell viability after only 5 hr. Only a certain subclass of the aminoglycosides is active in causing high levels of mistranslation in eucaryotic cell-free systems. We were therefore interested in testing a variety of aminoglycosides with respect to their effect on the growth of Tetrahymena. The data in Table 2A compare the inhibition of net growth by a variety of aminoglycosides. Lividomycin B, paromomycin, paromamine, gentamicin A and a commercial preparation of kanamycin (which contains 10% kanamycin C as determined by paper chromatography) are particularly inhibitory, while the other aminoglycosides (including kanamycins A and 8) are comparatively inactive. The data in Table 28 compare paromomycin, paromamine, neomycin and neamine at equimolar concentrations. Neamine and paromamine are fragments of neomycin and paromomycin, respec-
Cdl 330
Table 2. The Effect of TetrahymenaO
of Aminoglycoside
on the Growth
(PM)
% Inhibition Net Growth
167 141 310 141 310 177 177 177 172 172 172
98 97.5 93 4 3 31 17 5 28 2 9
141 154
0 6
141 141 141 141
97.5 13 4 0
Concentration Antibiotic
Antibiotics
of
A
I
0
4
8
I
12 TIME
Figure
1. Effect
of Paromomycin
Cells were grown (O+) Control; pM paromomycin; pM paromomycin.
Table
1. The Effect
Paromomycin Concentration WV
1
16
20
24
(hrs.)
in SPP medium in shaking cultures at 30°C. (O-O) 10 AM paromomycin; (A-A) 25 (A-A) 50 $I paromomycin; (U+) 100
Time of Exposure
(hr)
0
A B CIA (A,B,C)b A B Cb
0
on Cell Growth
of Paromomycin
Lividomycin Paromomycin Paromamine Neomycin Neamine Gentamicin Gentamicin Gentamicin Kanamycin Kanamycin Kanamycin Kanamycin Streptomycin Butirosin
on Cell Viability
Viable
Cells
0
0 5 25
23,100 61,000 1,450,000
50
0 5 25
23,100 65,600 65,000
500
0 5 25
23,100 400
February
Mistranslation
1978, Copyright
Q 1978 by MIT
in a Eucaryotic
Edward Palmer and James M. Wilhelm Department of Microbiology University of Rochester School of Medicine and Dentistry Rochester. New York 14642
Summary Previous work from our laboratory has demonstrated that a subclass of the aminoglycoside antibiotics, those containing the drug fragment paromamine, stimulates mistranslatlon in cellfree protein-synthesizing systems derived from eucaryotlc cells. We report here experiments which show that the ciliate Tetrahymena thermophila (formerly T. pyriformis, syngen 1) is sensltlve to the paromamlne-containing aminoglycoside antibiotics. The drugs are active with respect to growth inhibition, inhibition of protein synthesis In the whole organism, inhibition of protein synthesis in vitro and the stimulation of mistranslation in cell-free protein-synthesizing systems. Because of their misreading properties, these drugs may be useful in isolating and prop agating strains carrying mutatfons which can be translationally suppressed (that Is, missense and nonsense mutations). Introduction Gorini and his colleagues have demonstrated that streptomycin can suppress ‘a number of auxotrophic mutations in E. coli (for review, see Gorini, 1974). This phenomenon has been called phenotypic suppression and most probably results from the mistranslation of RNA, which occurs in the presence of this antibiotic (Davies, Gilbert and Gorini, 1964; Friedman and Weinstein, 1964). Previous work has shown that streptomycin is inactive with cytoplasmic ribosomes derived from eucaryotic organisms (Weinstein, Ochoa and Friedman, 1966; Friedman, Berezney and Weinstein, 1968). There are a large number of other aminoglycoside antibiotics whose structures are detailed elsewhere (for example, Benveniste and Davies, 1973), and Wilhelm, Pettit and Jessop (1978) have recently identified a specific subclass of the aminoglycosides which can stimulate misreading in cellfree protein-synthesizing systems derived from eucaryotic cells. These particular aminoglycosides are distinguished by the fact that they all contain the drug fragment paromamine (or 3’-deoxyparomamine). Paromamaine is a rather simple two-ring molecule, consisting of D-glucosamine linked to an aminocycylitol, 2-deoxystreptamine.
Organism
It would be extremely useful to distinguish a genetically manipulable eucaryotic organism which is sensitive to these agents. Utilizing these drugs, one could potentially isolate and propagate strains carrying mutations which can be translationally suppressed (nonsense and missense mutations). We have found that the ciliate Tetrahymena thermophila is sensitive to the paromamine-containing aminoglycoside antibiotics, and report experiments designed to examine the action of these antibiotics on protein synthesis in this organism.
Results We were interested in distinguishing a eucaryotic organism which is sensitive in vivo to aminoglycoside antibiotics known to stimulate eucaryotic ribosomes to mistranslate RNA in cell-free systems (Wilhelm et al., 1978).
Growth Is Sensitive to Aminoglycosides Of the drugs which are active in eucaryotic systems, paromomycin is the most readily available. The data of Figure 1 show that the growth of Tetrahymena thermophila is extremely sensitive to paromomycin. Increasing amounts of the drug increasingly inhibit growth until it is completely eliminated in 100 PM paromomycin. We were able to determine the effects of paromomycin on cell viability by measuring the number of cells which formed clones when singly dispersed in medium without drug after various lengths of exposure to paromomycin. The data in Table 1 indicate that while 50 PM paromomycin virtually eliminates growth, it does not decrease cell viability even after 25 hr. On the other hand, 500 PM paromomycin drastically reduces cell viability after only 5 hr. Only a certain subclass of the aminoglycosides is active in causing high levels of mistranslation in eucaryotic cell-free systems. We were therefore interested in testing a variety of aminoglycosides with respect to their effect on the growth of Tetrahymena. The data in Table 2A compare the inhibition of net growth by a variety of aminoglycosides. Lividomycin B, paromomycin, paromamine, gentamicin A and a commercial preparation of kanamycin (which contains 10% kanamycin C as determined by paper chromatography) are particularly inhibitory, while the other aminoglycosides (including kanamycins A and 8) are comparatively inactive. The data in Table 28 compare paromomycin, paromamine, neomycin and neamine at equimolar concentrations. Neamine and paromamine are fragments of neomycin and paromomycin, respec-
Cdl 330
Table 2. The Effect of TetrahymenaO
of Aminoglycoside
on the Growth
(PM)
% Inhibition Net Growth
167 141 310 141 310 177 177 177 172 172 172
98 97.5 93 4 3 31 17 5 28 2 9
141 154
0 6
141 141 141 141
97.5 13 4 0
Concentration Antibiotic
Antibiotics
of
A
I
0
4
8
I
12 TIME
Figure
1. Effect
of Paromomycin
Cells were grown (O+) Control; pM paromomycin; pM paromomycin.
Table
1. The Effect
Paromomycin Concentration WV
1
16
20
24
(hrs.)
in SPP medium in shaking cultures at 30°C. (O-O) 10 AM paromomycin; (A-A) 25 (A-A) 50 $I paromomycin; (U+) 100
Time of Exposure
(hr)
0
A B CIA (A,B,C)b A B Cb
0
on Cell Growth
of Paromomycin
Lividomycin Paromomycin Paromamine Neomycin Neamine Gentamicin Gentamicin Gentamicin Kanamycin Kanamycin Kanamycin Kanamycin Streptomycin Butirosin
on Cell Viability
Viable
Cells
0
0 5 25
23,100 61,000 1,450,000
50
0 5 25
23,100 65,600 65,000
500
0 5 25
23,100 400
