OIG'-G
Molec. gen. Genet. 151, 49-56 (11977)
© by Springer-Verlag 1977
Extra-Chromosomal Inheritance of Rhodamine 6G Resistance in Saccharomyces cerevisiae Giovanna Carignani*, William E. Lancashire and David E. Griffiths Department of Molecular Sciences, University of Warwick, Coventry CV4 7AL, U.K,
Summary. Rhodamine 6G was found to be a specific inhibitor of aerobic growth of yeast, having no effect on fermentative growth. A single step spontaneous mutant ofS. c e r e v i s i a e resistant to rhodamine 6G was isolated, which showed cross-resistance to the ATPase inhibitors venturicidin and triethyltin, to the uncoupler 1799, to bongkrekic acid and to cycloheximide, but not to oligomycin or to the inhibitors of mitochondrial protein synthesis, chloramphenicol and erythromycin, The genetic analysis of this mutant showed that both nuclear and cytoplasmic (but apparently not mitochondrial) factors may be involved in the determination of the mutation. The behaviour. is discussed as a possible function for 2 micron circular (omicron) DNA.
systems, particularly A D P / A T P translocation (Gear, 1974), which we have previously demonstrated to be associated with extranuclear genetic determinants (Cain et al., 1974). This paper, therefore, describes the isolation and genetic characterisation of mutants resistant to rhodamine 6G, and evidence is presented which implicates a nuclear and cytoplasmic genetic control of resistance. The results will be discussed in terms of a D N A species having plasmid and episomal properties and may simultaneously b e associated with both nuclear and mitochondrial genomes and/or their expression, and therefore constituting an important link in the complex nucleo-cytoplasmic interactions which are involved in the biogenesis of functional energy conserving membranes constituting the complete mitochondrion,
Introduction Biochemical genetic studies of yeast mutants resistant to inhibitors of mitochondrial metabolism have shown that many of the resistance determinants are located on mitochondrial D N A (Avner and Griffiths, 1973; Lancashire and Griffiths, 1975b). However, one particular class of mutants showing simultaneous resistance to triethyltin and venturicidin, whilst exhibiting genetic behaviour associated with mitochondrial genome determinants, did not appear to be linked to mitochondrial D N A (Lancashire and Griffiths, 1975a, b), although strong genetic and biochemical interactions were found to occur between the two systems (Lancashire, 1974). In a search for other mutants with similar genetic behaviour, the compound rhodamine 6G was selected because of its reported biochemical interaction with mitochondrial transport * Present address." Istituto di Chimica Biologica dell Universita di Padova, via Marzolo 3, Padova, Italy
Materials and Methods Yeast Stratus
Haploid strains D22 a ad2p+, D6 c~arg met p+o)+ and D41 c~ur his p*o) + used in this study were obtained from Dr. D. Wilkie. Diploid strains DL211 (R6G•) and DL212 (R6Gs) are mitotic segregants from the cross D22/72 x D41. Media
The followingcomplex media were used in this study: Glycerol medium (N3): 1% yeast extract, 1% peptone, 3% glycerol, 0.05 M Na, K phosphate buffer pH 6.25. Glucose medium (NO): as above but 2% glucose instead of glycerol. 2.3% agar was added to the media for making plates. Preparation of drug-containing media and the source of antibiotics and inhibitors have been described previously (Lancashire and Griffiths, 1975a). In addition, rhodamine 6G was obtained from Fisons Scientific Instruments, rhodamine 3G from Phase Separations Ltd, and rhodamines B and S from BDH Chemicals Ltd.
50
G. Carignani et a l . Rhodamine 6G Resistance in Yeast
Isolation of Resistant Mutants Parental drug-sensitive strain D22 was harvested after growth to late logarithmic phase in glycerol medium. Cells were plated at a cell density of 10 s cells/plate on N3 medium containing rhodamine 6G (50 lag/ml or 100 gg/ml). Resistant colonies were picked off after 1-2 weeks incubation at 30 ° and after subcloning twice on glycerol medium (N3) were stocked on glucose medium slants.
1969) between the petite and grande tester strain D41, followed by determination of percentage petites in the zygote population. This value corrected for spontaneous production of petites in the control cross D22/72 x D41 is the value taken for suppressivity.
A bbre via tions p+/p pO
Determination of Drug Resistance The resistance or sensitivity of yeast colonies was determined on solid media by the drop-out procedure or velveteen replica plating techniques as used previously (Lancashire and Griffiths, 1975a).
Identification of Drug Resistance Markers in Petzte Strains The resistance of petite strains, which cannot be assayed directly due to their inability to grown on non-fermentable carbon sources, was determined using the "marker-rescue" technique described previously (Lancashire and Griffiths, 1975a). This involves the mating of petites to the drug sensitive grande strain D41 and subsequently testing the drug resistance of the resultant diploids,
= grande/cytoplasmic petite strains = cytoplasmic petite mutant that is deleted of all mitochondrial DNA, i.e. rho ° R6GR/R6G s =rhodamine 6G resistant or rhodamine 6G sensitive phenotypes TETR/TET s =triethyltin resistant or triethyltin sensitive phenotypes VENR/VEN s ~ venturicldin resistant or venturicidin sensitive pheno types 1799 =bis (hexafluoroacetonyl) acetone RR/Rs, TR/T s and vR/vS =resistant/sensitive genotypes for rhodamine 6G, triethyltin and venturicidin, respectively. R°T ° and V ° states in which the resistance/sensitive determinants are absent or deleted.
Results
Genetic Techniques
Effects of Rhodamine Compounds" on Yeast Growth
The mating of yeast strains on minimal medium, sporulation, random spore analysis and quantitative random diploid analysis were carried out as previously (Avner and Griffiths, 1973).
Biochemical studies on isolated mitochondria have indicated that rhodamine 6G may have an interaction site on the inner membrane, thereby interfering with mitochondrial metabolism (Gear, 1974). As with previous biochemical genetic studies with yeast resistant mutants it is important to establish that the growth inhibitors characteristics of rhodamine 6G are consistent with this biochemical action. This was investigated by examining the effects of varying concentrations of the compound on yeast growth utilising oxidisable (ethanol or glycerol) and fermentable carbon sources. The results in Table 1 indicate that on solid media rhodamine 6G inhibits growth of strains D22 and D41 at low concentrations when glycerol is used as carbon source, whereas considerably higher concentrations are required to inhibit growth on glucose. Similar results are observed in liquid culture, where growth
b~duction of Petites by Ethidium Bromide Yeast cells were grown in 10 ml glucose medium for 24 h (suspension A). An inoculum of A (0.1 ml) was put into 10 ml of glucose containing 10 pg/ml ethidium bromide and incubated 24 h (suspension B). Suspension B was further subcultured in 10 pg/ml ethidium bromide (suspension C) followed by one further subculture in glucose medmm alone (suspension D). At each stage cells were washed with sterile distilled water followed by plating on glucose medium for determination of petite frequency by tetrazolium overlay.
Determination of Suppressivity Value Suppressivity of petite strains (Ephrussi and Grandchamp, 1965) was determined by formation of synchronous zygotes (Fowell,
Table 1. Tolerance of wild type strains to different Rhodamines on fermentable (glucose) and non-fermentable (glycerol) growth substrates Strain
D22 D6 D41
Tolerance to Inhibitors (gg/ml) Rhodamine 6G
Rhodamine 3G
Rhodamme B
Rhodamine S
Glucose
Glycerol
Glucose
Glycerol
Glucose
Glycerol
Glucose
Glycerol
40 40 40
5 40 10
50 200 200
10 100 20
200 200 200
200 200 200
50 50 50
50 50 50
Strains were assayed for tolerance to inhibitors on solid media as described previously (Lancashire and Griffiths, 1975a). The following concentrations of inhibitors were used: Rhodamine 6G 1, 5, 10, 20 and 40 gg/ml; Rhodamine 3G and Rhodamine B 10, 20, 50, 100 and 200 pg/ml; Rhodamlne 10, 20 and 50 gg/ml.
G. Carignani et al.: Rhodamine 6G Resistance in Yeast
on ethanol and the aerobic phase of growth on glucose are inhibited. This indicates that rhodamine 6G has an in vivo mode of action directed towards mitochondrial metabolism. The effects of rhodamine B, rhodamine 3G and rhodamine S are also shown in the table, and it is seen that none of them have the same specificity as rhodamine 6G. Rhodamines B and S have no inhibitory effect on yeast growth and rhodamine 3G inhibits growth well when yeast is growing both oxidatively and fermentatively, indicating a nonmitochondrial locus of action.
Isolation and Characterisation of Rhodamine 6G Resistant Mutants Rhodamine resistant mutants were isolated from the sensitive parent strain D22 by the procedure described in the methods section. A total of 20 mutants were isolated, 14 from 50 ~tg/ml and 6 from 100 ~tg/ml. The spontaneous frequencies of occurrence of resistant colonies were 2 x 10 -v and 10 -7 on 50 and 100 gg/ml respectively. As shown in Table 2, most of the mutants were stable with respect to their rhodamine resistance, many of the mutants being specifically resistant to 100 I~g/ml rhodamine 6G. No resistance to rhoda-
Table2. Tolerance of resistant mutants to Rhodalnine 6G and Rhodamine 3G on a non-fermentable substrate Strain
D22 D22/R501 D22/R502 D22/R504 D22/R505 D22/R506 D22/R507 D22/R508 D22/R509 D22/R 510 D22/R511 D22/R512 D22/R513 D22/R514 D22/R101 D22/R 102 D22/R 103 D22/72 D22/R 105 D22/R 106
Tolerance to Inhibitors Rhodamine 6G (.ug/ml)
Rhodamine 3G 0tg/ml)
5 50 50 50 50 50 10 20 I0 10 50 50 100 50 20 50 50 100 100 50
20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20
Strains were assayed for resistance to inhibitors on solid glycerol media. The concentration of the inhibitors in the media were: 10, 20, 50, 100 and 200gg/ml.
51
mine 3G was observed. Subcloning experiments revealed that the rhodamine resistance was not lost spontaneously during vegetative growth in any of the mutants.
Cross-Resistance with the Inhibitors of Yeast Growth To further substantiate the specificity of rhodamine 6G resistant mutants, they were tested for crossresistance to a variety of compounds having uncoupling and/or inhibitory modes of action on mitochondrial metabolism, in particular triethyltin, chloramphenicol, cycloheximide and erythromycin. As can be seen in Table 3, none of the rhodamine 6G mutants exhibit cross-resistance to the ATP synthesis inhibitor oligomycin, or to the inhibitors of mitochondrial protein synthesis chloramphenicol and erythromycin. Many of the mutants are highly resistant to the ATPase inhibitors triethyltin and venturicidin, and also the uncoupler 1799, the A D P / A T P translocase inhibitor, bongkrekic acid, and cycloheximide, an inhibitor of cytoplasmic protein synthesis. Many mutants were also resistant to hexachlorophene which has been shown to be a potent inhibitor of adenine nucleotide translocation across the mitochondrial membrane [Al-lami, A.H., unpublished studies]. The cross-resistance characteristics of the rhodamine mutants are essentially similar to patterns of resistance observed previously with mutants isolated as resistant to trialkyltin salts and venturicidin (Lancashire and Griffiths, 1975a, b).
Genetic Analysis of Rhodamine Resistance In order to determine the mode of inheritance of the rhodamine resistance mutations, genetic manipulations were carried out to test for the known criteria for distinguishing nuclear and cytoplasmic (mitochondrial) mutants (Coen et al., 1970).
Mitotic Segregation When rhodamine resistant strains were mated with suitable sensitive strains and the diploid zygotic progeny analyzed for tolerance to the drug, a clear mitotic segregation of resistant and sensitive colony types was observed in several of the mutants, for example strain D22/72 in Table 4. The transmission values varied widely in repeats of the same cross and the explanation for this is not known. A high polarity of the resistance distribution is observed in most
G. Carignani et al. : Rhodamine 6G Resistance in Yeast
52
Table 3. Cross resistance of rhodamine 6G mutants to other inhibitors Strains
Tolerances to inhibitors Rhodamine 6G
D2 D22 D22 D22 D22 D22 D22 D22 D22 D22 D22 D22 D22 D22 D22 D22 D22 D22 D22 D22
'R501 'R502 'R504 'R505 R506 R507 R508 'R509 R510 'R511 'R512 'R513 'R514 R101 R102 'R103 '72 'R105 R106
Oligomycin (gg/ml)
Triethyltin
Venturicidin
Erythromycin
Chloramphenicol
Cycloheximide
(gg/ml)
(gg/ml)
(~tg/ml)
(mg/ml)
(mg/ml)
(~tg/ml)
5 50 50 50 50 50 10 20 10 10 50 50 100 50 20 50 50 100 100 50
Molec. gen. Genet. 151, 49-56 (11977)
© by Springer-Verlag 1977
Extra-Chromosomal Inheritance of Rhodamine 6G Resistance in Saccharomyces cerevisiae Giovanna Carignani*, William E. Lancashire and David E. Griffiths Department of Molecular Sciences, University of Warwick, Coventry CV4 7AL, U.K,
Summary. Rhodamine 6G was found to be a specific inhibitor of aerobic growth of yeast, having no effect on fermentative growth. A single step spontaneous mutant ofS. c e r e v i s i a e resistant to rhodamine 6G was isolated, which showed cross-resistance to the ATPase inhibitors venturicidin and triethyltin, to the uncoupler 1799, to bongkrekic acid and to cycloheximide, but not to oligomycin or to the inhibitors of mitochondrial protein synthesis, chloramphenicol and erythromycin, The genetic analysis of this mutant showed that both nuclear and cytoplasmic (but apparently not mitochondrial) factors may be involved in the determination of the mutation. The behaviour. is discussed as a possible function for 2 micron circular (omicron) DNA.
systems, particularly A D P / A T P translocation (Gear, 1974), which we have previously demonstrated to be associated with extranuclear genetic determinants (Cain et al., 1974). This paper, therefore, describes the isolation and genetic characterisation of mutants resistant to rhodamine 6G, and evidence is presented which implicates a nuclear and cytoplasmic genetic control of resistance. The results will be discussed in terms of a D N A species having plasmid and episomal properties and may simultaneously b e associated with both nuclear and mitochondrial genomes and/or their expression, and therefore constituting an important link in the complex nucleo-cytoplasmic interactions which are involved in the biogenesis of functional energy conserving membranes constituting the complete mitochondrion,
Introduction Biochemical genetic studies of yeast mutants resistant to inhibitors of mitochondrial metabolism have shown that many of the resistance determinants are located on mitochondrial D N A (Avner and Griffiths, 1973; Lancashire and Griffiths, 1975b). However, one particular class of mutants showing simultaneous resistance to triethyltin and venturicidin, whilst exhibiting genetic behaviour associated with mitochondrial genome determinants, did not appear to be linked to mitochondrial D N A (Lancashire and Griffiths, 1975a, b), although strong genetic and biochemical interactions were found to occur between the two systems (Lancashire, 1974). In a search for other mutants with similar genetic behaviour, the compound rhodamine 6G was selected because of its reported biochemical interaction with mitochondrial transport * Present address." Istituto di Chimica Biologica dell Universita di Padova, via Marzolo 3, Padova, Italy
Materials and Methods Yeast Stratus
Haploid strains D22 a ad2p+, D6 c~arg met p+o)+ and D41 c~ur his p*o) + used in this study were obtained from Dr. D. Wilkie. Diploid strains DL211 (R6G•) and DL212 (R6Gs) are mitotic segregants from the cross D22/72 x D41. Media
The followingcomplex media were used in this study: Glycerol medium (N3): 1% yeast extract, 1% peptone, 3% glycerol, 0.05 M Na, K phosphate buffer pH 6.25. Glucose medium (NO): as above but 2% glucose instead of glycerol. 2.3% agar was added to the media for making plates. Preparation of drug-containing media and the source of antibiotics and inhibitors have been described previously (Lancashire and Griffiths, 1975a). In addition, rhodamine 6G was obtained from Fisons Scientific Instruments, rhodamine 3G from Phase Separations Ltd, and rhodamines B and S from BDH Chemicals Ltd.
50
G. Carignani et a l . Rhodamine 6G Resistance in Yeast
Isolation of Resistant Mutants Parental drug-sensitive strain D22 was harvested after growth to late logarithmic phase in glycerol medium. Cells were plated at a cell density of 10 s cells/plate on N3 medium containing rhodamine 6G (50 lag/ml or 100 gg/ml). Resistant colonies were picked off after 1-2 weeks incubation at 30 ° and after subcloning twice on glycerol medium (N3) were stocked on glucose medium slants.
1969) between the petite and grande tester strain D41, followed by determination of percentage petites in the zygote population. This value corrected for spontaneous production of petites in the control cross D22/72 x D41 is the value taken for suppressivity.
A bbre via tions p+/p pO
Determination of Drug Resistance The resistance or sensitivity of yeast colonies was determined on solid media by the drop-out procedure or velveteen replica plating techniques as used previously (Lancashire and Griffiths, 1975a).
Identification of Drug Resistance Markers in Petzte Strains The resistance of petite strains, which cannot be assayed directly due to their inability to grown on non-fermentable carbon sources, was determined using the "marker-rescue" technique described previously (Lancashire and Griffiths, 1975a). This involves the mating of petites to the drug sensitive grande strain D41 and subsequently testing the drug resistance of the resultant diploids,
= grande/cytoplasmic petite strains = cytoplasmic petite mutant that is deleted of all mitochondrial DNA, i.e. rho ° R6GR/R6G s =rhodamine 6G resistant or rhodamine 6G sensitive phenotypes TETR/TET s =triethyltin resistant or triethyltin sensitive phenotypes VENR/VEN s ~ venturicldin resistant or venturicidin sensitive pheno types 1799 =bis (hexafluoroacetonyl) acetone RR/Rs, TR/T s and vR/vS =resistant/sensitive genotypes for rhodamine 6G, triethyltin and venturicidin, respectively. R°T ° and V ° states in which the resistance/sensitive determinants are absent or deleted.
Results
Genetic Techniques
Effects of Rhodamine Compounds" on Yeast Growth
The mating of yeast strains on minimal medium, sporulation, random spore analysis and quantitative random diploid analysis were carried out as previously (Avner and Griffiths, 1973).
Biochemical studies on isolated mitochondria have indicated that rhodamine 6G may have an interaction site on the inner membrane, thereby interfering with mitochondrial metabolism (Gear, 1974). As with previous biochemical genetic studies with yeast resistant mutants it is important to establish that the growth inhibitors characteristics of rhodamine 6G are consistent with this biochemical action. This was investigated by examining the effects of varying concentrations of the compound on yeast growth utilising oxidisable (ethanol or glycerol) and fermentable carbon sources. The results in Table 1 indicate that on solid media rhodamine 6G inhibits growth of strains D22 and D41 at low concentrations when glycerol is used as carbon source, whereas considerably higher concentrations are required to inhibit growth on glucose. Similar results are observed in liquid culture, where growth
b~duction of Petites by Ethidium Bromide Yeast cells were grown in 10 ml glucose medium for 24 h (suspension A). An inoculum of A (0.1 ml) was put into 10 ml of glucose containing 10 pg/ml ethidium bromide and incubated 24 h (suspension B). Suspension B was further subcultured in 10 pg/ml ethidium bromide (suspension C) followed by one further subculture in glucose medmm alone (suspension D). At each stage cells were washed with sterile distilled water followed by plating on glucose medium for determination of petite frequency by tetrazolium overlay.
Determination of Suppressivity Value Suppressivity of petite strains (Ephrussi and Grandchamp, 1965) was determined by formation of synchronous zygotes (Fowell,
Table 1. Tolerance of wild type strains to different Rhodamines on fermentable (glucose) and non-fermentable (glycerol) growth substrates Strain
D22 D6 D41
Tolerance to Inhibitors (gg/ml) Rhodamine 6G
Rhodamine 3G
Rhodamme B
Rhodamine S
Glucose
Glycerol
Glucose
Glycerol
Glucose
Glycerol
Glucose
Glycerol
40 40 40
5 40 10
50 200 200
10 100 20
200 200 200
200 200 200
50 50 50
50 50 50
Strains were assayed for tolerance to inhibitors on solid media as described previously (Lancashire and Griffiths, 1975a). The following concentrations of inhibitors were used: Rhodamine 6G 1, 5, 10, 20 and 40 gg/ml; Rhodamine 3G and Rhodamine B 10, 20, 50, 100 and 200 pg/ml; Rhodamlne 10, 20 and 50 gg/ml.
G. Carignani et al.: Rhodamine 6G Resistance in Yeast
on ethanol and the aerobic phase of growth on glucose are inhibited. This indicates that rhodamine 6G has an in vivo mode of action directed towards mitochondrial metabolism. The effects of rhodamine B, rhodamine 3G and rhodamine S are also shown in the table, and it is seen that none of them have the same specificity as rhodamine 6G. Rhodamines B and S have no inhibitory effect on yeast growth and rhodamine 3G inhibits growth well when yeast is growing both oxidatively and fermentatively, indicating a nonmitochondrial locus of action.
Isolation and Characterisation of Rhodamine 6G Resistant Mutants Rhodamine resistant mutants were isolated from the sensitive parent strain D22 by the procedure described in the methods section. A total of 20 mutants were isolated, 14 from 50 ~tg/ml and 6 from 100 ~tg/ml. The spontaneous frequencies of occurrence of resistant colonies were 2 x 10 -v and 10 -7 on 50 and 100 gg/ml respectively. As shown in Table 2, most of the mutants were stable with respect to their rhodamine resistance, many of the mutants being specifically resistant to 100 I~g/ml rhodamine 6G. No resistance to rhoda-
Table2. Tolerance of resistant mutants to Rhodalnine 6G and Rhodamine 3G on a non-fermentable substrate Strain
D22 D22/R501 D22/R502 D22/R504 D22/R505 D22/R506 D22/R507 D22/R508 D22/R509 D22/R 510 D22/R511 D22/R512 D22/R513 D22/R514 D22/R101 D22/R 102 D22/R 103 D22/72 D22/R 105 D22/R 106
Tolerance to Inhibitors Rhodamine 6G (.ug/ml)
Rhodamine 3G 0tg/ml)
5 50 50 50 50 50 10 20 I0 10 50 50 100 50 20 50 50 100 100 50
20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20
Strains were assayed for resistance to inhibitors on solid glycerol media. The concentration of the inhibitors in the media were: 10, 20, 50, 100 and 200gg/ml.
51
mine 3G was observed. Subcloning experiments revealed that the rhodamine resistance was not lost spontaneously during vegetative growth in any of the mutants.
Cross-Resistance with the Inhibitors of Yeast Growth To further substantiate the specificity of rhodamine 6G resistant mutants, they were tested for crossresistance to a variety of compounds having uncoupling and/or inhibitory modes of action on mitochondrial metabolism, in particular triethyltin, chloramphenicol, cycloheximide and erythromycin. As can be seen in Table 3, none of the rhodamine 6G mutants exhibit cross-resistance to the ATP synthesis inhibitor oligomycin, or to the inhibitors of mitochondrial protein synthesis chloramphenicol and erythromycin. Many of the mutants are highly resistant to the ATPase inhibitors triethyltin and venturicidin, and also the uncoupler 1799, the A D P / A T P translocase inhibitor, bongkrekic acid, and cycloheximide, an inhibitor of cytoplasmic protein synthesis. Many mutants were also resistant to hexachlorophene which has been shown to be a potent inhibitor of adenine nucleotide translocation across the mitochondrial membrane [Al-lami, A.H., unpublished studies]. The cross-resistance characteristics of the rhodamine mutants are essentially similar to patterns of resistance observed previously with mutants isolated as resistant to trialkyltin salts and venturicidin (Lancashire and Griffiths, 1975a, b).
Genetic Analysis of Rhodamine Resistance In order to determine the mode of inheritance of the rhodamine resistance mutations, genetic manipulations were carried out to test for the known criteria for distinguishing nuclear and cytoplasmic (mitochondrial) mutants (Coen et al., 1970).
Mitotic Segregation When rhodamine resistant strains were mated with suitable sensitive strains and the diploid zygotic progeny analyzed for tolerance to the drug, a clear mitotic segregation of resistant and sensitive colony types was observed in several of the mutants, for example strain D22/72 in Table 4. The transmission values varied widely in repeats of the same cross and the explanation for this is not known. A high polarity of the resistance distribution is observed in most
G. Carignani et al. : Rhodamine 6G Resistance in Yeast
52
Table 3. Cross resistance of rhodamine 6G mutants to other inhibitors Strains
Tolerances to inhibitors Rhodamine 6G
D2 D22 D22 D22 D22 D22 D22 D22 D22 D22 D22 D22 D22 D22 D22 D22 D22 D22 D22 D22
'R501 'R502 'R504 'R505 R506 R507 R508 'R509 R510 'R511 'R512 'R513 'R514 R101 R102 'R103 '72 'R105 R106
Oligomycin (gg/ml)
Triethyltin
Venturicidin
Erythromycin
Chloramphenicol
Cycloheximide
(gg/ml)
(gg/ml)
(~tg/ml)
(mg/ml)
(mg/ml)
(~tg/ml)
5 50 50 50 50 50 10 20 10 10 50 50 100 50 20 50 50 100 100 50
