Limits...
Sinefungin resistance of Saccharomyces cerevisiae arising from Sam3 mutations that inactivate the AdoMet transporter or from increased expression of AdoMet synthase plus mRNA cap guanine-N7 methyltransferase.

Zheng S, Shuman S, Schwer B - Nucleic Acids Res. (2007)

Bottom Line: Thus, Sam3 is a tunable determinant of sinefungin potency.Insights to the intracellular action of sinefungin stem from the finding that increased gene dosage of yeast AdoMet synthase plus cap guanine-N7 methyltransferase afforded greater resistance to sinefungin than either enzyme alone.These results are consistent with the proposal that mRNA cap methylation is a principal target of sinefungin's bioactivity.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology Program, Sloan-Kettering Institute and Microbiology, Weill Cornell Medical College, New York, NY 10065, USA.

ABSTRACT
The S-adenosylmethionine (AdoMet) analog sinefungin is a natural product antibiotic that inhibits nucleic acid methyltransferases and arrests the growth of unicellular eukarya and eukaryal viruses. The basis for the particular sensitivity of fungi and protozoa to sinefungin is not known. Here we report the isolation and characterization of spontaneous sinefungin-resistant mutants of the budding yeast Saccharomyces cerevisiae. In all cases, sinefungin resistance was attributable to a loss-of-function mutation in Sam3, the yeast high-affinity AdoMet transporter. Overexpression of wild-type Sam3 increased the sensitivity of yeast to growth inhibition by sinefungin. Thus, Sam3 is a tunable determinant of sinefungin potency. The shared ability of protozoan parasites to import AdoMet might determine sinefungin's anti-infective spectrum. Insights to the intracellular action of sinefungin stem from the finding that increased gene dosage of yeast AdoMet synthase plus cap guanine-N7 methyltransferase afforded greater resistance to sinefungin than either enzyme alone. These results are consistent with the proposal that mRNA cap methylation is a principal target of sinefungin's bioactivity.

Show MeSH

Related in: MedlinePlus

Sinefungin resistance of sfr and sam3Δ strains. The indicated yeast strains were grown in YPD medium at 30°C until A600 reached ∼0.7. The cells were harvested by centrifugation and suspended in water. Serial 10-fold dilutions were prepared and aliquots (2 µl) were spotted on an unsupplemented agar plate (‘none’) and on YPD agar plates onto which 150 µl aliquots of a sinefungin solution (0.5, 1, 2 or 20 mM) had been applied and spread. If one assumes that the applied drug is evenly distributed through the depth of the agar plate, then these doses correspond to net sinefungin concentrations of 3, 6, 12 and 120 µM, respectively. The plates were photographed after incubation for 2 days at 30°C.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2175321&req=5

Figure 3: Sinefungin resistance of sfr and sam3Δ strains. The indicated yeast strains were grown in YPD medium at 30°C until A600 reached ∼0.7. The cells were harvested by centrifugation and suspended in water. Serial 10-fold dilutions were prepared and aliquots (2 µl) were spotted on an unsupplemented agar plate (‘none’) and on YPD agar plates onto which 150 µl aliquots of a sinefungin solution (0.5, 1, 2 or 20 mM) had been applied and spread. If one assumes that the applied drug is evenly distributed through the depth of the agar plate, then these doses correspond to net sinefungin concentrations of 3, 6, 12 and 120 µM, respectively. The plates were photographed after incubation for 2 days at 30°C.

Mentions: To better quantify sinefungin resistance and simultaneously compare the sensitivities of various mutants strains, we applied 150 µl aliquots of 0.5, 1, 2 and 20 mM sinefungin stock solutions to the surfaces of YPD agar plates (containing 25 ml of medium) and allowed the drug to adsorb into the agar before spotting serial dilutions of wild-type, sfr, and sam3Δ cells. (If one assumes that the applied drug is evenly distributed through the z-plane of the agar plate, then these doses correspond to net sinefungin concentrations of 3, 6, 12 and 120 µM, respectively.) Wild-type yeast cells failed to grow on plates that had received ≥0.5 mM sinefungin (Figure 3). Back titration of the applied dose revealed that 150 µl of a 0.2 mM sinefungin solution sufficed to prevent growth of the wild-type yeast strain on a YPD agar plate (data not shown). The sam3Δ strain was impervious to the 20 mM dose of sinefungin, which translates into at least 100-fold resistance compared to the wild-type strain (Figure 3). This result signifies that sinefungin is transported into yeast cells by the same permease that transports AdoMet. The sfr-1 and sfr-3 strains phenocopied sam3Δ with respect to resistance to the highest level of sinefungin (Figure 3). However, growth of the sfr-2 and sfr-4 strains, though resistant to the 0.5 mM sinefungin dose, was slowed at the 2 mM dose and inhibited fully by the 20 mM dose (Figure 3). These results highlight heterogeneity of the resistance phenotypes of different sfr strains.Figure 3.


Sinefungin resistance of Saccharomyces cerevisiae arising from Sam3 mutations that inactivate the AdoMet transporter or from increased expression of AdoMet synthase plus mRNA cap guanine-N7 methyltransferase.

Zheng S, Shuman S, Schwer B - Nucleic Acids Res. (2007)

Sinefungin resistance of sfr and sam3Δ strains. The indicated yeast strains were grown in YPD medium at 30°C until A600 reached ∼0.7. The cells were harvested by centrifugation and suspended in water. Serial 10-fold dilutions were prepared and aliquots (2 µl) were spotted on an unsupplemented agar plate (‘none’) and on YPD agar plates onto which 150 µl aliquots of a sinefungin solution (0.5, 1, 2 or 20 mM) had been applied and spread. If one assumes that the applied drug is evenly distributed through the depth of the agar plate, then these doses correspond to net sinefungin concentrations of 3, 6, 12 and 120 µM, respectively. The plates were photographed after incubation for 2 days at 30°C.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175321&req=5

Figure 3: Sinefungin resistance of sfr and sam3Δ strains. The indicated yeast strains were grown in YPD medium at 30°C until A600 reached ∼0.7. The cells were harvested by centrifugation and suspended in water. Serial 10-fold dilutions were prepared and aliquots (2 µl) were spotted on an unsupplemented agar plate (‘none’) and on YPD agar plates onto which 150 µl aliquots of a sinefungin solution (0.5, 1, 2 or 20 mM) had been applied and spread. If one assumes that the applied drug is evenly distributed through the depth of the agar plate, then these doses correspond to net sinefungin concentrations of 3, 6, 12 and 120 µM, respectively. The plates were photographed after incubation for 2 days at 30°C.
Mentions: To better quantify sinefungin resistance and simultaneously compare the sensitivities of various mutants strains, we applied 150 µl aliquots of 0.5, 1, 2 and 20 mM sinefungin stock solutions to the surfaces of YPD agar plates (containing 25 ml of medium) and allowed the drug to adsorb into the agar before spotting serial dilutions of wild-type, sfr, and sam3Δ cells. (If one assumes that the applied drug is evenly distributed through the z-plane of the agar plate, then these doses correspond to net sinefungin concentrations of 3, 6, 12 and 120 µM, respectively.) Wild-type yeast cells failed to grow on plates that had received ≥0.5 mM sinefungin (Figure 3). Back titration of the applied dose revealed that 150 µl of a 0.2 mM sinefungin solution sufficed to prevent growth of the wild-type yeast strain on a YPD agar plate (data not shown). The sam3Δ strain was impervious to the 20 mM dose of sinefungin, which translates into at least 100-fold resistance compared to the wild-type strain (Figure 3). This result signifies that sinefungin is transported into yeast cells by the same permease that transports AdoMet. The sfr-1 and sfr-3 strains phenocopied sam3Δ with respect to resistance to the highest level of sinefungin (Figure 3). However, growth of the sfr-2 and sfr-4 strains, though resistant to the 0.5 mM sinefungin dose, was slowed at the 2 mM dose and inhibited fully by the 20 mM dose (Figure 3). These results highlight heterogeneity of the resistance phenotypes of different sfr strains.Figure 3.

Bottom Line: Thus, Sam3 is a tunable determinant of sinefungin potency.Insights to the intracellular action of sinefungin stem from the finding that increased gene dosage of yeast AdoMet synthase plus cap guanine-N7 methyltransferase afforded greater resistance to sinefungin than either enzyme alone.These results are consistent with the proposal that mRNA cap methylation is a principal target of sinefungin's bioactivity.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology Program, Sloan-Kettering Institute and Microbiology, Weill Cornell Medical College, New York, NY 10065, USA.

ABSTRACT
The S-adenosylmethionine (AdoMet) analog sinefungin is a natural product antibiotic that inhibits nucleic acid methyltransferases and arrests the growth of unicellular eukarya and eukaryal viruses. The basis for the particular sensitivity of fungi and protozoa to sinefungin is not known. Here we report the isolation and characterization of spontaneous sinefungin-resistant mutants of the budding yeast Saccharomyces cerevisiae. In all cases, sinefungin resistance was attributable to a loss-of-function mutation in Sam3, the yeast high-affinity AdoMet transporter. Overexpression of wild-type Sam3 increased the sensitivity of yeast to growth inhibition by sinefungin. Thus, Sam3 is a tunable determinant of sinefungin potency. The shared ability of protozoan parasites to import AdoMet might determine sinefungin's anti-infective spectrum. Insights to the intracellular action of sinefungin stem from the finding that increased gene dosage of yeast AdoMet synthase plus cap guanine-N7 methyltransferase afforded greater resistance to sinefungin than either enzyme alone. These results are consistent with the proposal that mRNA cap methylation is a principal target of sinefungin's bioactivity.

Show MeSH
Related in: MedlinePlus