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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.

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Related in: MedlinePlus

SAM3 restores sinefungin sensitivity to yeast sfr strains. The indicated yeast strains were transformed with either the control CEN TRP1 plasmid or a derivative bearing wild-type SAM3. Cells were grown in Trp− medium at 30°C until A600 reached ∼0.7, then harvested by centrifugation and suspended in water. Serial 10-fold dilutions were prepared and aliquots (2 µl) were spotted on an unsupplemented Trp− agar plate (‘no drug’) and on a Trp− plate onto which 150 µl of a 0.5 mM sinefungin solution had been applied and spread. The plates were photographed after incubation for 3 days at 30°C.
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Figure 4: SAM3 restores sinefungin sensitivity to yeast sfr strains. The indicated yeast strains were transformed with either the control CEN TRP1 plasmid or a derivative bearing wild-type SAM3. Cells were grown in Trp− medium at 30°C until A600 reached ∼0.7, then harvested by centrifugation and suspended in water. Serial 10-fold dilutions were prepared and aliquots (2 µl) were spotted on an unsupplemented Trp− agar plate (‘no drug’) and on a Trp− plate onto which 150 µl of a 0.5 mM sinefungin solution had been applied and spread. The plates were photographed after incubation for 3 days at 30°C.

Mentions: To test whether the sfr mutations were allelic to SAM3, the sfr and sam3Δ strains were transformed with a CEN TRP1 plasmid bearing the wild-type SAM3 gene under the control of its native promoter. The strains were transformed in parallel with the empty CEN TRP1 plasmid vector. The SAM3 plasmid restored sinefungin sensitivity to the sam3Δ strain (Figure 4). The sfr-1, sfr-2, sfr-3, and sfr-4 strains were also rendered sinefungin-sensitive by the plasmid-borne SAM3 gene (Figure 4), implying that the sfr mutations are recessive and allelic to SAM3. The results are not consistent with the sfr mutations affecting a regulator of SAM3 expression or function, insofar as a single copy of the SAM3 gene on a plasmid would not be expected to restore Sam3 activity in such a scenario.Figure 4.


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)

SAM3 restores sinefungin sensitivity to yeast sfr strains. The indicated yeast strains were transformed with either the control CEN TRP1 plasmid or a derivative bearing wild-type SAM3. Cells were grown in Trp− medium at 30°C until A600 reached ∼0.7, then harvested by centrifugation and suspended in water. Serial 10-fold dilutions were prepared and aliquots (2 µl) were spotted on an unsupplemented Trp− agar plate (‘no drug’) and on a Trp− plate onto which 150 µl of a 0.5 mM sinefungin solution had been applied and spread. The plates were photographed after incubation for 3 days at 30°C.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: SAM3 restores sinefungin sensitivity to yeast sfr strains. The indicated yeast strains were transformed with either the control CEN TRP1 plasmid or a derivative bearing wild-type SAM3. Cells were grown in Trp− medium at 30°C until A600 reached ∼0.7, then harvested by centrifugation and suspended in water. Serial 10-fold dilutions were prepared and aliquots (2 µl) were spotted on an unsupplemented Trp− agar plate (‘no drug’) and on a Trp− plate onto which 150 µl of a 0.5 mM sinefungin solution had been applied and spread. The plates were photographed after incubation for 3 days at 30°C.
Mentions: To test whether the sfr mutations were allelic to SAM3, the sfr and sam3Δ strains were transformed with a CEN TRP1 plasmid bearing the wild-type SAM3 gene under the control of its native promoter. The strains were transformed in parallel with the empty CEN TRP1 plasmid vector. The SAM3 plasmid restored sinefungin sensitivity to the sam3Δ strain (Figure 4). The sfr-1, sfr-2, sfr-3, and sfr-4 strains were also rendered sinefungin-sensitive by the plasmid-borne SAM3 gene (Figure 4), implying that the sfr mutations are recessive and allelic to SAM3. The results are not consistent with the sfr mutations affecting a regulator of SAM3 expression or function, insofar as a single copy of the SAM3 gene on a plasmid would not be expected to restore Sam3 activity in such a scenario.Figure 4.

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