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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

Sinefungin phenotype of sfr sam3Δ strains. sam3Δ cells transformed with a CEN TRP1 vector containing the genes specified at left were grown in Trp− 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 Trp− agar plate (‘no drug’) and on Trp− plates onto which 150 µl of a 0.5 mM or 2 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 5: Sinefungin phenotype of sfr sam3Δ strains. sam3Δ cells transformed with a CEN TRP1 vector containing the genes specified at left were grown in Trp− 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 Trp− agar plate (‘no drug’) and on Trp− plates onto which 150 µl of a 0.5 mM or 2 mM sinefungin solution had been applied and spread. The plates were photographed after incubation for 3 days at 30°C.

Mentions: To check that the SAM3 alleles obtained from these four sfr strains were indeed functionally compromised, we cloned them into CEN TRP1 plasmids and then used these vectors, and a wild-type SAM3 control, to transform the sam3Δ strain. The transformants were tested for growth on Trp− plates that had been overlaid with 0.5 or 2 mM sinefungin. The wild-type SAM3 gene restored sinefungin-sensitivity, but the empty vector control did not, nor did any of the four mutated sam3 genes from the sfr strains (Figure 5).Figure 5.


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 phenotype of sfr sam3Δ strains. sam3Δ cells transformed with a CEN TRP1 vector containing the genes specified at left were grown in Trp− 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 Trp− agar plate (‘no drug’) and on Trp− plates onto which 150 µl of a 0.5 mM or 2 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 5: Sinefungin phenotype of sfr sam3Δ strains. sam3Δ cells transformed with a CEN TRP1 vector containing the genes specified at left were grown in Trp− 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 Trp− agar plate (‘no drug’) and on Trp− plates onto which 150 µl of a 0.5 mM or 2 mM sinefungin solution had been applied and spread. The plates were photographed after incubation for 3 days at 30°C.
Mentions: To check that the SAM3 alleles obtained from these four sfr strains were indeed functionally compromised, we cloned them into CEN TRP1 plasmids and then used these vectors, and a wild-type SAM3 control, to transform the sam3Δ strain. The transformants were tested for growth on Trp− plates that had been overlaid with 0.5 or 2 mM sinefungin. The wild-type SAM3 gene restored sinefungin-sensitivity, but the empty vector control did not, nor did any of the four mutated sam3 genes from the sfr strains (Figure 5).Figure 5.

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