A common telomeric gene silencing assay is affected by nucleotide metabolism.

Marlies P Rossmann, Weijun Luo, Olga Tsaponina, Andrei Chabes, Bruce Stillman

Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.

Molecular cell 2011 Apr 8

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Telomere-associated position-effect variegation (TPEV) in budding yeast has been used as a model for understanding epigenetic inheritance and gene silencing. A widely used assay to identify mutants with improper TPEV employs the URA3 gene at the telomere of chromosome VII-L that can be counterselected with 5-fluoroorotic acid (5-FOA). 5-FOA resistance has been inferred to represent lack of transcription of URA3 and therefore to represent heterochromatin-induced gene silencing. For two genes implicated in telomere silencing, POL30 and DOT1, we show that the URA3 telomere reporter assay does not reflect their role in heterochromatin formation. Rather, an imbalance in ribonucleotide reductase (RNR), which is induced by 5-FOA, and the specific promoter of URA3 fused to ADH4 at telomere VII-L are jointly responsible for the variegated phenotype. We conclude that metabolic changes caused by the drug employed and certain mutants being studied are incompatible with the use of certain prototrophic markers for TPEV. Copyright © 2011 Elsevier Inc. All rights reserved.

Citation

Marlies P Rossmann, Weijun Luo, Olga Tsaponina, Andrei Chabes, Bruce Stillman. A common telomeric gene silencing assay is affected by nucleotide metabolism. Molecular cell. 2011 Apr 8;42(1):127-36