title: A common telomeric gene silencing assay is affected by nucleotide metabolism.
abstract: 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.
authors.affiliation: Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.
authors.forename: Marlies P
authors.initials: MP
authors.lastname: Rossmann
authors.forename: Weijun
authors.initials: W
authors.lastname: Luo
authors.forename: Olga
authors.initials: O
authors.lastname: Tsaponina
authors.forename: Andrei
authors.initials: A
authors.lastname: Chabes
authors.forename: Bruce
authors.initials: B
authors.lastname: Stillman
mesh_terms: D034961:Antigens, Nuclear / Q000235:genetics / Q000378:metabolism; D055012:Chromosomal Position Effects; D020868:Gene Silencing; D005800:Genes, Fungal; D011495:Histone-Lysine N-Methyltransferase / Q000235:genetics / Q000378:metabolism; D008957:Models, Genetic; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D009963:Orotic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen; D012264:Ribonucleotide Reductases / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D016615:Telomere / Q000235:genetics* / Q000378:metabolism*
chemical_list: D034961:Antigens, Nuclear; D009687:Nuclear Proteins; C546594:POL30 protein, S cerevisiae; D018809:Proliferating Cell Nuclear Antigen; D029701:Saccharomyces cerevisiae Proteins; C554167:URA3 protein, S cerevisiae; D009963:Orotic Acid; C001242:5-fluoroorotic acid; D012264:Ribonucleotide Reductases; C416235:Dot1 protein, S cerevisiae; D011495:Histone-Lysine N-Methyltransferase