Chemical-Induced Read-Through at Premature Termination Codons Determined by a Rapid Dual-Fluorescence System Based on S. cerevisiae.
Nonsense mutations generate in-frame stop codons in mRNA leading to a premature arrest of translation. Functional consequences of premature termination codons (PTCs) include the synthesis of truncated proteins with loss of protein function causing severe inherited or acquired diseases. A therapeutic approach has been recently developed that is based on the use of chemical agents with the ability to suppress PTCs (read-through) restoring the synthesis of a functional full-length protein. Research interest for compounds able to induce read-through requires an efficient high throughput large scale screening system. We present a rapid, sensitive and quantitative method based on a dual-fluorescence reporter expressed in the yeast Saccharomyces cerevisiae to monitor and quantitate read-through at PTCs. We have shown that our novel system works equally well in detecting read-through at all three PTCs UGA, UAG and UAA.
Chemistry Department, University of Bari, Bari, Italy.
Emiliano
E
Altamura
Department of Life Sciences and Biotechnology, Biochemistry and Molecular Biology Section, University of Ferrara, Ferrara, Italy.
Monica
M
Borgatti
Department of Life Sciences and Biotechnology, Biochemistry and Molecular Biology Section, University of Ferrara, Ferrara, Italy.
Alessia
A
Finotti
Department of Life Sciences and Biotechnology, Biochemistry and Molecular Biology Section, University of Ferrara, Ferrara, Italy.
Jessica
J
Gasparello
Department of Life Sciences and Biotechnology, Biochemistry and Molecular Biology Section, University of Ferrara, Ferrara, Italy.
Roberto
R
Gambari
Institute of Biomembranes and Bioenergetics, National Researches Council, Bari, Italy.
Mariangela
M
Spinelli
Institute of Biomembranes and Bioenergetics, National Researches Council, Bari, Italy.
Rosa
R
Castaldo
Institute of Biomembranes and Bioenergetics, National Researches Council, Bari, Italy.
Nicola
N
Altamura
D018389:Codon, Nonsense; D018388:Codon, Terminator / Q000235:genetics*; D005453:Fluorescence; D017930:Genes, Reporter; D006801:Humans; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013489:Suppression, Genetic
D018389:Codon, Nonsense; D018388:Codon, Terminator; D012333:RNA, Messenger