New vectors for simplified construction of BrdU-Incorporating strains ofSaccharomyces cerevisiae
| Autor: | Oscar M. Aparicio, Christopher J. Viggiani |
|---|---|
| Rok vydání: | 2006 |
| Předmět: |
DNA Replication
Genetic Vectors Saccharomyces cerevisiae Gene Dosage Bioengineering Polymerase Chain Reaction Thymidine Kinase Applied Microbiology and Biotechnology Biochemistry chemistry.chemical_compound Gene Expression Regulation Fungal Genetics URA3 DNA Fungal Nucleotide salvage Selectable marker biology DNA replication biology.organism_classification Molecular biology Cell biology Bromodeoxyuridine chemistry Thymidine kinase Thymidine Biotechnology |
| Zdroj: | Yeast. 23:1045-1051 |
| ISSN: | 1097-0061 0749-503X |
| DOI: | 10.1002/yea.1406 |
| Popis: | The thymidine analogue BrdU is a powerful tool for analysing nucleotide incorporation in studies of DNA replication or repair. S. cerevisiae lacks the thymidine salvage pathway that enables efficient cellular uptake and incorporation of thymidine analogues into DNA. Recent in vivo reconstitution of this pathway in yeast by high-level expression of Herpes simplex virus thymidine kinase (HSV-TK) or combined expression of HSV-TK and human equilibrative nucleoside transporter (hENT1) has enabled analysis of BrdU incorporation in yeast. While the BrdU incorporation systems are highly valuable, the construction and use of strains utilizing these systems can be complicated by specific requirements of the available systems. We have created a set of vectors that simplify the construction and use of BrdU-Incorporating (BrdU-Inc) strains of budding yeast. Each vector in the set contains HSV-TK and hENT1 under the control of promoters that express constitutively, and one of four different selectable markers (HIS3, TRP1, LEU2 or URA3) for genomic integration. With these BrdU-Inc vectors, one-step integration of a single copy produces yeast that efficiently incorporate BrdU upon its addition to the medium. These vectors ease strain construction and maintenance, thereby facilitating routine use of BrdU for analysis in yeast. |
| Databáze: | OpenAIRE |
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