TheURA5 gene encoding orotate-phosphoribosyl transferase of the yeastKluyveromyces lactis: Cloning, sequencing and use as a selectable marker
✍ Scribed by Bai, Xiyuan; Larsen, Michael; Meinhardt, Friedhelm
- Publisher
- John Wiley and Sons
- Year
- 1999
- Tongue
- English
- Weight
- 264 KB
- Volume
- 15
- Category
- Article
- ISSN
- 0749-503X
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✦ Synopsis
A pair of degenerate primers was used for amplification and cloning of an internal fragment of the K. lactis URA5 gene. Primers were designed on the basis of highly conserved motifs within protein sequences predicted for URA5 genes from several microorganisms. Using the amplified fragment as a probe, we finally cloned and sequenced a 1•9 kb chromosomal fragment containing the orotate-phosphoribosyltransferase-encoding URA5 gene and an incomplete open reading frame strikingly similiar to SEC65 of Saccharomyces cerevisiae and other yeasts, in which the gene encodes a subunit of the signal recognition particle. Uracil-requiring mutants of K. lactis CBS 683 were selected on media containing 5-fluoro-orotic acid and used as recipients in transformation experiments using K. lactis URA5 as the selectable marker, thereby proving functionality of the cloned gene.