The URA5 gene encoding orotate-phosphoribosyl transferase of the yeast Kluyveromyces lactis: Cloning, sequencing and use as a selectable marker

A pair of degenerate primers was used for amplification and cloning of an i nternal fragment of the K. lactis URA5 gene. Primers were designed on the b asis 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 1.9kb chromosomal fragment containing t he orotate-phosphoribosyltransferase-encoding URA5 gene and an incomplete o pen reading frame strikingly similar to SEC65 of Saccharomyces cerevisiae a nd other yeasts, in which the gene encodes a subunit of the signal recognit ion particle. Uracil-requiring mutants of K. lactis CBS 683 were selected o n media containing 5-fluoro-orotic acid and used as recipients in transform ation experiments using K. lactis URA5 as the selectable marker, thereby pr oving functionality of the cloned gene. The sequence presented here has bee n submitted to the EMBL data library under Accession No. AJ001358. Copyrigh t (C) 1999 John Wiley & Sons, Ltd.