Cloning and sequencing of the ura3 and ura5 genes, and isolation and characterization of uracil auxotrophs of the fungus Mortierella alpina 1S-4

• Published in: Bioscience, biotechnology, and biochemistry biotechnology, and biochemistry, 2004-02, Vol.68 (2), p.277-285
• Main Authors: Takeno, S. (Kyoto Univ. (Japan)), Sakuradani, E, Murata, S, Inohara Ochiai, M, Kawashima, H, Ashikari, T, Shimizu, S
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; AUXOTROPHY; Base Sequence; Biological and medical sciences; Blotting, Southern; CLONING; Cloning, Molecular; Culture Media; DNA, Complementary - biosynthesis; DNA, Complementary - genetics; DNA, Fungal - biosynthesis; DNA, Fungal - genetics; Fundamental and applied biological sciences. Psychology; Gene Library; GENES; Genes, Fungal - genetics; ISOLATION; Molecular Sequence Data; MORTIERELLA; Mortierella - genetics; Mortierella - metabolism; Mortierella alpina; MUTANTS; Mutation - genetics; NUCLEOTIDE SEQUENCE; Oils - metabolism; Orotate Phosphoribosyltransferase - genetics; Orotidine-5'-Phosphate Decarboxylase - genetics; TRANSFERASES; ura3 gene; ura5 gene; Uracil - chemistry; Uracil - metabolism; uracil auxotrophy; URACIL DERIVATIVES
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.68.277

The oil-producing fungus Mortierella alpina 1S-4 is an industrial strain. In order to prepare host strains for a transformation system for this fungus, six uracil auxotrophs were obtained by means of random mutation with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). When the activities of orotate phosphoribosyl transferase (OPRTase, EC 2.4.2.10) and orotidine-5′-phosphate decarboxylase (OMPdecase, EC 4.1.1.23) were examined in the mutants and wild strain, OPRTase activity was found to be completely absent in all mutants, on the other hand, OMPdecase activity was intact. The genomic DNA and cDNA of the ura5 gene encoding OPRTase and the ura3 gene encoding OMPdecase were cloned and sequenced. The Ura5p deduced amino acid sequence of this fungus showed highest similarity to that of Vibrio cholerae classed among prokaryote. Furthermore, the mutational points in the ura5 genes of two selected mutants were identified; a base-replacement and a base-insertion.