Disruption of a Yeast ADE6 Gene Homolog in Ustilago maydis

Abstract

A putative homolog of the Sacharromyces cereviseae ADE6 and Escherichia coli purL genes is identified near a multigenic complex, which contains two genes, sid1 and sid2, involved in a siderophore biosynthetic pathway inUstilago maydis. The putative ADE6 homolog was mutated by targeted gene disruption. The resulting mutant strains demonstrated a requirement for exogenous adenine, indicating that the U. maydis ade6 homolog is required for purine biosynthesis. Authors M. L. Heidenreich, A. D. Budde, An Zhiqiang, and S. A. Leong This regular paper is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol55/iss1/10 40 Fungal Genetics Reports Disruption of a Yeast ADE6 Gene Homolog in Ustilago maydis Heidenreich, M. L., Budde, A. D., Zhiqiang, An,and Leong, S. A. Department of Bacteriology, University of Wisconsin, Madison, WI 53706. USDA, ARS CCRU, Madison, WI 53726. Department of Plant Pathology, University of Wisconsin, Madison, WI, 53706 Fungal Genetics Reports 55:40-43 A putative homolog of the Sacharromyces cereviseae ADE6 and Escherichia coli purL genes is identified near a multigenic complex, which contains two genes, sid1 and sid2, involved in a siderophore biosynthetic pathway in Ustilago maydis. The putative ADE6 homolog was mutated by targeted gene disruption. The resulting mutant strains demonstrated a requirement for exogenous adenine, indicating that the U. maydis ade6 homolog is required for purine biosynthesis. Ustilago maydis is the causal agent of corn smut disease. Under conditions of iron stress, this fungus produces cyclic peptides, siderophores, for the purpose of iron acquisition (Leong and Winkelmann, 1998). The limits of this gene cluster were investigated by systematically analyzing the sequence of the flanking DNA. The Ustilago genomic sequence of the region downstream of sid1 sequence showed a predicted 1402 amino acid polypeptide encoding a probable ade6 gene (http://mips.gsf.de/genre/proj/ustilago/singleGeneReport.html?entry=um05162), and having 55.7% similarity to the translated purL gene of E. coliB, and 54.2% similarity with the translated ADE6 gene of Saccharomyces cerevisiae. These genes encode formylglycineamide ribonucleotide synthetase, which catalyzes the fourth step in the purine biosynthetic pathway (Schendel et. al., 1989). Information contained in the S. cerevisiae Genbank sequence submission 557019 indicated that disruption of this gene leads to an adenine-requiring phenotype. To determine whether the predicted ade6 gene is required for purine biosynthesis, the gene was disrupted by insertion of a cassette encoding hygromycin phosphotransferase. Materials and Methods The HindlII-NruI fragment containing the 5’ region of the putative ade6 gene was derived from an 8.2 kb HindIII fragment of pSid1, a cosmid clone that contains a Sau3A partial digest of a region of the genome of Ustilago strain 518 (Wang et. al., 1989), Table 1. The 8.2 kb HindIII fragment was initially cloned into pUC18 followed by deletion of SmaI-NruI and internal NruI-NruI fragments to yield the 2.5 kb cloned HindIII-NruI insert (Fig. 1). Plasmid DNA isolation from E. coli was performed using the alkaline lysis protocol (Maniatis et. al., 1982). U. maydis chromosomal DNA isolation was performed by the glass bead technique (Voisard et. al., 1993). Restriction enzyme digestions were carried out as suggested by the manufacturer (New England Biolabs). E. coli transformation was carried out using the calcium shock method (Maniatis et. al., 1982). U. maydis transformation was performed as described (Voisard et. al., 1993). Radiolabeling, DNA ligation and synthesis were carried out as using standard procedures (Maniatis et. al., 1982). Colony and Southern hybridizations were performed as described (Holden et. al., 1989). The translated sequences were aligned pairwise using the Lipman Pearson Method in Lasergene 7.1 (DNAstar, Madison) and by multiple alignment using Clustal W in Lasergene 7.1 (DNAstar, Madison) with the translated sequence of ade6 generated in this study, a hypothetical Ade6 protein in the Ustilago genome (http://mips.gsf.de/genre/proj/ustilago/singleGeneReport.html?entry=um05162), E. coli PurL, and yeast Ade6. Strain Relevant Characteristics Source U. maydis #521 (FGSC 9914) wild type a1b1 Robin Holliday, National Institute for Medical Research, Mill Hill, Great Britain U. maydis #518 (FGSC 9914) wild type a2b2 Robin Holliday, National Institute for Medical Research, Mill Hill, Great Britain Table 1. Fungal Strains 1 Heidenreich et al.: Disruption of a Yeast ADE6 Gene Homolog in Ustilago maydis Published by New Prairie Press, 2017