Gene cluster involved in melanin biosynthesis of the filamentous fungus Alternaria alternata

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Abstract
The filamentous fungus Alternaria alternata produces melanin, a black pigment, from acetate via 1,8-dihydroxynaphthalene. To isolate a fungal gene required for melanin biosynthesis, we transformed an A. alternata Brm1- (light brown) mutant with the DNA of a wild-type strain genomic library constructed by use of a cosmid carrying the hygromycin B phosphotransferase gene. When hygromycin B-resistant transformants were screened for melanin production, 1 of 1,363 transformants appeared to regain melanin production, as judged by black pigmentation of the cultured mycelia. The cosmid, named pMBR1, was recovered by packaging nuclear DNA of the melanin-producing transformant into lambda phage. The gene on pMBR1 that enables the Brm1- mutant to produce melanin was designated BRM1. In addition to the BRM1 gene, pMBR1 was found to carry two more genes involved in melanin biosynthesis. These two genes, designated ALM and BRM2, transformed A. alternata Alm- (albino) and Brm2- (brown) mutants, respectively, to the wild-type phenotype. The three genes are located within a ca. 30-kb genomic region in the order ALM-BRM1-BRM2. Analysis of the gene transcripts indicated approximate sizes of 7.2, 4.0, and 0.9 kb for ALM, BRM1, and BRM2, respectively. The BRM1 and BRM2 transcripts are generated from the same strand, but the ALM transcript is generated from the opposite strand. The three mRNA species accumulate in cultured mycelia of the wild-type strain synchronously with mycelial melanization. The essential roles of the three genes in melanin biosynthesis were confirmed by transformation-mediated gene disruption experiments.