Characterization of the Aspergillus nidulans biotin biosynthetic gene cluster and use of the bioDA gene as a new transformation marker.

The genes involved in the biosynthesis of biotin were identified in the hyphal fungus Aspergillus nidulans through homology searches and complementation of Escherichia coli biotin-auxotrophic mutants. Whereas the 7,8-diaminopelargonic acid synthase and dethiobiotin synthetase are encoded by distinct genes in bacteria and the yeast Saccharomyces cerevisiae, both activities are performed in A. nidulans by a single enzyme, encoded by the bifunctional gene bioDA. Such a bifunctional bioDA gene is a genetic feature common to numerous members of the ascomycete filamentous fungi and basidiomycetes, as well as in plants and oömycota. However, unlike in other eukaryota, the three bio genes contributing to the four enzymatic steps from pimeloyl-CoA to biotin are organized in a gene cluster in pezizomycotina. The A. nidulans auxotrophic mutants biA1, biA2 and biA3 were all found to have mutations in the 7,8-diaminopelargonic acid synthase domain of the bioDA gene. Although biotin auxotrophy is an inconvenient marker in classical genetic manipulations due to cross-feeding of biotin, transformation of the biA1 mutant with the bioDA gene from either A. nidulans or Aspergillus fumigatus led to the recovery of well-defined biotin-prototrophic colonies. The usefulness of bioDA gene as a novel and robust transformation marker was demonstrated in co-transformation experiments with a green fluorescent protein reporter, and in the efficient deletion of the laccase (yA) gene via homologous recombination in a mutant lacking non-homologous end-joining activity.

Malignant transformation of DMBA/TPA-induced papillomas and nevi in the skin of mice selectively lacking retinoid-X-receptor alpha in epidermal keratinocytes.

Retinoid-X-receptor alpha (RXRalpha), a member of the nuclear receptor (NR) superfamily, is a ligand-dependent transcriptional regulatory factor. It plays a crucial role in NR signalling through heterodimerization with some 15 NRs. We investigated the role of RXRalpha and its partners on mouse skin tumor formation and malignant progression upon topical DMBA/TPA treatment. In mutants selectively ablated for RXRalpha in keratinocytes, epidermal tumors increased in size and number, and frequently progressed to carcinomas. As keratinocyte-selective peroxisome proliferator-activated receptor gamma (PPARgamma) ablation had similar effects, RXRalpha/PPARgamma heterodimers most probably mediate epidermal tumor suppression. Keratinocyte-selective RXRalpha-null and vitamin-D-receptor null mice also exhibited more numerous dermal melanocytic growths (nevi) than control mice, but only nevi from RXRalpha mutant mice progressed to invasive human-melanoma-like tumors. Distinct RXRalpha-mediated molecular events appear therefore to be involved, in keratinocytes, in cell-autonomous suppression of epidermal tumorigenesis and malignant progression, and in non-cell-autonomous suppression of nevi formation and progression. Our study emphasizes the crucial role of keratinocytes in chemically induced epidermal and melanocytic tumorigenesis, and raises the possibility that they could play a similar role in UV-induced tumorigenesis, notably in nevi formation and progression to melanoma.