The echinocandin-type antimycotic mulundocandin and its derivatives are produced by the filamentous fungus Aspergillus sydowii (strain FH2551). These agents have been considered as a potential drug to treat immunocompromised patients who suffer from severe opportunistic fungal infections. In order to generate strains with a modified mulundocandin biosynthesis, we developed molecular tools for genetic engineering of A. sydowii as an alternative to conventional strain improvement procedures. For our experiments, we used strain FH2551, which was discriminated from other Aspergillus strains by determining the sequence of the two internal transcribed spacers (ITS1 and ITS2) of the rDNA locus. In addition, the electrophoretic karyotype of A. sydowii was established using pulsed-field gel electrophoresis (PFGE), leading to a calculated genomic size of about 40 Mb. For gene mapping, chromosomes were subjected to PFGE either unrestricted or after incubation with rare cutting enzymes and probed with heterologous genes. Using the bacterial hygromycin B phosphotransferase gene as a selectable marker for transformation of A. sydowii, we generated transformants with single and multiple copies of plasmid DNA. Subsequently, the heterologous lacZ and gfp genes were efficiently transferred and expressed in A. sydowii. The majority of lacZ-transformants showed more than 6 pkat beta-galactosidase activity/mg protein, while the control strains had no significant background activity. Fluorescence microscopy of gfp-transformants demonstrated that the green-fluorescent protein is present in a stable and active form in the cytoplasm of vegetative hyphae and conidiospores.
von Hippel-Lindau (VHL) disease is a pleiotropic disorder featuring a variety of malignant and benign tumors of the eye, central nervous system, kidney, and adrenal gland. Recently the VHL gene has been identified in the chromosomal region 3p25-26. Prognosis and successful management of VHL patients and their descendants depend on unambiguous diagnosis. Due to recurrent hemangioblastomas, a29-year-old patient without familial history of VHL disease was diagnosed to be at risk for the disease. Histopathological examination of a small renal mass identified a clear cell tumor with a G1 grading. Genetic characterization of the germline and of the renal tumor was performed. Polymerase chain reaction/single strand conformation polymorphism (PCR/SSCP) analysis with primers from the VHL gene identified a deletion of a single nucleotide in exon 2 in the patient's germline and in the tumor, but not in the DNA of his parents. This deletion therefore must be a de novo mutation. Comparative genome hybridization (CGH) and fluorescence in situ hybridization (FISH) analysis of the G1 tumor with differentially labelled yeast artifical chromosome (YAC) clones showed loss of 3p and of the 3p26 signals, respectively. In conclusion, we identified a de novo germline mutation in the VHL gene of a young patient and a somatic chromosome 3p loss at the homologous chromosome 3 in his renal tumor. Our results suggest a recessive mode of inactivation of the VHL gene, providing solid evidence for its tumor-suppressor gene characteristics. Our data show the diagnostic potential of genetic testing, especially in patients without VHL family history. Furthermore, the findings of homozygous inactivation of the VHL gene in a G1 tumor support the notion that the inactivation of the VHL gene is an early event in tumorigenesis of renal cell carcinoma.
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