03787nam a2200157 a 450000100080000000500110000800800410001910000190006024500700007926000160014930000110016550000650017652033530024165000250359465000100361912450912003-05-21       2000    bl uuuu  m   00u1 u    #d1 aANDRADE, A. C.  aABC transportes and multidrug resistance in aspergillus nidulans.  a2000.c2000  a157 p.  aThesis (Doctor) - Wageningen Universiteit, Wageningen, 2000.  aThe ATP-binding cassette (ABC) transporters comprise a large and multifuncional family of proteins. ABC transporters are present from archae-bacteria to man but became especiallly known for their involvement in multidrug resistance (MDR) in tumour cells. MDR in often accompanied by a massive overproduction of ABC transporters. ABC transporters are also involved in various human diseases such as cystic fibrosis, adrenoleukodystrophy, the Tangier disease and familial high-density lipoprotein deficiency. Furthermore, they play a role as peptide transporters in antigen presentation. Thhe majority of the ABC transporters in higher organisms consists of two transmenbrane domains (TMD), each with six predicted membrane spanning regions, and two nucleotide binding domain can be either located at the amino terminus or at the carboxy terminous of the polypeptide, yielding proteins with a [TMD-NBF]2 or [NBF-TMD]2 configuration. In chapter 1 an overview of the remarkable variety of cellular functions that these proteins can perforn in all living cells , is presented. The main goal  of the studies presented in this thesis was to understand the role of ABC-transporter proteins in MDR of the filamentous fungus Aspergillus nidulans. Genetic and biochemical data previously generated in the laboratory of phytopathology, demonstrate that resistance to azole fungicides in laboratory-generated ima (imazalil-resistant) mutans is based on an increased energy-dependent efflux mechanism that prevents intracellular accumulation of the fungicide. This observation, provided the basis for the present work. Similar efflux mechanisms are described for human cancer cells with a MDR phenotype. MDR in cancer cells is conferred by overexpression of the human ABC transporter MDR1 or P-glycoprotein. Therefore, our research focused on a search for ABC-transporter homologues in the A. nidulans genome. These studies resulted in the characterization of the first two ABC transporter genes (atrA and atrB) of the ATP-binding cassette superfamily from a filamentous fungus (chapter 2). In a addition, we report the caracterization of five additional ABC-transporter-encoding (atrC-atrG) genes from this fungus (chapter 3 and 5). By now, the superfamily of ABC transporters has more than a thousand members indentified. Hence it comproses the largest protein family known to date, and many additional atr genes are expected to occur in the genome of A. nidulans. The role the identified atr genes in MDR was studied by expression analysis after drug treatment and assessing the sensivity of genetically-engineered deletion and overexpression mutants of atr genes ( chapters 2-6). Expression of atr genes was also analyzed in the ima mutants of A. nidulans (chapter 5). Biochemical ewxperiments confirmed that altered sensitivity to fungicides observeds in the ar deletion and overexpression mutants can be ascribed to differential accumulation of the compounds in fungal mycelium (chapters 3,. 4 and 6). Attebtion was also focussed on a putative role of atr genes in secretion of endogenous secondary metabolites. More Specifically, we have tested the hypothesis whether atr genes play a role in penicillin production (chapter 3). In chapter 7, the results obtained in our studies and relevant aspects that may apply of other filamentous fungi are discussed.  aAspergillus Nidulans  aFungo