Final Activity Report Summary - MUTANAL (Identification and mutational analysis of the nucleobase and nucleoside transporters of Aspergillus nidulans implicated in the transport of ... drugs)
The research objectives for the period from the 1st to the 12th months of the project were (i) the determination of the substrate specificity of seven nucleobase transporters (Perm1-Perm7) belonging to a distinct NCS1 (Nucleobase/Cation Symporter 1) transporter family and two nucleoside (ENT, CNT) transporters of Aspergillus nidulans and (ii) isolation of the nucleoside CNT and one of the nucleobase transporter genes in order to subject them to mutational analysis for the purpose to reveal the structure-function relationship of these enzymes and their substrate-binding centre.
Substrate specificities of five of the seven nucleobase transporters and one of the nucleoside transporters were determined by growth tests of deletion mutants and reinforced by developmental Northern analysis and uptake experiments. Perm1 was identified as exclusive allantoin transporter on the basis of growth test. perm1 deletion mutant was not able to grow when allantoin was applied as sole N-source in the media, while wild type grew normally. The result was reinforced by Northern analysis, which indicated that perm1 is regulated by its substrate: the presence of allantoin in the growth media affected the expression level of perm1 gene positively. Perm4 proved to be a high-affinity, high-capacity uracil permease of germinating conidia on the basis of growth test of deleted perm4 mutant on the toxic 5-fluoro uracil and kinetic uptake experiments, reinforced by developmental Northern analysis. perm4 is not expressed in resting conidiospores, is transcriptionally activated and reaches a peak during the isotrophic growth phase of conidiospore germination, and stays at a basic low level in mycelium.
Transcriptional expression is correlated with uracil transport activity. Perm5 is supposed to be involved in thyamine transport on the basis of Northern analysis. Expression level of perm5 is increased from a low basic level to a very high level in response to the presence of thyamine in the media during the isotrophic growth phase of conidiospore germination and stays at a high level in mycelium. Perm6 is involved in guanine transport on the basis of growth test of perm6 deletion mutant, where guanine was applied as sole N-source. perm3 is expressed similarly to perm4, although at an extremely low level, perm3 deletion mutant shows growth ability in presence of the toxic 5-fluoro uracil and kinetic experiments confirmed that Perm3 has minor role in uracil uptake since it is a low-affinity and low-capacity permease. Function of Perm2 and 7 remained unclear. Amongst the nucleoside type transporters, function of CNT was determined as adenosine and exclusive uridine permease on the basis of growth test of cnt deletion mutant on toxic analogues. According to the proposed project we started to develop the random mutagenesis on the cnt and perm1 genes.
Substrate specificities of five of the seven nucleobase transporters and one of the nucleoside transporters were determined by growth tests of deletion mutants and reinforced by developmental Northern analysis and uptake experiments. Perm1 was identified as exclusive allantoin transporter on the basis of growth test. perm1 deletion mutant was not able to grow when allantoin was applied as sole N-source in the media, while wild type grew normally. The result was reinforced by Northern analysis, which indicated that perm1 is regulated by its substrate: the presence of allantoin in the growth media affected the expression level of perm1 gene positively. Perm4 proved to be a high-affinity, high-capacity uracil permease of germinating conidia on the basis of growth test of deleted perm4 mutant on the toxic 5-fluoro uracil and kinetic uptake experiments, reinforced by developmental Northern analysis. perm4 is not expressed in resting conidiospores, is transcriptionally activated and reaches a peak during the isotrophic growth phase of conidiospore germination, and stays at a basic low level in mycelium.
Transcriptional expression is correlated with uracil transport activity. Perm5 is supposed to be involved in thyamine transport on the basis of Northern analysis. Expression level of perm5 is increased from a low basic level to a very high level in response to the presence of thyamine in the media during the isotrophic growth phase of conidiospore germination and stays at a high level in mycelium. Perm6 is involved in guanine transport on the basis of growth test of perm6 deletion mutant, where guanine was applied as sole N-source. perm3 is expressed similarly to perm4, although at an extremely low level, perm3 deletion mutant shows growth ability in presence of the toxic 5-fluoro uracil and kinetic experiments confirmed that Perm3 has minor role in uracil uptake since it is a low-affinity and low-capacity permease. Function of Perm2 and 7 remained unclear. Amongst the nucleoside type transporters, function of CNT was determined as adenosine and exclusive uridine permease on the basis of growth test of cnt deletion mutant on toxic analogues. According to the proposed project we started to develop the random mutagenesis on the cnt and perm1 genes.