published_or_final_version / Microbiology / Master / Master of Medical Sciences

Nine mutagen sensitive (mus) mutants of Aspergillus nidulans were analyzed for effects on recombination. These mus show increased sensitivity to various mutagens; several were UV-sensitive. The majority were sterile in homozygous crosses homozygous mus diploids were essential to test for mitotic recombination. / Two distinct tests were used: intergenic recombination between colour mutations and their centromeres; and, intragenic recombination between two distinguishable adE alleles. Both hyper- and hypo-rec mus mutations were identified, with 15- to 20-fold differences between them. Three mutations musN, musO, and musQ had greatly elevated recombination frequencies. Only one, musL significantly reduced recombination but consequently increased malsegregation. / Analysis of the genotype of ad$ sp{+}$ recombinants, from musN and musL diploids, revealed qualitative differences from mus$ sp{+}$. musN enhanced mitotic crossing over but not gene conversion; while musL reduced the frequency of simple convertants but increased that of complex homozygous ad$ sp{+}$ genotypes. Mitotic conversion and crossing over, generally associated with each other, were uniquely affected in such mus diploids.

Información extraída del <a href="http://www.cindefi.org.ar/?page_id=61&language=es">sitio web del CINDEFI</a>

No description available.

No description available.

In this study, several aspects of nitrate assimilation and transport have been studied using the filamentous fungus Aspergillus nidulans, which has been shown to be safe laboratory organism as judged by it's pathogenicity towards insect larvae. In silico analysis of the A. nidulans genome sequence, identified two putative genes designated cnxL and cnxK that might be involved in molybdenum cofactor (a component of nitrate reductase) biosynthesis as well as two putative nitrate reductases encoding genes niaB and niaC. All four genes are hitherto unknown. Although many features of these proteins provided clues of functionality, biochemical and genetical approaches employed in this present study failed to elicit expression of any of these four genes. A NrtA protein structure model was developed based on residue homology with the E. coli GlpT a protein, the structure of which has been solved. The results of thiol cross-linking of three double cysteine mutants in four NrtA essential residues, R87, R368, N168 and N459, indicated that the molecular distance between R87 and R368 is ~ 0.4 Å, R368 and N168 ~ 6.2 Å, R87 and N459 is ~ 2.2 Å. Another important observation was the change in the confirmation of Tm 2 and Tm 8 in the presence of nitrate. This shift resulted in an increase of ~ 2 Å gap between the residues R87 and R368. Distances between amino acid residue pairs estimated using such molecular rulers contradicted the NrtA existing model. Cysteine-scanning mutagenesis studies were extended to the generation of a library of single cysteine mutants of NrtA residues spanning Tm 2 and Tm 8. The majority of single cysteine mutants possessed wild type NrtA protein expression levels but unfortunately most were found to be loss-of-function. Consequently, thiol chemistry of this crop of mutants was not perused. Attempts were also made to overexpress and crystallise the bacterial nitrate transporters but none of the transporter tested proved to be a successful candidate for crystallisation. In this regard, bacterial nitrate transporters, NarU (E. coli), Nar (Bacillus cereus), NarK1 and NarK2 (Pseudomonas aeruginosa) and NarK2 (Thermus thermophilus) fused with GFP were expressed in E. coli and used in crystallisation trials. Although this approach has proved successful for a number of membrane proteins, unfortunately was not helpful with regard to the purification of any of the above bacterial nitrate transporters to yield protein expression levels required for successful protein crystallography. Finally, the effects of potential nitrate transport inhibitors were studied on net nitrate transport by NrtA and NrtB proteins of A. nidulans. The results indicated that chlorate had more of an inhibitory effect on NrtA net nitrate transport than that by NrtB. Chlorite and sulphite equally affected net nitrate transport by either NrtA or NrtB proteins while caesium strongly inhibited the net nitrate transport by NrtB transporter.

No description available.

Submerged cultures of Aspergillus flavus and Aspergillus niger were grown in a medium containing avidin, a substance which serves as an inhibitor of the conversion of acetate to malonate. Control cultures were grown without the addition of avidin.Analysis of the fatty acids produced by cultures grown in a medium containing avidin gave an increase in C16 fatty acids and a decrease in C18 fatty acids, suggesting that malonate plays an important role in the elongation of long chain fatty acids in these organisms. This effect was observed for five to fifteen hours.Evidence of the conversion of palmitate to stearate to oleate to linoleate was presented. That the conversion of oleate to linoleate involves a desaturase which is highly specific is suggested by the observation that, although there are a number of monoenoic acids present in these organisms, the only dienoic acid found was linoleic acid.

Human interleukin 6 (hIL6) is a multifunctional cytokine effecting the function and proliferation of many cell types. The further understanding of hIL6 and its possible medical applications rely on the availability of this protein. The filamentous fungus Aspergillus oryzae is an important industrial organism and is used for the large scale production of many enzymes. As this fungus has an impressive secretory output it was decided to attempt to produce hIL6 in this organism. The initial work was based on the development of a gene transfer system for A. oryzae in order that the hIL6 gene could be introduced to the organism. A transformation system based on the homologous nitrate reductase gene is described. This system yielded up to 800 transformants per µg plasmid DNA. Additionally, the adaptation of the transformation system based on the A. nidulans anuiS gene and the use of other transformation systems is reported. In order to ensure that the hIL6 gene was efficiently transcribed it was considered important that homologous control regions from highly produced and regulated A. oryzae genes were linked to the hIL6 gene. Therefore the genes encoding glucoamylase and a-amylase were isolated from A. oryzae. A method for the purification of A. oryzae Si nuclease is described and the amino acid sequence of the N terminus is reported. A. oryzae produces large amounts of extracellular proteases, a feature unlikely to be attractive in a heterologous host. Therefore the production of protease production in A. oryzae was studied. A method is described for the selection of protease deficient mutants. Using this method two protease mutants were isolated and these have been characterized. One mutation designated prtA2 protects against the degradation of hJL6 in vitro. The A. oryzae alkaline protease gene was isolated and mutagenised and the attempts made to produce specific protease mutant by reverse genetics are described. A system is described where by A. oryzae can be engineered to produce relatively high levels of hIL6. Using gene fusion constructs transformants producing in the range of 1 mg per Litre have been isolated. This system is heterologous, recommendations for increasing hIL6 production are included.

No description available.