Identifying target proteins of the CreB deubiquitination enzyme in the fungus Aspergillus nidulans.

Carbon catabolite repression in A. nidulans is a regulatory system which allows the organism to utilize the most preferable carbon source by repressing the expression of genes encoding enzymes utilizing alternative carbon sources. A ubiquitination pathway was shown to be one of the key mechanisms which regulate carbon source utilization, when creB was found to encode a deubiquitinating enzyme. Strains containing mutations in creB show loss of repression for some metabolic pathways in carbon catabolite repressing conditions, and also grow very poorly on several sole carbon sources such as quinate and proline, suggesting CreB plays multiple roles in the cell. This work describes the analysis of the interaction of CreB with CreA, and with PrnB and QutD. Various epitope-tagged versions of CreA were expressed in A. nidulans, and an internally located HA-epitope tag was found to allow detection of CreA using Western analysis. A diploid strain was constructed between strains containing HA-tagged CreA and FLAG-tagged CreB. When CreB was immunoprecipitated, HA-tagged CreA was also precipitated in the diploid, indicating that CreA and CreB are present in a complex in vivo. To determine whether CreA is a ubiquitinated protein, a version of CreA that was tagged with both an HA epitope and a His-tag was expressed in A. nidulans, and protein extracts were precipitated with an UbiQapture™-Q matrix. Western analysis was used to show that CreA was present in the precipitate. These findings suggest that CreA is a ubiquitinated protein, and a target of the CreB deubiquitination enzyme. To determine whether the proline permease (PrnB) is a direct substrate of CreB, plasmids to express epitope-tagged versions of PrnB were constructed and introduced into the prnB mutant strain. No tagged protein could be detected by Western analysis, even when these constructs were over-expressed from the gpdA promoter. However, a construct to express an HA epitope tagged version of quinate permease (QutD) fully complemented the qutD mutant strain, and HA-tagged QutD could be easily detected in Western analysis when probed with the anti-HA monoclonal antibody. A diploid strain was made between a complementing transformant and a strain expressing a FLAG-tagged CreB construct. When QutDHA was immunoprecipitated, CreBFLAG was detected in the immunoprecipitate of the diploid. A proportion of QutDHA was also co-precipitated in the diploid when CreBFLAG was immunoprecipitated. Thus, CreB is present in a complex with QutD in vivo. Further results showed that the concentration of QutD in the cell is lower in a creB null mutant background than in the wild-type background, indicating that deubiquitination is required to prevent protein turnover. Northern analysis of mRNA showed that the failure of creB mutant strains to grow on quinate medium was not due to a failure of transcriptional induction of qutD, as the amount of mRNA was not lower in a creB1937 mutant background compared to the wild-type. Furthermore, experiments were undertaken that showed that QutD is a ubiquitinated protein. These findings suggest that quinate permease is regulated through deubiquitination involving the CreB deubiquitination protein in A. nidulans. In addition to the candidate protein approach asking whether CreA is a substrate of CreB, a proteomics approach was also used to identify proteins that interact with CreA. However, no clear interacting proteins were identified using this approach.Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 200

Polyunsaturated fatty acids production by Schizochytrium sp. isolated from mangrove

Five Schizochytrium strains (N-1, N-2, N-5, N-6, and N-9) were isolated from fallen, senescent leaves of mangrove tree (Kandelia candel) in Hong Kong. The fungi were cultivated in glucose yeast extract medium containing 60 g of glucose, 10 g of yeast extract and 1 L of 15‰ artificial seawater, initial pH 6.0, with shaking for 52 hr at 25ºC. Biomass yields of 5 isolates ranged from 10.8 to 13.2 g/l. Isolate N-2 yielding the highest dried cell mass at 13.2 g/l and isolate N-9 grew poorly with 10.8 g/l of biomass. EPA (Eicosapentaenoic acid, 20:5n-3) yield was low in most strains, while DHA (Docosahexaenoic acid, 22:6n-3) was high on the same medium. The contents of DHA in biomass varied: 174.9, 203.6, 186.1, 171.3 and 157.9 mg/g of dried-biomass for Schizochytrium isolate N-1, N-2, N-5, N-6, and N-9, respectively. Isolate N-2 had the highest proportion of DHA in fatty acid profile with 15:0, 28.7%; 16:0, 21.3%; 18:0, 0.9%; 18:3, 0.2%; 20:4, 0.3%; 20:5, 0.9%; 22:4, 6.7%; 22:6, 36.1%; and others, 9.3%. The salinity range for growth of Schizochytrium isolates was from 0-30‰ with optimum salinity for growth between 20-30‰

Effects of Polymer Impregnation on Properties of Bamboo

This research is targeted to investigate the effects of polymer impregnation on the properties of bamboo. Polymer impregnation can improve adhesion of flattened bamboo and also improve the resistance of polymer-impregnated bamboo to fungi. During the flattening bamboo process, linseed oil was used as a processing aid. Two different kinds of solvents, methanol and ethanol, were used to extract linseed oil. Weights of samples before and after extraction were compared. Ethanol is a better solvent than methanol to extract linseed oil since weight loss is higher. The flattened bamboo samples were adhered together by phenol-formaldehyde (PF) adhesive or polymeric diphenylmethane diisocyanate (MDI) adhesive. The adhesion test was performed in shear mode. Sample extracted with ethanol and adhered by MDI gave a maximum shear stress of 5.84 MPa while samples extracted with methanol and adhered by PF gave only 2.45 MPa. A higher percentage of wood failure was observed in samples with higher adhesion strength. In durability to fungi, a polymer-wood composite was made by impregnation of methyl methacrylate in the wood under vacuum. The composite showed better resistance to fungi, especially, in nodes. Nodes have a lower densities and less effective packing of fibers than internodes. Therefore monomers can diffuse into node and then polymerize. More polymer loading gives better durability to fungi

Effects of Polymer Impregnation on Properties of Bamboo

ABSTRACT This research is targeted to investigate the effects of polymer impregnation on the properties of bamboo. Polymer impregnation can improve adhesion of flattened bamboo and also improve the resistance of polymer-impregnated bamboo to fungi. During the flattening bamboo process, linseed oil was used as a processing aid. Two different kinds of solvents, methanol and ethanol, were used to extract linseed oil. Weights of samples before and after extraction were compared. Ethanol is a better solvent than methanol to extract linseed oil since weight loss is higher. The flattened bamboo samples were adhered together by phenol-formaldehyde (PF) adhesive or polymeric diphenylmethane diisocyanate (MDI) adhesive. The adhesion test was performed in shear mode. Sample extracted with ethanol and adhered by MDI gave a maximum shear stress of 5.84 MPa while samples extracted with methanol and adhered by PF gave only 2.45 MPa. A higher percentage of wood failure was observed in samples with higher adhesion strength. In durability to fungi, a polymer-wood composite was made by impregnation of methyl methacrylate in the wood under vacuum. The composite showed better resistance to fungi, especially, in nodes. Nodes have a lower densities and less effective packing of fibers than internodes. Therefore monomers can diffuse into node and then polymerize. More polymer loading gives better durability to fungi