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This dissertation, Cloning and Characterization of Putative Molecular Targets of Penicillium Marneffei Identified by Random Genome Exploration by Yee-lam, Elim, Cheung, 張以琳, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled Cloning and characterization of putative molecular targets of Penicillium marneffei identified by random genome exploration Submitted by Cheung Yee Lam Elim for the degree of Master of Philosophy at the University of Hong Kong in December 2002 Penicillium marneffei is a dimorphic fungus causing intracellular infection of the reticulo-endothelial system in humans and bamboo rats. It is endemic in Southeast Asia and causes opportunistic infection in about 10% of AIDS patients in this region. The absence of an identified sexual stage of P. marneffei, the potentially infectious nature of the mould phase conidia, and the difficulty of achieving a complete yeast form of the tissue-invasive phase in vitro have markedly impaired the understanding of the molecular biology and biochemical mechanisms in the thermal dimorphic switching and host-microbe interactions of this fungus. Its thermal dimorphism and the propensity to cause infection in the AIDS patients makes it an ideal candidate for genomic analysis which may provide crucial information in understanding the morphogenesis of the fungus and its interaction with the immune system in pathogenesis. Pulsed field gel electrophoresis of the genomic DNA of P. marneffei showed the presence of three (5.0 Mb, 4.0 Mb, and 2.2 Mb) or more chromosomes. Results obtained from telomeric fingerprinting revealed the presence of 6-12 bands, suggesting that there were chromosomes of similar sizes. The genome size of P. marneffei was hence about 17.8-26.2 Mb. Random exploration of the genome of P. marneffei yielded 2303 random sequence tags (RSTs), corresponding to 10-15% of the genome. The G+C content of the genome is 48.8%. 11.7%, 6.3%, and 17.4% of the RSTs showed homology to yeast-specific sequences, fungus non-yeast sequences, and sequences common to both forms respectively. Analysis of the RSTs revealed genes for information transfer (ribosomal protein genes, tRNA synthetase subunits, translation initiation and elongation factors), metabolism, and compartmentalization, (several multi-drug resistance protein genes and homologues of fluconazole resistance gene). The presence of genes encoding pheromone homologues and ankyrin repeat- containing proteins of other fungi and algae strongly suggests the presence of a sexual stage that presumably exists in environmental conditions. Random exploration of the P. marneffei genome revealed sequences that encode putative methionine aminopeptidase (Met-AP2), acid protease, phospholipase B, and DnaJ. These represent putative virulence factors and molecular targets for antifungal development. Comparison of the cDNA sequences and the genomic DNA sequences revealed that there are six and two introns in MAP2 gene and phospholipase B gene respectively, whereas the acid protease and dnaJ genes contain no intron. None of the four proteins were immunogenic. Characterizations of the functions of Met-AP2 and acid protease were carried out. For Met-AP2, complementation experiments showed that the function of Met-AP in Met-AP deficient E. coli can be complemented by Met-AP2 of P. marneffei. As for the acid protease, results from zymography and protease assay using azocasein as substrate revealed that the purified acid protease functioned best at pH 5. DOI: 10.5353/th_b2664260 Subjects: Penicillium -

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