Secondary metabolism in some members of the genus <I>Aspergillus</i> includes the production of aflatoxin, a secondary metabolite that is both immunosuppressive and carcinogenic to animals and humans. <I>A. flavus</i>, in particular, is well known for its ability to contaminate stored food products and to accumulate high levels of aflatoxin in these products. The fungus is also able to infect plants such as corn, cotton, peanuts, and tree nuts long before harvest. Extensive research has been conducted on <I>A. flavus</i> in areas such as population biology, ecology, pathogenicity, genetics, and genomics. The aflatoxin biosynthetic cluster has been sequenced, and researchers have identified regulatory mechanisms involved in the control of aflatoxin production. In spite of the large volume of research in this area, however, many unanswered questions remain concerning the genetic regulation of aflatoxin production and the molecular signals that intimately associate the synthesis of aflatoxin with specific environmental and nutritional conditions. In an effort to identify genes whose expression is affected by growth and aflatoxin production, a wild type strain of <I>A. flavus</i> was compared to a <I>fadA<sup>G42R</sup></i> mutant strain using a cDNA microarray enriched for genes temporally differentially expressed with respect to aflatoxin production. HPLC measurements confirmed that the wild type strain produced high levels of aflatoxin, while the <I>fadA<sup>G42R</sup></i> mutant strain was unable to synthesize any detectable level of the toxin. Hierarchical clustering of differentially expressed genes produced three clusters of genes induced in the wild type strain that contained known aflatoxin genes as well as other genes with no established role in secondary metabolism. A large cluster of genes induced in the <I>fadA<sup>G42R</sup></i> mutant strain included 15 ribosomal genes as well as genes showing homology to an alkyl hydroperoxide reductase (Ahp1p) from <I>Saccharomyces cerevisiae</i> involved in protection against oxidative stress. In order to further characterize this gene in <I>A. flavus</i>, designated <I>ahpA</i>, and its corresponding homolog in <I>A. nidulans</i>, the two genes were overexpressed in a <I>S. cerevisiae AHP1Ä</i> mutant strain. Transformants were tested against a range of concentrations of tert-butyl hydroperoxide (TBHP) to determine whether the genes from <I>A. flavus</i> and <I>A. nidulans</i> could protect against the sensitivity to TBHP observed in the mutant strain. Results indicated that although the protection was not as effective as that conferred by the native <I>AHP1</i> gene, <I>ahpA</i> and the <I>A. nidulans AHP1</i> homolog were able to protect the yeast cells against toxicity induced by exposure to TBHP.
| Identifer | oai:union.ndltd.org:NCSU/oai:NCSU:etd-07302004-202428 |
| Date | 02 August 2004 |
| Creators | Schwartzburg, Kimberly Ann |
| Contributors | Dr. Margaret Daub, Dr. Ralph Dean, Dr. Gary Payne |
| Publisher | NCSU |
| Source Sets | North Carolina State University |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://www.lib.ncsu.edu/theses/available/etd-07302004-202428/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to NC State University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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