Approaches to identify candidate genes for resistance to facial eczema disease in sheep

Description: xiv, 303 p. : ill. (some col.) : 30 cm. + 1 CD-ROM (4 3/4 in.) Notes: "June 2007" University of Otago department: Biochemistry. Thesis (Ph. D.)--University of Otago, 2007. Includes bibliographical references.Facial eczema disease (FE) is a secondary photosensitisation disease of ruminants caused by exposure to the mycotoxin sporidesmin. Resistance to FE has a significant genetic component and previous research has included a whole genome scan and investigation of candidate genes. The aim of this study was to use multiple approaches to identify genes associated with resistance to FE. ABC transporters have been considered as putative candidate genes for FE since the yeast ABC transporter, PDR5, was found to modulate sensitivity to sporidesmin in Saccharomyces cerevisiae. A previous study had shown that hepatic expression of the ovine ABC transporter, ABCB1, was induced following exposure to sporidesmin but only in resistant animals (Longley (1998) PhD Thesis, University of Otago). In the present study, using qRT-PCR, a difference in the expression of ABCB1 between resistant and susceptible animals was not confirmed. It is concluded that ABCB 1 is not likely to be a candidate gene for FE. As the full genome sequences for several mammalian species are now available, phylogenetic analyses were used to identify the most likely mammalian ortholog of the yeast PDR5 protein. This analysis found that the yeast PDR5 protein was most closely related to the mammalian ABCG sub-family. The human ABCG sub-family has five members one of which, ABCG2, is a known xenobiotic transporter. Comparative mapping of ABCG2 indicated that it co-localised to a region of the sheep genome weakly associated with resistance to FE. The full-length sequence of ovine ABCG2 was determined and two synonymous polymorphisms were found. These two polymorphisms, together with an intronic SNP were genotyped across a panel of selection-line animals. The allele frequencies of the intronic SNP were found to be significantly different between the selection lines, providing evidence for the association of ABCG2 with resistance to FE. The hepatic expression of ABCG2 was examined but no differential expression between the selection-lines was observed. Global gene expression profiling via microarray analysis was undertaken as a novel approach to identify candidate genes. Differences in gene expression were examined between naive and sporidesmin-dosed resistant and susceptible animals using a bovine cDNA microarray. A small number of differentially expressed genes were identified. Follow-up studies found that there were a relatively high number of errors in EST identity. Eight differentially expressed genes were selected for confirmation by Northern analysis. Six of these genes were shown to be differentially expressed, but neither the patterns nor the magnitude of the differential expression reflected that observed on the microarray. One of the six genes identified as differentially expressed was catalase, which has previously been implicated in resistance to FE. This finding validates the approach taken using gene expression profiling to identify candidate genes. The final approach used in this study necessitated the development and characterisation of an in vitro system for studying sporidesmin toxicity. The system chosen was a human hepatoma cell line, HepG2. To date the only effective treatment for FE is the prophylactic administration of high levels of zinc sulphate. The mechanism of protection by zinc is unknown, but zinc is known to be a potent modulator of gene expression. Conceptually, any genes modulated by zinc are possible candidates for resistance to FE. It was shown that zinc pre-treatment could protect HepG2 cells against sporidesmin-induced cytotoxicity. Equivalent protection was provided by the addition of zinc in the presence of the transcriptional inhibitor actinomycin D, suggesting that the mechanism of zinc protection is independent of de novo gene transcription. Overall, the goal of this project was to find genes to assist selection of sheep resistant to FE. Toward this goal, this research has identified several new candidate genes and avenues for investigation