The involvement of the multiple drug resistance family in facial eczema resistance
Facial Eczema (FE) is a disease of ruminants that was first reported in New Zealand at the beginning of this century. Today this disease is considered the most important mycotoxin affecting the sheep industry, causing an estimated 75 million dollars per year in lost revenue. FE is a secondary photosensitisation disease arising from liver dysfunction. The liver damage is caused by a mycotoxin, sporidesmin, produced in the spores of the saprophytic fungus Pithomyces chartarum. The cytotoxicity of sporidesmin is postulated to arise from its production of free radicals. The reduced (dithiol) form of sporidesmin undergoes autoxidation in a reaction that generates superoxide radicals (O2} It is the increased generation of these free radicals, overwhelming the ability of the cellular enzymes to reduce and detoxify them, that is the cause of sporidesmin toxicity. Differences among Romney sheep in their response to sporidesmin has been used to select lines which vary markedly in their resistance to the toxin. The biochemical and genetic basis of these differences is not known. The main research focus thus far has been on the study of antioxidant enzymes and their role in resistance to FE. A further candidate, the multiple drug resistant (MDR1) protein, has been implicated in cross resistance to many lipophilic cytotoxic compounds. Studies have indicated this phenomenon is due to decreased drug accumulation in resistant cells. This research was aimed at studying the role of MDRl in resistance to sporidesmin. With the discovery of a biological or genetic marker for FE resistance current selection techniques could be replaced by marker assisted selection. The sheep MDR1 gene was cloned and sequenced which enabled the design of both sheep specific probes and primers for the mapping of MDR1 and analysis of its segregation with FE resistance. The sheep .MDR1 gene showed high sequence and structural homology to other MDR family members, including 12 potential transmembrane spanning domains and consensus sequence for 2 nucleotide binding domains. The MDR1 gene was placed on the sheep genetic linkage map using RFLP analysis. Its localisation on sheep chromosome 4 is orthologous to the mapping of the human MDR genes on chromosome 7 and the mouse mdr family on chromosome 5. To analyse MDR1 segregation a highly polymorphic MDR1-linked microsatellite marker was isolated and screened across pedigrees in which resistance to FE was segregating. Results showed no linkage of the MDR1 marker with resistance or susceptibility to FE, suggesting that a structural change in the MDR1 gene per se was not linked to resistance. The amplification in this analysis of up to six allelic bands, segregating as two haplotypes, suggested the presence of at least three members in the sheep MDR family. Fragments were amplified from a second, MDR2, family member and used to map the gene adjacent to MDR1 on sheep chromosome 4. Promoter elements have previously been documented that differentially regulate MDR1. MDR1 mRNA levels were therefore studied in both resistant and susceptible animals using quantitative RT-PCR. Variation was detected in the level of MDR1 expression between resistant and susceptible animal samples from both hepatic and leukocyte cells. An increase in MDR1 expression was recorded in resistant cells exposed to sporidesmin, wheras susceptible cells showed no significant change in MDR1 expression on exposure to the toxin. These results suggest that resistant cells are able to induce the overexpression of MDR1 on exposure to sporidesmin. The mechanism by which this occurs or the proteins involved in the interaction are yet to be determined. In conclusion this research has demonstrated the sheep MDR family is composed of at least two gene members with high sequence homology to characterised MDR genes from other mammals. Hepatic overexpression of at least one of these, MDR1, is associated with resistance to sporidesmin. The differential expression in leukocyte cells may be exploited in an improved animal selection test that eliminates the need to expose valuable livestock to sporidesmin.
Advisor: Crawford, Allan
Degree Name: Doctor of Philosophy
Degree Discipline: Biochemistry
Publisher: University of Otago
Research Type: Thesis