Changes in gene expression reveal pathways to galactose sensitivity in GALT-null Drosophila melanogaster. Público
Hang, Darwin Benjamin (2012)
Published
Abstract
Classic galactosemia is an autosomal recessive metabolic disorder with short and long term complications as a result of severe reduction in galactose-1-phosphate uridylyl transferase (GALT) activity. GALT is the second enzyme in the Leloir pathway of galactose metabolism. The mechanisms of both the short and long term outcomes are unknown. Our lab generated the first GALT-null animal model that displays acute and long term outcomes reminiscent of the human disease.
We chose to perform a gene expression based microarray experiment as an open minded approach to understand the changes that occur when GALT-null and WT larvae are exposed to galactose. GALT-null and WT L1 larvae were placed on galactose free food and then switched to either galactose free or galactose containing food. Analysis was done using Gene Ontology (GO). When both genotypes were exposed to galactose, there was enrichment for GO terms Glutathione S-Transferase (GST) and oxidation reduction processes. The number of genes involved in these processes was greater in the GALT-null larvae compared to the WT larvae, indicating that there may be differences in oxidative stress level or response in the GALT-null compared to the WT larvae after 12 hours of galactose exposure. Comparison of the gene expression profiles of the GALT-null and WT larvae raised on glucose showed increases in the amount of differentially expressed genes involved in mitochondrial, neurological, and metabolic processes.
The microarray data revealed that two GST genes showed a large increase in expression when both the GALT-null and WT larva were exposed to galactose. GSTD6 and GSTE7 have been shown to increase their expression as part of the Drosophila response to oxidative stress. Real Time PCR for these genes validated the microarray results. Both genes showed elevated expression when larvae were exposed to galactose, indicating that dietary galactose exposure induced oxidative stress. GSTD6 in particular showed a greater increase in expression level when the GALT-null larvae were exposed to galactose than when the WT larvae were exposed to galactose. In contrast, the reduction of galactokinase (GALK, the first enzyme in the Leloir pathway) activity had no significant effect on the expression of these genes.
Table of Contents
Chapter 1: Introduction 1
Chapter 1 References 5
Chapter 2: Gene expression analysis of GALT-null and WT larvae on glucose and galactose containing diets provides insight into pathways of the acute and long term outcomes in GALT-null Drosophila melanogaster 10
Materials and Methods 8
Results and Discussion 10
Conclusions 17
Supplementary Tables 19
Chapter 2 References 35
Chapter 3: Oxidative stress contributes to outcome severity in a Drosophila melanogaster model of classic galactosemia 37
Abstract 40
Introduction 41
Results 44
Discussion 58
Methods 65
Supplementary Data 67
Chapter 3 References 70
Chapter 4: The expression levels of GSTD6 and GSTE7 are not significantly altered by the reduction of GALK activity. 80
Methods 82
Results and Discussion 83
Supplementary Figure 86
Chapter 4 References 89
Chapter 5: Conclusion 90
Chapter 5 References 93
Appendix: UDP-galactose 4'-epimerase activities toward UDP-Gal and UDP-GalNAc play different roles in the development of Drosophila melanogaster. 94
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