Project description:Alcoholism is a complex disorder determined by interactions between genetic and environmental risk factors. Drosophila represents a powerful model system to dissect the genetic architecture of alcohol sensitivity, as large numbers of flies can readily be reared in defined genetic backgrounds and under controlled environmental conditions. Furthermore, flies exposed to ethanol undergo physiological and behavioral changes that resemble human alcohol intoxication, including loss of postural control, sedation, and development of tolerance. We performed artificial selection for alcohol sensitivity for 25 generations and created duplicate selection lines that are either highly sensitive or resistant to ethanol exposure along with unselected control lines. We used whole genome expression analysis to identify 1,678 probe sets with different expression levels between the divergent lines, pooled across replicates, at a false discovery rate of q < 0.001. We assessed to what extent genes with altered transcriptional regulation might be causally associated with ethanol sensitivity by measuring alcohol sensitivity of 37 co-isogenic P-element insertional mutations in 35 candidate genes, and found that 32 of these mutants differed in sensitivity to ethanol exposure from their co-isogenic controls. Furthermore, 23 of these novel genes have human orthologues. Combining whole genome expression profiling with selection for genetically divergent lines is an effective approach for identifying candidate genes that affect complex traits, such as alcohol sensitivity. Because of evolutionary conservation of function, it is likely that human orthologues of genes affecting alcohol sensitivity in Drosophila may contribute to alcohol-associated phenotypes in humans. Keywords: artificial selection, whole genome expression profiling
Project description:The National Institute on Alcohol Abuse and Alcoholism has estimated that approximately 14 million people in the United States suffer from alcoholism. Alcohol sensitivity, the development of tolerance to alcohol and susceptibility to addiction vary in the population. Whereas environmental factors, such as stress and social experience, contribute to individual variation in sensitivity to chronic alcohol consumption, genetic factors have also been implicated. However, genetic polymorphisms that predispose to alcoholism remain largely unknown due to extensive genetic and environmental variation in human populations. Drosophila, however, allows studies on genetically identical individuals in controlled environments. Although addiction to alcohol has not been demonstrated in Drosophila, flies show responses to alcohol exposure that resemble human intoxication, including hyperactivity, loss of postural control, sedation, and exposure-dependent development of tolerance. We assessed whole-genome transcriptional responses following alcohol exposure and demonstrate immediate down-regulation of olfactory sensitivity and, concomitant with development of tolerance, altered transcription of enzymes associated with fatty acid biosynthesis. Our results identify key enzymes in conserved metabolic pathways that may contribute to human alcohol sensitivity. Keywords: Drosophila, model system, alcohol sensitivity, tolerance
Project description:Alcoholism is a complex disorder determined by interactions between genetic and environmental risk factors. Drosophila represents a powerful model system to dissect the genetic architecture of alcohol sensitivity, as large numbers of flies can readily be reared in defined genetic backgrounds and under controlled environmental conditions. Furthermore, flies exposed to ethanol undergo physiological and behavioral changes that resemble human alcohol intoxication, including loss of postural control, sedation, and development of tolerance. We performed artificial selection for alcohol sensitivity for 25 generations and created duplicate selection lines that are either highly sensitive or resistant to ethanol exposure along with unselected control lines. We used whole genome expression analysis to identify 1,678 probe sets with different expression levels between the divergent lines, pooled across replicates, at a false discovery rate of q < 0.001. We assessed to what extent genes with altered transcriptional regulation might be causally associated with ethanol sensitivity by measuring alcohol sensitivity of 37 co-isogenic P-element insertional mutations in 35 candidate genes, and found that 32 of these mutants differed in sensitivity to ethanol exposure from their co-isogenic controls. Furthermore, 23 of these novel genes have human orthologues. Combining whole genome expression profiling with selection for genetically divergent lines is an effective approach for identifying candidate genes that affect complex traits, such as alcohol sensitivity. Because of evolutionary conservation of function, it is likely that human orthologues of genes affecting alcohol sensitivity in Drosophila may contribute to alcohol-associated phenotypes in humans. Experiment Overall Design: Starting with flies from Raleigh natural population (see material and methods) we performed artificial selection for alcohol sensitivity for 35 generation. In each generations we scored 60 males and females, separately, from each line (resistant, sensitive, and control)using inebriometer, and the 20 highest-scoring flies from the resistant lines and the 20 lowest-scoring flies from the sensitive lines were selected as parents for the next generation. Control line flies were scored each generation and 20 random flies were used as parents. Experiment Overall Design: At generation 25, two replicates of 15 three-five day old virgin males and females were collected from each selection line. Total RNA was extracted from the 24 samples . Biotinylated cRNA probes were hybridized to high density oligonucleotide microarrays (Affymetrix, Inc. Drosophila GeneChip 2.0) and visualized with a streptavidin-phycoerythrin conjugate, as described in the Affymetrix GeneChip Expression Analysis Technical Manual (2000), using internal references for quantification. The quantitative estimate of expression of each probe set is the Signal (Sig) metric, as described in the Affymetrix Microarray Suite, Version 5.0.
Project description:Thomas Hunt Morgan and colleagues identified variation in gene copy number in Drosophila in the 1920s and 1930s and linked such variation to phenotypic differences [Bridges, C. B. (1936) Science 83, 210]. Yet the extent of variation in the number of chromosomes, chromosomal regions, or gene copies, and the importance of this variation within species, remain poorly understood. Here, we focus on copy-number variation in Drosophila melanogaster. We characterize copy-number polymorphism (CNP) across genomic regions, and we contrast patterns to infer the evolutionary processes acting on this variation. Copy-number variation in D. melanogaster is non-randomly distributed, presumably due to a mutational bias produced by tandem repeats or other mechanisms. Comparisons of coding and noncoding CNPs, however, reveal a strong effect of purifying selection in the removal of structural variation from functionally constrained regions. Most patterns of CNP in D. melanogaster suggest that negative selection and mutational biases are the primary agents responsible for shaping structural variation. Keywords: comparative genomic hybridization