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.
Sr-CIV UP in 'ethanol resistant' vs 'wild type'
CG9837 DOWN in 'ethanol resistant' vs 'wild type'
LysP DOWN in 'ethanol resistant' vs 'wild type'
(Sample) organism/Drosophila melanogaster
(Factor value) PHENOTYPE/ethanol sensitive
CG33337 UP in 'ethanol resistant' vs 'wild type'
CG13313 UP in 'ethanol sensitive' vs 'wild type'
CG17752 DOWN in 'ethanol sensitive' vs 'wild type'
CG32751 DOWN in 'ethanol resistant' vs 'wild type'
Cyp28a5 DOWN in 'ethanol resistant' vs 'wild type'
CG15661 UP in 'ethanol sensitive' vs 'wild type'
CG11034 DOWN in 'ethanol resistant' vs 'wild type'
PGRP-SC1b DOWN in 'ethanol resistant' vs 'wild type'
CG5724 DOWN in 'ethanol sensitive' vs 'wild type'
Analysis of 'ethanol sensitive' vs 'wild type'
CG18417 UP in 'ethanol resistant' vs 'wild type'
Cyp6a17 UP in 'ethanol resistant' vs 'wild type'
CG30083 DOWN in 'ethanol sensitive' vs 'wild type'
(Factor value) PHENOTYPE/wild type genotype
CG6012 DOWN in 'ethanol sensitive' vs 'wild type'
CG10559 DOWN in 'ethanol resistant' vs 'wild type'
CG33109 DOWN in 'ethanol resistant' vs 'wild type'
CG6385 DOWN in 'ethanol resistant' vs 'wild type'
Cyp6a23 DOWN in 'ethanol resistant' vs 'wild type'
Analysis of 'ethanol resistant' vs 'wild type'
(Factor value) PHENOTYPE/ethanol resistant
Jon66Ci DOWN in 'ethanol sensitive' vs 'wild type'
CG11034 DOWN in 'ethanol sensitive' vs 'wild type'